JP2003079369A - Breast-specific gene and protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polypeptide of a human breast-specific gene, a nucleic acid encoding the same, a procedure for recombinant production of the polypeptide, use of these as a diagnostic marker of breast cancer and as a reagent for determining metastasis, a method for screening an antibody and an antagonist to a polypeptide of a breast cancer-specific gene and a therapeutic use thereof. SOLUTION: Disclosed is an isolated polynucleotide containing a member selected from a group consisting of the follow: a polynucleotide encoding the same polypeptide as a polypeptide encoded by a polynucleotide having a specific sequence; a polynucleotide encoding the same mature polypeptide as a polypeptide encoded by a human gene having a coding part containing a DNA having at least 90% identity with a DNA included in a clone having another specific sequence; and a polynucleotide hybridizable to the polynucleotide and having at least 70% identity with the polynucleotide.

Description

Description: BACKGROUND OF THE INVENTION [0001] The present invention relates to a newly identified
A polynucleotide, such a polynucleotide
Polypeptides and breast disorders, especially breast cancer
For the detection of breast cancer metastases
It relates to the use of polynucleotides and polypeptides. Book
The invention further relates to the production and function of the polypeptides of the invention.
Regarding the inhibition of 20 breast-specific genes of the present invention
Are sometimes referred to as "BSG1", "BSG1"
2 "etc. [0002] The mammary gland is subject to various easily detectable disorders.
Provided. Detection is usually achieved by inspection consisting of palpation
Can be done. Unfortunately, there is little regular self-examination
Are not occupied, so many breast lumps
Discovered only by consultation. Fine gauge suspect cysts
Aspiration with a needle is another fairly common diagnostic
It is an implementation. Mammography or dry radiography of the breast
Woven X-ray) is yet another diagnostic practice. Lesion area
Biopsy of suspect areas is the best method of diagnostic testing
You. [0003] Many types of tumors that affect the mammary gland
And cysts. Fibroadenoma is the most common benign breast
Tumor. Pathological entities include cyst disease and
Following carcinoma and then third. these
Tumors are most often found in young people and are usually
Be recognized. Because do you feel encapsulated
It is. The most common benign condition of fibrous cysts is
It is a common female breast disease, with about 20% premenopausal
Happens to women. Lipomas of the breast are also common, and
These are benign in nature. Breast carcinoma in a woman
Is the most common malignant condition among women and affects women.
It has the highest mortality rate among all cancers. One of women
At one point in life, 15 women in the United States
One person develops breast cancer in the sex. The reported year
The incidence between 1947 and 100,000 of the population
70 per woman, 72.5 for whites in 1969
From 47.8 to 60.1 for black and black
Rose. The annual mortality rate from 1930 to the present
Remains fairly constant, female population 100,000
About 23 people. Breast cancer is rare in men
But when it happens, it is usually not recognized until late,
Therefore the outcome of the treatment is not good. In women, the breast
Carcinomas are rarely seen before the age of 30, and the incidence is
It rises rapidly after menopause. For this reason, postmenopausal milk
Until the lump in the bunch is proven to be otherwise,
Should be considered. [0004] One aspect of the present invention relates to:
According to the human breast-specific gene of the present invention transcribed
Specific to RNA or DNA corresponding to such RNA
Contains a nucleic acid molecule of sufficient length to hybridize
A nucleic acid probe is provided. [0005] According to another aspect of the invention, host-derived support is provided.
Transcribed from the human breast-specific gene of the present invention
RNA or DNA corresponding to such RNA
By detecting the presence, breast cancer formation and breast cancer
Methods and products for diagnosing metastasis of are provided. According to yet another aspect of the present invention, a host-derived
In the sample corresponding to the breast-specific gene of the present invention.
Detects altered levels of the peptide, thereby
Elevated levels of polypeptides may indicate a diagnosis of breast cancer
To diagnose breast cancer formation and breast cancer metastasis
Methods and products are provided. [0007] According to another aspect of the present invention, mRNA,
DNA, cDNA, genomic DNA, and their
Antisense analogs and biologically active and diagnostic
Humans containing those fragments useful for ablation or treatment
An isolated polynucleic acid encoding a breast-specific polypeptide
A nucleotide is provided. [0008] According to yet another aspect of the present invention, the sequence listing
A human breast-specific gene comprising the polynucleotide of claim
Provided. According to a further aspect of the present invention, polynucs
Novel polypeptide encoded by leotide, and
Biologically active and diagnostically or therapeutically useful
Of fragments, analogs and derivatives of
You. According to a still further aspect of the present invention, the invention
Recombinant prokaryotic host cells and
And / or a recombinant eukaryotic host cell,
Facilitates the expression of quality and subsequent recovery of the protein
Under recombinant conditions, including the step of culturing
Provided are methods for producing such polypeptides
Is done. According to a still further aspect of the present invention,
Antibodies specific for such polypeptides,
Provides antibodies that can be used to detect breast cancer metastasis
Is done. According to another aspect of the invention, one or more books
Use polypeptides of the invention to treat breast cancer
And a method for converting this polypeptide to this polypeptide
Compounds that interact with the peptide (for example, the polypeptides of the present invention)
Compounds that inhibit or activate
There is provided a method for using According to yet another aspect of the invention, one or more
Of the polynucleotide and / or polypeptide of the present invention
Inhibits activation of the drug, for therapeutic use, for example, in the treatment of breast cancer
Screen for detecting compounds that can be used in
Is provided. According to a still further aspect of the present invention,
Such a polypeptide, or such a polypeptide
Encoding polynucleotides can be used for scientific research, DNA
In vitro for synthesis and production of DNA vectors
A method is provided for utilizing for a purpose. These and other aspects of the present invention are described herein.
It should be apparent to those skilled in the art from the teachings herein. Means for Solving the Problems In one aspect, the present invention
The invention relates to an isolated polynucleotide, comprising: (a) the polynucleotide according to FIG. 1 (SEQ ID NO: 1);
A polynucleotide encoding the same polypeptide as: (b) one of FIGS. 2-20 (SEQ ID NOs: 2-20)
DNA having at least 90% identity to the DNA of
Mature polymorphs identical to human genes having a coding portion containing
A polynucleotide encoding the peptide; (c) hybridizing to the polynucleotide of (a).
And at least 70% of the polynucleotide
A polynucleotide having identity; and (d) less DNA contained in the deposited clone.
A coding portion containing DNA having 90% identity with both
Encodes the same mature polypeptide as the human gene
A member selected from the group consisting of:
, Including isolated polynucleotides. In a preferred embodiment, the human genetic gene is
Offspring may contain DNA contained in the deposited clone.
You. In a further preferred embodiment, the above method
Member is the same as the polynucleotide of FIG. 1 (SEQ ID NO: 1)
Can be a polynucleotide encoding a polypeptide of the invention
You. In another aspect, the present invention provides
The present invention relates to a vector containing a nucleotide. In another aspect, the present invention provides the vector
To transformed or transfected host cells
Related. In another aspect, the present invention provides the above-described vector
A polypeptide comprising the step of genetically manipulating cells with
And a method for producing a cell capable of expressing the peptide. In another aspect, the present invention provides the above-described host cell
From the cells, the polynucleotide encoded by the polynucleotide
Producing polypeptides, including the step of expressing the polypeptides
About how to live. In another aspect, the invention relates to a polypeptide
And (i) a polypeptide encoded by a human gene
The DNA of the human gene is shown in FIG.
Nos. 2 to 20) contain at least 90% of the same DNA.
(Ii) a deduced amino acid sequence shown in FIG. 1 (SEQ ID NO: 1);
A polypeptide having a sequence, as well as fragments thereof;
Analogs and derivatives; and (iii) the coding region is included in the deposited clone.
DNA having at least 90% identity to the DNA
A polypeptide encoded by a human gene comprising A,
And fragments, analogs and polypeptides of polypeptides.
And a derivative comprising a member selected from the group consisting of
Related to repeptides. [0024] In a preferred embodiment, the polyp
The predicted amino acid sequence shown in FIG. 1 (SEQ ID NO: 1)
May be provided. [0025] In another aspect, the present invention provides the above-mentioned polypeptide.
Related to antibodies against the peptide. [0026] In another aspect, the present invention provides the above-mentioned polypeptide.
The present invention relates to a compound that inhibits activation of a peptide. [0027] In another aspect, the invention provides a therapeutically effective amount
Administering the above compound to a patient.
Patients with a need to inhibit foreign gene proteins
To a method for treating. In a preferred embodiment, the compound is
A polypeptide, and a therapeutically effective amount of the compound is
Providing the DNA encoding the polypeptide to the
Administered by expressing this polypeptide in vivo
Can be done. In another aspect, the invention provides a therapeutically effective amount
Administering the above polypeptide to a patient,
Treating patients in need of breast-specific gene proteins
About how to. [0030] In another aspect, the present invention relates to a method comprising administering to a host
A method for diagnosing breast disorders, comprising:
Transcription of human genes in samples from major non-breast tissues
In the step of measuring, the gene corresponds to the sequence shown in FIGS.
No. 1-20) DNA selected from the group consisting of DNA
Containing DNA having at least 90% identity to
Has a transcript, whereby transcription is performed in the breast
Indicating a disorder of the subject. In a preferred embodiment, the transfer is
The presence of altered levels of RNA transcribed from the gene
Can be measured by detecting In a further preferred embodiment, the transfer is
DNA complementary to RNA transcribed from the human gene
Measured by detecting the presence of an altered level of
Can be In a further preferred embodiment, the transfer is
The presence of altered levels of the above human gene expression products
It can be measured by issuing. [0034] In another aspect, the present invention relates to a method comprising administering to a host
A method for measuring breast damage comprising: B
An antibody specific to the SG antigen or an epitope portion thereof,
Contacting with a host-derived liquid sample; in the sample
The presence of altered levels of the BSG gene product
A process comprising: [0035] In another aspect, the present invention provides
A method for identifying antagonists to peptides
And the following: polypeptides as natural substrates and screens
Simultaneous or sequential on labeled compounds to be labeled
Contacting in any order; and said protein
Treatment in a manner sufficient to inhibit the binding of
Determine if the therapeutic drug effectively competes with the natural substrate
A process comprising the steps of: BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings illustrate embodiments of the present invention.
Of the invention, and is encompassed by the claims.
It is not meant to limit the scope of the light. FIG. 1 shows the entirety of the breast-specific gene 1 of the present invention.
Long cDNA sequence. FIG. 2 shows the partial cDNA sequence of the present invention and
And the corresponding amino acid sequence of breast-specific gene 2
It is. FIG. 3 shows the partial cDNA sequence of the present invention and
4 is the predicted amino acid sequence of breast-specific gene 3. FIG. 4 shows the partial cDNA sequence of the present invention and
With the corresponding deduced amino acid sequence of breast-specific gene 4
is there. FIG. 5 shows the part of the breast-specific gene 5 of the present invention.
2 is a cDNA sequence. FIG. 6 shows the partial cDNA sequence of the present invention and
4 is a predicted amino acid sequence of breast-specific gene 6. FIG. 7 shows the part of the breast-specific gene 7 of the present invention.
2 is a cDNA sequence. FIG. 8 shows the part of the breast-specific gene 8 of the present invention.
2 is a cDNA sequence. FIG. 9 shows the part of the breast-specific gene 9 of the present invention.
2 is a cDNA sequence. FIG. 10 shows the breast-specific gene 10 of the present invention.
Is a partial cDNA sequence. FIG. 11 shows the breast-specific gene 11 of the present invention.
Is a partial cDNA sequence. FIG. 12 shows the breast-specific gene 12 of the present invention.
Is a partial cDNA sequence. FIG. 13 shows the breast-specific gene 13 of the present invention.
Is a partial cDNA sequence. FIG. 14 shows the breast-specific gene 14 of the present invention.
Is a partial cDNA sequence. FIG. 15 shows the breast-specific gene 15 of the present invention.
Is a partial cDNA sequence. FIG. 16 shows the breast specific gene 16 of the present invention.
Is a partial cDNA sequence. FIG. 17 shows the breast-specific gene 17 of the present invention.
Is a partial cDNA sequence. FIG. 18 shows the breast-specific gene 18 of the present invention.
Is a partial cDNA sequence. FIG. 19 shows the breast-specific gene 19 of the present invention.
Is a partial cDNA sequence. FIG. 20 shows the breast-specific gene 20 of the present invention.
Is a partial cDNA sequence. The term “breast-specific gene” refers to such a
The gene is expressed mainly in breast-derived tissue, and
Una gene is expressed in cells derived from tissues other than breast
Means that it can be done. However, the expression of such genes
At present, breast-derived tissue is better than non-breast-derived tissue
Significantly higher. According to one aspect of the present invention, the estimation of FIG.
Mature polypeptide having amino acid sequence (SEQ ID NO: 1)
And its fragments, analogs and derivatives
A polynucleotide encoding a body is provided. According to a further aspect of the invention, FIGS.
0 (SEQ ID NO: 2 to 20)
And at least 90% identical (preferably
Or at least 95% identical, and most preferably
At least 97% or 100% identical)
Mature polypeptide identical to the human gene with the coding moiety
A polynucleotide encoding the peptide is provided.
You. According to yet another aspect of the present invention, the code
Part is ATCC Accession No. 97175 (1995
(Deposited on June 2) and one of the polynucleotides
At least 90% identical (preferably at least 95%
Identical, and most preferably at least 97% or 1
00% identical) to the human gene containing the polynucleotide
A polynucleotide encoding a mature polypeptide
Provided. According to yet another aspect of the present invention, the code
Part is the polynucleus of FIGS. 1 to 20 (SEQ ID NOS: 1 to 20)
At least 90% identical to one of the otide sequences (preferably
Are at least 95% identical, and most preferably
At least 97% or 100% identical).
MRNA transcribed from the coding portion of human genes
Or its corresponding cDNA)
A nucleotide probe is provided. According to the present invention, the code part is
20 (SEQ ID NOS: 2 to 20)
The analogs, derivatives, and other such polypeptides
And at least 90% identical (preferably
Are at least 95% identical, and more preferably less
At least 97% or 100% identical) DNA sequence
Mature poly-encoded by the coding part of the human gene
Regarding peptides. The present invention also relates to the mature form of FIG. 1 (SEQ ID NO: 1).
Polypeptides, and the hula of such polypeptides
Segment, analogs, and derivatives. The present invention further provides that the code portion
C Accession No. 97175 (Deposited on June 2, 1995)
At least 90% with one of the polynucleotides encompassed by
% Identical (preferably at least 95% identical, and
More preferably at least 97% or 100% identical
The same) encoded by a human gene including DNA
For mature polypeptides. According to one aspect of the invention, FIG.
Mature polypeptide having the deduced amino acid sequence of No. 1)
Or a fragment, analog or derivative thereof
The isolated nucleic acid (polynucleotide) encoding
Provided. The polynucleotide of the present invention may be in the form of RNA.
Or DNA (this DNA is cDNA, genomic DN
A, and synthetic DNA). DN
A can be double-stranded or single-stranded, and if single-stranded
Contains the coding or non-coding (antisense) strand.
You can. The coding sequence encoding the mature polypeptide is:
Figures 1-20 (SEQ ID NOs: 1-20) or deposited claw
May contain DNA that is identical to the coding sequence of the
Or the coding sequence is not redundant or degenerate in the genetic code.
As a result, the coding sequence is shown in FIGS.
20) DNA or gene containing the deposited cDNA
Coding for the same mature polypeptide as the coding sequence of
Coding sequence. The polypeptide encoding the mature polypeptide of the present invention
Renucleotides can include, but are not limited to:
Not: only the coding sequence for the mature polypeptide;
Peptide coding sequence and leader sequence or secretion
Additional coding such as sequence or proprotein sequence
Coding sequence of the mature polypeptide (and
Additional coding sequences as well as non-coding sequences (eg
For example, coding sequences for introns or mature polypeptides.
Sequence 5 'and / or 3' non-coding sequences). Thus, the term “encoding a polypeptide”
"Polynucleotide" refers to the polypeptide coding sequence only.
And a further coding sequence
And / or a polynucleotide comprising a non-coding sequence
Including. The present invention further provides a mature polypeptide of the present invention.
Encoding fragments, analogs, and derivatives of
The present invention relates to variants of the polynucleotide described above.
Polynucleotide variants are naturally occurring polynucleotides.
Allelic variants or polynucleotides present in
It may be a non-naturally occurring variant. Accordingly, the present invention relates to the present invention as described above.
Polynucleotide encoding the same mature polypeptide as
And variants of such polynucleotides.
No. This variant is a fragment of the polypeptide of the invention.
Encodes a derivative, derivative or analog. Like this
Nucleotide variants include deletion mutants, substitution mutants, and
Includes addition or insertion variants. The polynucleotide of the present invention has its code
The DNA whose sequence is shown in FIGS.
Naturally occurring allelic variants of human genes, including
Coding sequence or DNA copy in the deposited clone
Code sequence. As is known in the art,
A gene variant is a substitution, deletion, or substitution of one or more nucleotides.
Or may have an additional function of the encoded polypeptide
Forms of polynucleotide sequences that do not substantially alter the sequence
It is a state. The present invention also provides a mature polypeptide code.
The sequence is identical from the host cell in the same reading frame.
Polynucleotides that support polypeptide expression and secretion
Peptide sequences (eg, regulate the transport of polypeptides from cells)
Leader sequence that functions as a secretory sequence for
Includes polynucleotides that can be fused. Leader sequence
Is a preprotein, and
Leader to form mature forms of polypeptides
The sequence can be cleaved by the host cell. The polynucleus
Otides also add additional 5 'amino acids to the mature protein.
Can code for a proprotein with residues
You. The mature protein having a prosequence is a proprotein
Quality and inactive form of the protein
You. Once the prosequence is cleaved, the active mature protein
Quality remains. Thus, for example, the polynucleotides of the present invention
Is a mature protein or a protein with a prosequence
Quality, or pro and presequence (leader sequence)
It can encode a protein that has both. The polynucleotide of the present invention
To a marker sequence that allows for purification of the polypeptide
It may have the coding sequence fused in frame. marker
The sequence may be, in the case of a bacterial host, a sequence fused to a marker.
A pQE-9 vector that provides for purification of a mature polypeptide
Hex histidine tag supplied by Ah
Alternatively, for example, the marker sequence may be a mammalian host (eg,
If COS-7 cells are used, hemagglutination
Element (HA) tag. HA tag is Influence
For epitopes derived from the hemagglutinin protein
(Wilson, I. et al., Cell, 37: 767.
(1984)). The present invention further provides that at least 70
%, Preferably at least 90%, and more preferably
Here means that at least 95% identity is present
A polynucleotide hybridizing to the above polynucleotide
Regarding nucleotides. The present invention particularly relates to
Hybridize under stringent conditions to polynucleotides
It relates to a polynucleotide to be rediscovered. In this specification
The term "stringent conditions" is used to
Redidation is at least 95% between sequences and
Preferably with at least 97% identity
Means that only occurs. In a preferred embodiment
To hybridize to the polynucleotide described above in the present specification.
The polynucleotides to be used are shown in FIGS.
20) DNA or deposited cDNA (single or multiple)
Maturation of the present invention encoded by a coding sequence comprising
Have substantially the same biological function or activity as the polypeptide
Encodes the polypeptide to be retained. Alternatively, the polynucleotide may be
Hybridized to the polynucleotide described in
As noted above, it has identity and activity
At least 10 or not
Is 20 bases, preferably at least 30 bases, and
More preferably, it may have at least 50 bases. For example
A polynucleotide such as
As a probe (for example, the polynucleotide
Probe for collection or diagnostic probe) or
It can be used as a PCR primer. Accordingly, the present invention relates to FIGS.
1 to 20) by a human gene containing one DNA.
Polynucleotides encoding mature polypeptides to be encoded
At least 70%, preferably at least 9%
0% identity, and more preferably 95% identity
Having polynucleotides and fragments thereof
(This fragment should be at least 30 bases and preferably
Or at least 50 bases), as well as
Polypeptide encoded by such a polynucleotide
About. The partial sequence is added to the messenger RNA molecule.
This is a specific tag for this. The method in the form of cDNA
Probe the partial sequence for the complete sequence
CDNA clone corresponding to the full-length transcript
Identification and subsequent sequencing of the clone
Can be determined. Partial cDNA clones are also
Clones or regulatory and promoter regions,
Claw containing complete gene including xon and intron
Can be used as probes to identify proteins. Partial distribution of FIGS. 2 to 20 (SEQ ID NOs: 2 to 20)
Columns identify the corresponding full-length gene from which they are derived
Can be used for The partial sequence is nick tranceley
Labeling methods known to those skilled in the art (Basi
c Methods in Molecular Bio
logic, L .; G. FIG. Davis, M .; D. Dib
ner, and J.A. F. Batey Hen, Else
viar Press, NY, 1986)
Using polynucleotide kinase ³² End-labeled with P
Can be Lambda library prepared from human breast tissue
Are directly screened for the labeled sequence of interest.
Get or Bacterial Breasty
Libraries must be human to facilitate screening
It can be converted into pBluescript at once (St
ratagene Cloning Systems,
La Jolla, CA 92037). pBlu
For the script, see Sambrook et al., Mo.
rectangular Cloning-A Laborat
ory Manual, Cold Spring H
arbor Laboratory Press (1
989), page 1.20. Both methods are appropriate
It is well known in the art. Briefly, pBluesc
Have bacterial colonies containing the library in the rip
Bacterial lawn containing filter or lambda plaque (l
awn) is denatured and the DNA is fixed to the filter
Is done. Filters are provided by Davis et al.
Label using the hybridization conditions described.
The probe is hybridized to the probe. Lambda or
Partial sequence cloned into pBluescript
Evaluates background binding as a positive control
Hive for evaluation and for accurate clone identification
Adjust redistribution and wash stringency
Can be used to The obtained autoradiogram is
Compared to a duplicate plate of colonies or plaques;
Each of the exposed spots has a positive breach or
Respond to plaque. Colonies or plaques are selected
DNA is further analyzed and distributed.
Isolated from colonies for row determination. [0080] Positive cDNA clones were analyzed and
The amount of additional sequences they contain may be
Use the primer and the other primer derived from the vector
Determined using PCR. Larger than the original subarray
Clones with a unique vector-insert PCR product
Analysis by DNA digestion and DNA sequencing
Same as mRNA size determined by Zan blot analysis
Is determined to include direct or similar size inserts.
It is. Once one or more overlapping cDNA clones
Once defined, the complete sequence of the clone can be determined. Like
A new method is to use exonuclease III digestion.
(McCombie, WR, Kirkn
ess, E. , Fleming, J.A. T. , K
erlage, A.C. R. , Iovanisc
i, D. M. , And Martin-Gallar
do, R.D. , Methods, 3: 33-40,
1991). A series of deletion clones was generated, each of which
Are sequenced. The resulting duplicated sequence is highly degenerate
(Usually 3-5 overlapping sequences at each nucleotide position)
Are constructed in one contiguous sequence with
A reliable final sequence is obtained. DNA sequences (and their corresponding RNAs)
Sequence) can also be found in ATCC Accession No. 97175 (19
Deposited on June 2, 1995) and can be isolated therefrom.
Functional sequence, and its isolated DNA sequence (and
Fragment or part of the corresponding RNA sequence)
DNA (RNA) sequence encoding the same polypeptide
Sequences containing or containing the same DNA sequence
Including. The deposit (single or plural) referred to in this specification
Number) relates to the international recognition of deposits of microorganisms in patent procedures.
Maintained under the Budapest Treaty. These deposits
Is provided only as a convenience to those skilled in the art, and
That a deposit is required under 35 U.S.C. 112
I did not admit that. Polynuclei contained in the deposit
Otide sequences and polypeptides encoded thereby
The amino acid sequence of the peptide is incorporated herein by reference.
And with any description of the sequences herein
In case of conflict. Produce and use deposits
Or may require a license to sell,
And such license is granted by this specification.
Not necessarily. The present invention further provides at least 10 bases, preferably
Preferably it has at least 20 bases and more than 30
A polynucleotide which may have bases, the code
The part is converted from a human gene containing the DNA described above.
The RNA to be transcribed (and the D corresponding to such RNA
NA), and at least to it
Relates to polynucleotides having 70% identity. Therefore, the hybridization occurs as described above.
The polynucleotide sequences are described herein below.
Corresponding to the corresponding human for use in diagnostic assays.
Hybridize to the gene and detect its expression
Can be used to According to yet another aspect of the present invention, the host
Diagnostic assay to detect breast cancer micrometastases
Provided. Applicant has made an inference (reasonin) of the present invention.
g) to limit it to any particular scientific theory
But not in host cells other than breast-derived cells
The presence of active transcription of the breast specific gene of the invention may
It is considered an indicator of metastasis. This is true. What
If so, breast-specific genes can be found in all cells of the body
While they are found, their mRNA, cDNA
Transcription and expression products are mainly in the breast in unaffected individuals.
It is because it is limited to. But if breast cancer exists
Breast cancer cells migrate from the cancer to other cells,
As a result, these other cells are actively transferring breast-specific genes.
Transcription and expression are usually found in unaffected individuals
(Ie, transcription occurs in healthy individuals).
Higher than found in non-breast tissue). Breast origin
This amplified transcription or amplification in cells other than
Protein expression is an indicator of breast cancer metastasis
is there. In one example of such a diagnostic assay,
RNA sequences in samples from non-breast tissue
Detected by hybridization to the probe.
The sample contains nucleic acids or a mixture of nucleic acids, at least
One of them is the human breast-specific gene of the present invention or
Suspected to contain that fragment, they are
Transcribed and expressed in various tissues. So, for example
Assays to determine the presence of specific RNAs in cells
In an embodiment, RNA is first isolated from the cell.
You. Samples other than breast (blood, urine, saliva,
Including but not limited to tissue biopsy and autopsy material
(Not shown). Other than breast
Human breast features of the present invention in a sample obtained from
Increasing mRNA from heterologous genes or fragments thereof
Use of such a method to detect breadthed transcription
Are well within the purview of those skilled in the art from the teachings herein. The isolation of mRNA destroys cells and
Isolate total cellular RNA by differential centrifugation
Including. Once the total RNA is isolated, the mRNA
Is known to those skilled in the art as having a poly (A) tail (virtually
(Found at the 3 'end of the product mRNA molecule).
It is isolated using adenine nucleotide residues. Deo
Ori consisting only of xithymidine [oligo (dT)]
The gonucleotide is a cellulose packed in a small column.
And oligo (dT) cellulose. All details
As the preparation of vesicle RNA passes through such a column,
mRNA molecule is oligo (dT) with poly (A) tail
But the remaining RNA flows off the column. Next
Then, the bound mRNA is eluted from the column, and
Collected. [0092] Transcribed from the breast-specific gene of the present invention
One example of detecting an isolated mRNA is described herein.
As described above, the recovered mRNA is
Screening with oligonucleotide probes.
No. Such a probe is preferably analytically
It can be labeled with a detectable reagent or can be labeled
It is also understood that lobe identification is facilitated. useful
Radioactive, fluorescent dyes, or
Enzymes that can catalyze product formation include, but are not limited to
Not determined. [0092] Polynucleotide complementary to mRNA sequence (cDNA)
One example of detecting nucleotides is the polymerase chain reaction.
(PCR) is utilized with reverse transcriptase. PCR is D
Specific amplification of NA stretch or RNA stretch
Is a very powerful method for (Saiki et al., Na
cure, 234: 163-166 (1986)).
One application of this technology is in nucleic acid sequences present at low copy numbers.
Nucleic acid probe technology to grow a column to a detectable level
It is in the art. Many diagnostic and scientific applications of this method
Is H. A. PCR by Erlich (ed.)
Technology-Principles and
Applications for DNA Amp
ligation, Stockton Pres
s, USA, 1989, and A. Inis
(Eds.), PCR Protocols, Ac
ademic Press, San Diego,
USA, 1990. RT-PCR is a set of PCR and reverse transcriptase.
It is a match. Reverse transcriptase converts the cDNA molecule to the corresponding m
An enzyme produced from an RNA molecule. This is important
You. Because PCR amplifies nucleic acid molecules, especially DNA
And this DNA is isolated from a host-derived sample.
This is because it can be produced from the prepared mRNA. A specific example of an RT-PCR diagnostic assay is:
Removing the sample from the host tissue. This
Such samples are derived from tissues other than the breast, such as blood
I do. Therefore, one example of such a diagnostic assay is
Nuclear cell whole blood gradient isolation, total RNA extraction, total RNA RT
-PCR and agarose gel electrophoresis of PCR products
including. The PCR product may contain one or more breast-specific
RNA transcribed from the gene or its fragments
Contains complementary cDNA. More specifically, blood samples
And the whole blood is phosphate buffered to an equal volume
Mixed with saline, centrifuged and lymphocytes
And the granulocyte layer is carefully aspirated and phosphate buffered
Re-diluted in saline, and centrifuged again
It is. The supernatant is discarded and the pellet containing nucleated cells
Is a manufacturer (Tel-Test Inc., Friend)
RN as described by dswood, TX)
Used for RNA extraction using the azole B method. High specificity of the DNA sequence of the present invention
Oligonucleotide primers and probes are prepared
Is done. Probes should be at least 10 base pairs long, preferably
At least 30 base pairs in length, and most preferably
At least 50 base pairs long or longer. Reverse transcriptase
Reactions and PCR amplification are performed continuously without interruption
You. Taq polymerase is used during PCR and
PCR products are concentrated and all samples are
Tris-borate-EDTA agaro containing mbromide
Washed with sogel. According to another aspect of the invention, a non-breast set is provided.
Breast-specific ports of the invention in biological samples from tissue
By measuring the altered levels of peptides, milk
Methods for diagnosing tuft disorders, e.g., breast cancer, are provided.
You. Elevated levels of breast-specific polypeptides of the invention
Is the active transcription of the corresponding breast-specific gene product and
Shows expression. Breast-specific genes in host-derived samples
Updating used to detect polypeptide levels
Says are well known to those skilled in the art, and they
Muno assay, competitive binding assay, western block
Analysis, ELISA assay and "sandwich"
Including assays. Biological samples include tissue extracts,
Can include cell samples or biological fluids
Not limited. However, according to the present invention, biological
The pull is particularly free of breast tissue or cells. ELISA assays (Coligan et al.,
Current Protocols in Immu
Nology, 1 (2), Chapter 6, 1991)
First, an antibody specific to the breast-specific polypeptide of the invention
Preparation of a monoclonal antibody, preferably a monoclonal antibody.
No. In addition, reporter antibodies are
It is prepared by Reporter antibodies include radioactivity, fluorescence,
Or, in this example, horseradish peroxidase
A detectable reagent such as an enzyme is added. sample
Is removed from the host and the protein in the sample
Solid support (eg, polystyrene dish)
). Then, this dish
Any free protein binding sites on the
Incubation with non-specific proteins such as
Covered by The monoclonal antibody is then:
Incubated in the dish and during that time
Clonal antibody bound to polystyrene dish
Binds to breast-specific polypeptides. All unbound modules
Noclonal antibodies are washed away with a buffer. This and
Reporter linked to horseradish peroxidase
Antibodies are placed in the dish and, as a result,
Antibody bound to the breast-specific gene polypeptide
Binds to any monoclonal antibody. Then unbound
Are removed by washing. Next, Peru
The oxidase substrate is added to the dish and
When the amount of color development within the time is compared with the standard curve
Breast-specific present in a given volume of the patient's sample
It is a measured amount of polypeptide. [0098] A competition assay was performed for breast-specific polypeptides.
Used when antibody specific for conjugate is bound to solid support
Can be done. The breast-specific polypeptide is then labeled.
And labeled host-derived polypeptide samples
Is passed over a solid support and, for example, a liquid scintillator
Label detected by chromatography
Is the amount of breast-specific polypeptide in the sample.
May be involved. The "sandwich" assay is an ELISA
Similar to the assay. "Sandwich" assay smell
Breast specific polypeptide is passed over a solid support.
And binds to the antibody bound to the solid support. Next
At this point, the secondary antibody binds to the breast-specific polypeptide.
A tertiary antibody that is labeled and specific for the secondary antibody is then
Passed through the solid support and bound to a secondary antibody,
The amount can then be quantified. In another method, labeled breast-specific
Antibodies to the target polypeptide are used. One step up
In Say, the target molecule, if present, is immobilized
Then, it is incubated with the labeled antibody. Marked
The antibody binds to the immobilized target molecule. Unbound
After washing to remove molecules, the sample is
Assay for presence. In a two-step assay,
The immobilized target molecule is incubated with the unlabeled antibody.
Be baited. The labeled molecule-labeled antibody complex is present
If so, then a labeled secondary antibody specific for the unlabeled antibody
To join. Wash the sample and the presence of label
Assay. [0101] Specific for breast-specific gene proteins
Antibodies such as, for example, anti-idiotype antibodies
Labeled as above and tightly binds to breast cancer cells
And therefore its presence, to detect breast cancer
Can be used to detect cells. These antibodies can also be used, for example, in breast cancer cells.
Homing interactions that destroy them when they come in contact with
Method to target breast cancer cells
Can be used. This is a fact. Because this antibody
Is a gene specifically expressed in breast cancer
Specific and binding of the interaction reagent to the antibody
To cause direct transport of interacting reagents to the breast
It is. [0103] Antibodies of this type can also be used, for example, in the breast.
By labeling the antibody to facilitate scanning,
It can be used to perform in vivo imaging. Diagnostic imaging
One method for any breast to be imaged
Cancer cells, anti-breast labeled with a detectable marker
Contacting with a specific gene protein antibody.
In this method, the labeled antibody is used to
The reaction is performed under conditions that bind to proteins. In a specific example
Antibodies are used in breast (eg, breast cancer cells) and
It interacts with resin during such contact, resulting in
Imaging and breast visibility are amplified, breast disease
Or non-disease status. [0102] The markers used to label the antibodies
The choice will vary depending on the application. But the marker
The choice is easily determinable for a person skilled in the art. these
Labeled antibodies are used in immunoassays and histological
In applications, to detect the presence of the protein
Can be used. Labeled antibodies can be polyclonal or
Can be monoclonal. Changes in mRNA, cDNA or expression product
Normal levels in non-breast cells due to the presence of
Existence of breast-specific gene transcripts that are more active than the ones issued
Location is an important indicator of the presence of metastatic breast cancer. why
If the breast cancer cells are moving from the breast into the general circulation
This is because that. Therefore, this phenomenon has important clinical implications.
Have. Because the treatment of localized tumors is
Because it is completely different, in contrast to treatment. The disclosed 20 breast-specific genes
Incidentally, only the breast-specific gene 1 is the full-length gene. breast
Specific gene 1 is amyloid residue in human Alzheimer's disease
79% identical to the gene and 83% similar. milk
Tuft-specific gene 2 is human hydroxyindole-o-
30% identical to the methyltransferase gene,
And it is 48% similar. Breast-specific gene 3 is human
O-6-methylguanine-DNA methyl transferase
58% identical and 62% similar to
You. Breast-specific gene 4 has 3 in the mouse p120 gene.
4% identical and 65% similar. Breast specific
Gene 5 is human p70 ribosomal S6 kinase α-I
78% identical to the I gene and 89% similar
You. Breast-specific gene 6 is a human transcription factor NFATp
77% identical to the gene and 79% similar. As described above, the breast-specific gene of the present invention
Is used in the diagnosis of breast cancer formation and breast cancer metastasis.
Is a putative molecular marker. As shown in Table 1 below,
The presence of breast-specific genes indicates that normal breast, breast cancer,
And when tested in other cancer libraries,
The breast-specific gene of the present invention is contained in a breast cancer library.
Was found to be the most epidemic. This is the original
Ming gene detects breast cancer, as discussed earlier
That can be used for The table also shows the known genetics
Based on the homology of BSG1 to BSG6 to the offspring
Indicates constant identification. [Table 1] The above assay also demonstrates that bone marrow preserved prior to chemotherapy
Is contaminated by breast cancer cell micrometastases
Can be used to test for This assay
Blood cells from the bone marrow have been isolated and
In this method, the preserved bone marrow is
After the law, it is still possible to determine whether it is suitable for transplantation.
Make it work. The present invention further relates to mature polypeptides (eg,
BSG1 polypeptide), as well as such polypeptides.
For peptide fragments, analogs and derivatives
I do. The terms “fragment”, “derivative” and
And “analogs” are the polypeptides encoded by the gene of the present invention.
When referring to peptides, such polypeptides and essences
That retain the same biological function or activity
Means tide. Thus, the analog is an activated mature poly
For cleavage of the proprotein part to produce peptides
Includes proproteins that can be more activated. The polypeptide of the present invention is a recombinant polypeptide.
Tide, naturally occurring polypeptide, or synthetic polypeptide
And preferably a recombinant polypeptide. The polypeptide encoded by the gene of the present invention
Peptide fragments, derivatives, or analogs
(I) one or more amino acid residues therein are conservative or non-conservative;
Conservative amino acid residues (preferably conservative amino acid residues)
And substituted amino acid residues such
Is the amino acid residue encoded by this genetic code
Possible or impossible, or (ii) one or more thereof
The amino acid residue above contains a substituent, or (ii.
i) the polypeptide therein has a half-life of the polypeptide;
(Eg, polyethylene glycol
Fused to another compound, such as
(Iv) further amino acids therein are polypeptides (eg,
For example, leader or secretory sequences, or mature poly
The sequence used to purify the peptide or the proprotein
(Which may be a quality sequence).
Such fragments, derivatives, and analogs
Given the teachings herein, it is believed that they are within the purview of those skilled in the art.
It is. Polypeptides and Polynucleotides of the Invention
The tide is preferably provided in an isolated form; and
Preferably, it is uniformly purified. The term “isolated” means that a substance is
Environment (for example, the natural environment, if it occurs naturally)
Means that it has been removed from For example, alive
Naturally occurring polynucleotides in animals
Or polypeptide has not been isolated, but
Separated from some or all of the coexisting materials
The same polynucleotide or polypeptide
Have been. Such a polynucleotide is part of a vector.
And / or such a polynucleotide
Or polypeptide may be part of the composition, and
Such a vector or composition may be part of its natural environment.
In that it is not a part, it may still be isolated. The polypeptide of the present invention is shown in FIG.
1) the polypeptide (particularly the mature polypeptide);
At least 70% similarity (preferably 70% identity)
(SEQ ID NO: 1) for the polypeptide of FIG.
And more preferably at least 90% similarity
(More preferably at least 90% identity) in FIG.
And the polypeptide of FIG. 9 (SEQ ID NOs: 8 and 9)
And even more preferably at least 95%
Similarity (even more preferably 95% identity) is shown in FIG.
Polypeptide having the polypeptide of (SEQ ID NO: 1)
Containing a tide and also generally at least 30 amino acids
And more preferably contains at least 50 amino acids.
Such polypeptides having a polypeptide moiety such as
Including the portion of the peptide. As known to those skilled in the art, two polypeptides
The "similarity" between amino acids is the amino acid sequence of one polypeptide.
Sequence and its conservative amino acid substitutions in a second polypeptide
Is determined by comparison with the sequence of A fragment of the polypeptide of the present invention or
Is the corresponding full-length polypeptide by peptide synthesis.
Can be used to produce Therefore, this flag
Is an intermediate for producing the full-length polypeptide.
Can be used. Fragmentation of the polynucleotide of the present invention
Part or portion combines the full-length polynucleotide of the invention.
Can be used to generate The present invention also relates to the polynucleotide of the present invention.
Using the vector of the present invention
Host cells to be used
For the production of peptides. The host cell is a vector of the present invention, which comprises
For example, a cloning vector or an expression vector.
Genetically engineered (transduced)
Or transformed or transfected
Is done). Vectors include, for example, plasmids, viruses
It can be in the form of particles, phages, and the like. Manipulated host
Cells activate the promoter or select transformants.
Or to amplify breast-specific genes.
It can be cultured in a conventionally modified nutrient medium that has been modified.
Culture conditions (eg, temperature, pH, etc.)
Conditions previously used in the selected host cell, and
It will be clear to those skilled in the art. [0120] The polynucleotide of the present invention can be produced by a recombinant technique.
Can be used to produce a polypeptide. Subordinate
Thus, for example, a polynucleotide emits a polypeptide
Included in any one of a variety of expression vectors for expression
Can be Such vectors contain chromosomal DNA sequences,
Chromosomal and synthetic DNA sequences (eg, S
V40 derivatives; bacterial plasmids; phage DNA;
Baculovirus; yeast plasmid; plasmid and
Vector derived from a combination of phage DNA,
Rus DNA (eg, vaccinia, adenovirus, chicken
Pox virus, and pseudorabies)). But the inn
As long as it is replicable and viable in the Lord,
Any vector can be used. The appropriate DNA sequence may be identified by various procedures.
Can be inserted into the reactor. Generally, the DNA sequence
Appropriate restriction endonuclease sites by procedures known in the art
Inserted at the position (s). Such a procedure
And other procedures are considered to be within the purview of those skilled in the art. The DNA sequence in the expression vector
Appropriate expression control sequences (single or multiple) to direct the synthesis of
Number) (promoter). This
Representative examples of such promoters include:
Can be: LTR or SV40 promoter, E. coli. co
li lac or trp, λ phage P _L Promoter
And prokaryotic or eukaryotic cells or
It is known that they regulate gene expression in these viruses.
Other promoters that are. Expression vectors can also be translated
Ribosome binding site and transcription terminator for initiation
Containing Vectors can also be used to amplify expression
Suitable sequences. Furthermore, the expression vector is preferably in the form
Phenotypic traits for selection of transformed host cells (eg,
For eukaryotic cell cultures, dihydrofolate reductor
Ze or neomycin resistance, or col
i resistance to tetracycline or ampicillin
Containing one or more selectable marker genes that provide
You. A suitable DNA sequence as described herein above
Contains appropriate promoter or control sequences
The appropriate vector is transformed into an appropriate host and
It can be used to express protein. Representative examples of suitable hosts include the following:
Mention may be made: bacterial cells such as E. coli, St.
reptomyces, Salmonella type
fungus cells (eg, yeast); insect cells
Vesicles (eg, Drosophila S2 and Spo
doptera Sf9); animal cells (eg, CH
O, COS or Bowes melanoma); adenovirus
And plant cells. Selection of an appropriate host is described herein.
It is considered within the skill of the art from the teachings. More specifically, the present invention has also been broadly described above.
Encompasses a recombinant construct comprising one or more of the described sequences.
You. The construct is a vector (eg, a plasmid vector).
Or viral vectors) within this vector
The sequence of the present invention is inserted in the forward or reverse direction.
You. In a preferred aspect of this embodiment, the construct is
In addition, a regulatory sequence operably linked to the sequence (eg,
Including a promoter). Very many suitable vectors
Promoters and promoters are known to those of skill in the art, and
It is commercially available. The following vectors are provided as examples
You. Bacterial: pQE70, pQE60, pQE-9 (Q
iagen), pBS, pD10, phaescri
pt, psiX174, pbluescript S
K, pBSKS, pNH8A, pNH16a, pNH1
8A, pNH46A (Stratagene); ptr
c99a, pKK223-3, pKK233-3, pD
R540, pRIT5 (Pharmacia). Eukaryotic
Sex: pWLNEO, pSV2CAT, pOG44, pX
T1, pSG (Stratagene); pSVK3,
pBPV, pMSG, pSVL (Pharmaci
a). However, any other plasmid or vector
Even if they are replicable and viable in the host
As long as it can be used. The promoter region is CAT (chloramph
Enicol transferase) vector or selection
Any other desired vector using other vectors with
It can be selected from genes. Two suitable vectors are P
KK232-8 and pCM7. Especially well-known
LacI, lacZ, T
3, T7, gpt, λP _R , P _L And trp
It is. As eukaryotic promoters, CMV immediate-type,
HSV thymidine kinase, early SV40 and late SV
40, LTR derived from retrovirus, and mouse meta
Lothionein I. Appropriate vectors and
The choice of the motor is well within the level of ordinary skill in the art. In a further embodiment, the present invention relates to the above structure.
The present invention relates to host cells containing structures. The host cell is higher
Eukaryotic cells (eg, mammalian cells) or lower eukaryotes
Cells (eg, yeast cells) or
The primary cell can be a prokaryotic cell (eg, a bacterial cell)
You. Introduction of the construct into the host
Transfection, DEAE-dextran mediated tiger
By transfection or electroporation
(Davis, L., Dibner,
M. Battey, I .; , Basic Meth
ods in Molecular Biology,
(1986)). Conventional methods using constructs in host cells
To produce the gene product encoded by the recombinant sequence.
obtain. Alternatively, the polypeptides of the present invention
It can be produced synthetically by a tide synthesizer. Proteins include mammalian cells, yeast, and cells.
Under the control of appropriate promoters in fungi or other cells
Can be expressed. The cell-free translation system is also a DNA structure of the present invention.
Using RNA from architectural structures, such proteins
Can be used to produce quality. Prokaryotic host and
Suitable cloning vectors for use in eukaryotic hosts
And expression vectors are described in Sambrook et al., Mol.
eco Cloning: A Laborat
ory Manual, 2nd edition, ColdSpri
ng Harbor, N.M. Y. , (1989)
Indications are incorporated herein by reference).
ing. DNA encoding the polypeptide of the present invention
Transcription by higher eukaryotes enhances vector
Augmented by inserting sequences. Enhancers
Cis-acting element of DNA, usually about 10
About 300 bp, which acts on the promoter and
Increase transcription. As an example, bp100 of the replication origin
メ ガ 270 late stage SV40 enhancer, Cytomega
Lovirus early promoter enhancer, origin of replication
Late-stage polyoma enhancers and adenowis
Ruth enhancer. In general, the recombinant expression vector is a host cell
Origins and selectable markers that allow transformation of yeast
(For example, E. coli ampicillin resistance gene and
And S. cerevisiae TRP1 gene)
From highly expressed genes that direct transcription of structural sequences downstream
Including the promoter. Such promoters are
Glycolytic enzymes such as 3-phosphoglycerate quina
(PGK)), α-factor, acid phosphatase,
Is derived from the operon encoding the heat shock protein
obtain. The heterologous structural sequence includes a translation initiation sequence and a translation termination sequence.
Assembled in row and appropriate phase. If necessary, use different types of distribution.
The columns may have the desired characteristics (eg, safety of the expressed recombinant product).
N-terminal identification peptides that provide for simplified or simplified purification)
It can encode a fusion protein that includes a tide. Expression vectors useful for bacterial use are functional
Proper promoters and operable reading frames
The desired protein along with the initiation and translation termination signals
By inserting a structural DNA sequence encoding the protein
Be built. Vectors contain one or more phenotype selection markers
And maintenance of the vector and desired
Contains an origin of replication to provide amplification in the host
I do. As a suitable prokaryotic host for transformation,
E. FIG. coli, Bacillus subtilis,
Salmonella typhimurium,
Genus Pseudomonas, Streptomyc
Species within the genus es and Staphylococcus
Various species, but other species are also available for selection
I can be. As a representative, but not limiting, example,
Expression vectors useful for the use of bacteria are well-known cloning
Inheritance of vector pBR322 (ATCC 37017)
Selection plasmids derived from commercially available plasmids containing
And bacterial origins of replication. like this
Commercially available vectors include, for example, pKK223-3
(Pharmacia Fine Chemical
s, Uppsala, Sweden) and GEM1
(Promega Biotec, Madison,
WI, USA). These pBR32
2 The "backbone" portion is a suitable promoter and expressed
Combined with the structural arrangement to be performed. Transformation of a suitable host strain and suitable cells
Following growth of the host strain to density, the selected promoter
Appropriate means (eg, temperature shift or chemical induction).
And the cells are cultured for a further period of time.
It is. The cells are typically recovered by centrifugation.
Crushed by physical or chemical means, and
The crude extract obtained is retained for further purification.
You. Microbes used in protein expression
Cells are subjected to freeze-thaw cycles, sonication, mechanical disruption
Any convenient method, including disruption or use of cell lysing agents
Can be crushed by the method. Such methods are well known to those skilled in the art.
It is. [0138] Various mammalian cell culture systems are also recombinant.
And can be used to express proteins. Mammal
Examples of expression systems include Gluzman, Cell, 2
3: 175 (1981) Monkey kidney fibroblasts
COS-7 strain, and compatible vectors
Other cell lines (eg, C127, 3T3, CHO, He
La, and BHK cell lines). Mammalian expression
The vector contains an origin of replication, an appropriate promoter and
Hansers, as well as any necessary ribosome binding sites,
Polyadenylation sites, splice donor sites and splice sites
Rice acceptor site, transcription termination sequence, and 5 ′
Also includes ranking non-transcribed sequences. SV40 splice
DNA sequence derived from the site, and polyadenylation site
Are used to provide the necessary non-transcribed genetic elements.
Can be used. [0139] Breast-specific gene polypeptides include:
Recovered from the recombinant cell culture by a method comprising
Can be purified: ammonium sulfate precipitation or ethanol precipitation, acid extraction
Chromatography, anion or cation exchange chromatography,
Phosphocellulose chromatography, hydrophobic interaction
Chromatography, affinity chromatography
-, Hydroxylapatite chromatography, and
And lectin chromatography. Tampa if necessary
The refolding process of the
It can be used to complete the arrangement of the mature protein.
Finally, high performance liquid chromatography (HPLC)
Can be used for the final purification step. The polynucleotide of the present invention comprises the polynucleotide of the present invention.
Inflation into a marker sequence that allows purification of the peptide
May have the coding sequence fused. Marker sequence
One example of a vector, preferably a pQE-9 vector
Is a hexahistidine tag that can be supplied by
Is a polypeptide fused to a marker in the case of a bacterial host
Or a mammalian host (eg, COS)
-7 cells) is used, for example, a marker
The columns can be hemagglutinin (HA) tags. HA tag
Is an epidemic derived from influenza hemagglutinin
Corresponding to Tope (Wilson, I. et al., Cel
1, 37: 767 (1984)). [0141] The polypeptides of the present invention may be naturally occurring purified
Or is the product of a chemical synthesis procedure
Or prokaryotic or eukaryotic host (eg, culture
Bacteria, yeast, higher plants, insects, and mammalian cells
Vesicles) can be produced by recombinant techniques. Recombinant producer
Depending on the host used in turn, the polypeptide of the invention
Can be glycosylated or glycosylated
Maybe not. The polypeptides of the present invention also
Methionine amino acid residue. BSG1, and other breast specific genes,
And its protein products are useful for early detection of breast cancer.
Can be used for Because these are breast cancer
Is overexpressed. According to another aspect of the present invention, the breast of the present invention
Inhibits the action of specific genes or breast-specific proteins
Can be used to screen for
Essays are provided. The present invention blocks its biological effects.
Breast-specific gene tans with sufficient affinity to stop
Disclosed is a method for selecting a therapeutic agent that forms a complex with protein
I do. In this method, the protein is immobilized on a support.
With or with natural substrates and labeled therapeutics
Contacted in one of the sequential
Therapeutically effective in a manner sufficient to prevent binding to
Competition assays to determine if they compete with natural substrates
And various assays. [0144] In another embodiment, the substrate is immobilized on a support.
And labeled breast-specific polypeptides
And therapeutic agents (or unlabeled proteins and labeled
Contacted with both and bound to the substrate
If the amount of tuft-specific polypeptide is
Is determined to be reduced compared to the new assay.
You. Breast-specific polypeptides can be labeled with an antibody. [0145] Potential therapeutic compounds include anti-
Body and anti-idiotype antibodies
In some cases, oligonucleotides that bind to the polypeptide
Is included. Another example is the use of antisense technology for tuning.
Produced antisense construct, which blocks transcription.
Directed to breast-specific polynucleotides to stop
You. Antisense technology is based on triple helix formation or
Control gene expression via chisense DNA or RNA
Both of these methods can be used to
Based on the binding of nucleotides to DNA or RNA
Have been. For example, encoding a mature polypeptide of the invention
The 5 'coding portion of the polynucleotide sequence
Antisense RNA oligonucleotide of about 10 to 40 base pairs
Used to design otides. DNA oligonucleotide
Reotide is complementary to the region of the gene involved in transcription.
(Triple helix-Lee et al., Nucl.
Acids Res. , 6: 3073 (197
9); Cooney et al., Science, 241:
456 (1988); and Dervan et al., Sci.
ence, 251: 1360 (1991).
That the transcription and breast specific polynuclease
Inhibits the production of leotide. Antisense RNA oligo
Nucleotides hybridize to mRNA in vivo
And breast-specific gene polypeptides of mRNA molecules
Block translation to anti-sense (Oksense-Okan
o. Neurochem. , 56: 560
(1991); as antisense inhibitors of gene expression
Oligodeoxynucleotides, CRC Pres
s, Boca Raton, FL (198
8)). The above oligonucleotides are also delivered to cells
And thereby obtain antisense RNA or DN
A inhibits the production of breast-specific polypeptide
It can be expressed in vivo. Another example is binding to breast-specific polypeptides.
And occupy the active site, thereby
Active site to prevent normal biological activity
A small molecule that makes quality inaccessible. Examples of small molecules
Is a small peptide or peptide-like molecule,
It is not limited to these. These compounds are useful for treating breast cancer.
Can be used for Because these are the survival of breast cancer cells
Breast features in a manner sufficient to interfere with the natural functions required for
It interacts with the function of the different polypeptides. This
This is a fact. Because BSG and their tanks
The protein product is mainly expressed in breast cancer tissue, thus
This is because it is presumed that the formation of this state is decisive. The compounds are described, for example, hereinbelow.
With a pharmaceutically acceptable carrier as described
Can be used in products. The compounds of the present invention can be used in any suitable pharmaceutical carrier.
Can be used in combination with Such compositions are cured
A therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable
Includes carrier or excipient. Such a career
Saline, buffered saline, dextrose
Water, glycerol, ethanol, and their combinations
Combinations include, but are not limited to. Prescription
Should be adapted to the mode of administration. The present invention also relates to one of the pharmaceutical compositions of the present invention.
Pharmaceutical comprising one or more containers filled with one or more ingredients
Provide packs or kits. Such containers (single
Or more than one) regarding the manufacture of drugs or biological products
Specified by governmental agencies governing the manufacture, use, or sale of
Product labeling, which can be used in human
Institutions in the manufacture, use or sale of dosing
Represents authorization by In addition, the pharmaceutical composition may have other therapeutic
It can be used in combination with the compound. Pharmaceutical compositions may be, for example, oral, topical, static.
Intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, anal, or
It can be administered in a convenient manner by the intradermal route. Pharmaceutical composition
The substance is an amount effective for treating and / or preventing a particular condition.
Is administered. Generally, the pharmaceutical composition will have at least about 1
Administered in an amount of 0 μg / kg body weight, and often
They are administered in an amount not to exceed about 8 mg / kg body weight per day
Is done. In many cases, the dosage is about 10 μg / k daily
g to about 1 mg / kg body weight, administration route, symptoms
Etc. are considered. Breast-specific genes and compounds (these are
Is a polypeptide according to the invention.
Can be used by expressing such a polypeptide
You. This is often referred to as "gene therapy". Thus, for example, cells from a patient
A polynucleotide encoding a peptide (DNA or
RNA) can be manipulated ex vivo and then manipulated
The cells produced are the patients to be treated with this polypeptide
Provided to Such methods are well known in the art.
You. For example, a cell encodes a polypeptide of the invention.
The use of retroviral particles containing RNA
It can be operated by procedures known in the art. Similarly, cells can be used to produce polypeptides in vivo.
For expression of the peptide, for example, by procedures known in the art.
Can be manipulated in vivo. As known in the art,
Les containing RNA encoding the polypeptide of the present invention.
Producing cells for producing torovirus particles are
Polypep for manipulating cells in vivo and in vivo
It can be administered to a patient for expression of the tide. Such person
To administer the polypeptide of the invention by the method
And other methods are known to those skilled in the art from the teachings of the present invention.
it is obvious. For example, an expression vehicle for manipulating cells.
The virus is not a retrovirus (eg, adenovirus).
Ills). This should be paired with the appropriate delivery vehicle.
Once combined, they are used to manipulate cells in vivo.
Can be The retroviral plasmid described above in the present specification
Retroviruses from which the vector can be derived include Moro
Knee mouse leukemia virus, spleen necrosis virus, Rous
A retrovirus like the sarcoma virus, Harvey's sarcoma
Ils, chicken leukemia virus, gibbon monkey leukemia
Ills, human immunodeficiency virus, adenovirus, bone
Myeloproliferative sarcoma virus, and mammalian tumor virus
But not limited thereto. One embodiment
In the retroviral plasmid vector, the Moro
Derived from knee mouse leukemia virus. [0157] Vectors contain one or more promoters.
No. Suitable promoters that can be used include Mil.
ler et al., Biotechniques, Volume 7, 9
No. 980-990 (1989)
LTR; SV40 promoter;
Gallovirus (CMV) promoter, or any other
Promoters (eg, eukaryotic cell promoters)
(Histone, pol III, and β-actin pro
Including but not limited to motors)
But not limited to these. Other ui that can be used
Adenovirus promoter
, A thymidine kinase (TK) promoter, and B
19 parvovirus promoter, which
It is not limited to them. The choice of an appropriate promoter is
It will be apparent to those skilled in the art from the teachings contained in the specification. Nucleic acid sequence encoding the polypeptide of the present invention
The rows are under the control of a suitable promoter. Can be used
Suitable promoters include adenovirus promoters.
(Eg, the adenovirus major late promoter)
-); Or a heterologous promoter (eg, cytomegalo
Virus (CMV) promoter); RS virus (R
SV) promoter; an inducible promoter (eg, M
MT promoter, metallothionein promoter);
Heat shock promoter; albumin promoter; A
poAI promoter; human globin promoter;
Ilstymidine kinase promoter (eg,
Rupesthymidine kinase promoter); retrovirus
LTR (modified retrovirus LT described earlier herein)
R); β-actin promoter; and human growth
Hormone promoters, including but not limited to
Not. Promoters also encode polypeptides.
Can be a natural promoter that controls a gene. [0159] The retroviral plasmid vector
Transduce the packaging cell line to form a production cell line
Used to Packages that can be transfected
Examples of soaking cells include Miller, Human
Gene Therapy, Vol. 1, pp. 5-14 (1
990) (incorporated herein by reference in its entirety).
PE501, PA317, ψ-2, ψ-A
M, PA12, T19-14X, VT-19-17-H
2, ψCRE, ψCRIP, GP + E-86, GP + e
nvAm12, and the DAN cell line.
It is not limited to these. Vectors can be any vector known in the art.
The packaging cells can be transduced by any means.
Such means include electroporation,
Use of posomes and CaPO ₄ Precipitation
However, it is not limited to these. As an alternative, retro
Viral plasmid vector encapsulated in liposome
Or be bound to a lipid, and then
Can be administered. The production cell line encodes a polypeptide.
Infectious retrovirus containing nucleic acid sequence (s)
Produce svector particles. Then such a retro
Viral vector particles can be used in vitro or in vivo.
Either used to transduce eukaryotic cells
Can be The transduced eukaryotic cell is a polypeptide
Expressing the nucleic acid sequence (s) encoding
Eukaryotic cells that can be transduced include embryonic stem cells,
Embryonic sarcoma cells and hematopoietic stem cells, hepatocytes, fibroblasts
Vesicles, myoblasts, keratinocytes, endothelial cells, and trachea
Include, but are not limited to, epithelial cells. The present invention also relates to a breast-specific gene of the present invention.
For use as a diagnostic. For example, some diseases
The illness is caused by an inherited defective gene. For example, milk
Tuft-specific genes, CSG7 and CSG10,
Has reduced expression in breast cancer cells compared to expression in vesicles
It has been found that In addition, the remaining milk of the present invention
Tuft-specific genes are overexpressed in breast cancer. Subordinate
Thus, mutations in these genes can cause breast disorders (eg,
For example, breast cancer) can be detected. At the DNA level
Mutations in the breast-specific gene of the present invention can be performed by various techniques.
Can be detected by Nucleic acids used for diagnosis (genome
DNA, mRNA, etc.), blood, urine, saliva, tissue
Non-breast patient cells, such as biopsy and necropsy material
Can be obtained. Genomic DNA is used for direct detection
Or PCR (Saiki et al., Nature, 3).
24: 163-166 (1986)) prior to analysis.
It can be amplified enzymatically. RNA or cDNA can also be
It can be used for the same purpose. As an example, the present invention
PCR primers that are complementary to nucleic acids are
Identify and analyze mutations in specific polynucleotides
Can be used to For example, deletions and insertions
Size of amplified product compared to normal genotype
Can be detected. The point mutation is the amplified D
Breast-specific RNA or radioactivity radiolabeled for NA
Hybridizes to the labeled antisense DNA sequence
Of DNA after PCR amplification that can be identified by
Another good way to screen for segment mutations
A well-established method is to use single-stranded conformational polymorphisms (SSC
P) Analysis. The PCR product is ³² P-dCTP
10 cycles of re-amplification
To produce a 200-300 bp fragment
Digested with the appropriate restriction enzymes to produce
Denatured by heating for 5 minutes, then immersed in ice
It is. Then a non-denaturing gel (5% glycerol, 5%
Electrophoresis is carried out (Cryamide) (Glav
ac, D.C. And Dean, M .; , Human M
utation, 2: 404-414 (199
3)). Sequence Sequence between Reference Gene and "Variant"
Differences can be revealed by direct DNA sequencing.
You. In addition, the cloned DNA segment contains a specific D
Used as a probe to detect NA segments
Can be The sensitivity of this method was combined with PCR
Sometimes greatly amplified. For example, sequencing primers
Is the double-stranded PCR generated by the modified PCR
Used with product or single-stranded template molecule. Sequencing
Is a conventional procedure using radiolabeled nucleotides.
Or by automated sequencing procedures using fluorescent tags
Is performed. Genetic Testing Based on DNA Sequence Differences
Is the DNA in gels with or without denaturing agents
By detecting changes in the electrophoretic mobility of fragments
Can be achieved. Small sequence deletions and insertions, high resolution
Can be visualized by gel electrophoresis. Of different arrays
DNA fragments are run on denaturing formamide gradient gels.
Can be distinguished. Here the different DNA flags in the gel
The mobility of the element is determined by their specific melting temperature or fraction
In the gel at different locations according to the specific melting temperature
(See, for example, Myers et al., Science, 230:
1242 (1985)). Furthermore, the array
Changes (in certain small deletions) are caused by DNA migration
It is detected as a change in the degree pattern. A sequence change at a particular position may also
Ase protection assays (eg, RNase protection and S
1 Protection or chemical cleavage methods (eg, Cotton et al.,
PNAS, USA, 85: 4397-4401 (19
85))). Therefore, the detection of a specific DNA sequence is high.
Hybridization, RNase protection, chemical cleavage, direct
Direct DNA sequencing or the use of restriction enzymes (eg,
For example, restriction fragment length polymorphism (RFLP)) and Southern block
Can be achieved by methods such as The sequences of the present invention are also useful for chromosome identification.
It is. This sequence is located at a specific location on individual human chromosomes.
And specifically hybridize to that position
I can do it. In addition, we now identify specific sites on the chromosome
Need to be Currently available for labeling chromosome locations
Chromosome labeling based on real sequence data (repetitive polymorphism)
There are almost no chemical reagents. DNA to chromosome according to the invention
Mapping of these sequences to disease-related genes
This is an important first step in correlation with. [0167] Briefly, the sequence was converted from cDNA to P
Prepare CR primer (preferably 15-25 bp)
Can be mapped to chromosomes. 3 'untranslated region
Computer analysis of more than one in genomic DNA
Does not span exons, thus complicating the amplification process
Used to quickly select the primer to be used. Next
These primers contain individual human chromosomes
Used for PCR screening of somatic cell hybrids
You. Hybrid containing the human gene corresponding to the primer
Only yield amplified fragments. PCR mapping of somatic cell hybrids
To assign specific DNA to specific chromosomes
This is a quick procedure. The same oligonucleotide primer
Used in conjunction with the present invention, a fragment derived from a particular chromosome
Panel or a large genomic clone in a similar fashion
Pooling is used to determine partial localization (subloca).
lization can be achieved. Map to chromosomes
Other mapping strategies that can also be used to
For in situ hybridization, labeled
Using flow-sorted chromosomes
Prescreening and chromosome-specific cDNA libraries
For hybridization to construct libraries
Preselection by Metaphase chromosome spread of cDNA clones
In situ hybridization (FIS)
H) is used to provide accurate chromosomal locations in one step.
Can be used. This technique can be as short as 50 or 60 bases.
Can be used with new cDNAs. A review of this technology
And Verma et al., Human Chromos.
omes: a Manual of Basic T
techniques, Pergamon Press,
See New York (1988). Once the sequence has been mapped to a precise chromosomal location,
The physical location of the sequence on the chromosome
Can be correlated with the data. Such data, for example,
V. McKusick, Mendelian Inh
eritance inMan (Johns Hopk
ins University Welch Medi
available online from Cal Library
). Then, the same chromosome region
The relationship between the gene being dropped and the disease
Co-inheritance of physically adjacent genes). Next, the cD between affected and unaffected individuals
Need to determine differences in NA or genomic sequence
There is. Mutations are observed in some or all affected individuals
If this is not observed in any normal individual,
Mutations appear to be causative factors of the disease. Physical Mapping Techniques and Gene Mapping
At the current resolution of imaging technology, chromosomal regions associated with disease
The cDNAs located exactly in the region are 50 and 500
May be one of the potential causative genes. (this is,
1 megabase mapping resolution and 20kb warm
One gene). The polypeptide, its fragments and
Is another derivative, or their analog, or it
Cells expressing them produce antibodies against them
Can be used as an immunogen for These antibodies are
For example, polyclonal antibodies or monoclonal antibodies
Can be The present invention also relates to chimeric, single chain,
And humanized antibodies, and Fab fragments, or
Includes the products of the Fab expression library. In the field
Various procedures known in the art may be used to make such antibodies and fragments.
Can be used for the production of The polypeptide corresponding to the sequence of the present invention
Antibodies produced by direct injection of the polypeptide into animals.
Or by administration of the polypeptide to the animal.
Can be The animal is preferably non-human. Next
The antibody thus obtained is the polypeptide itself.
To join. In this way, the fragmentation of the polypeptide
Even sequences that encode only native polypeptides
Can be used to generate antibodies that bind to the entire drug.
Such antibodies then express the polypeptide.
Used to isolate a polypeptide from a tissue.
You. For preparation of monoclonal antibodies, cells
Any technique for providing antibodies produced by continuous culture of a strain
Surgery may be used. Examples include hybridoma technology
(Kohler and Milstein, 1975,
Nature, 256: 495-497), trio
Technology, human B cell hybridoma technology (Kozbo)
1983, Immunology Toda.
y 4: 72), and a human monoclonal antibody
EBV hybridoma technology (Cole et al.,
1985, Monoclonal Antibod
ies and Cancer Therapy, Al
an R. Liss, Inc. , Pp. 77-96).
Can be Techniques Described for Producing Single Chain Antibodies
(U.S. Pat. No. 4,946,778) shows the immunity of the present invention.
To generate a single-chain antibody to a non-genic polypeptide product
Can be adapted. Transgenic mice also
Can be used to generate a body. Antibodies can also be used, for example, to contact breast cancer cells.
Homing interaction reagents that destroy them when exposed
Method, can be used to target breast cancer cells.
You. This is a fact. Because the antibody is
This is because it is specific to the tuft-specific polypeptide. Mutual
The binding of the working reagent to the antibody binds the interacting reagent directly to the breast.
Let me carry it. This type of antibody is also useful, for example, in the pelvic region.
Label antibodies to facilitate area and breast scanning
Used to perform in vivo imaging
obtain. One method for diagnostic imaging is diagnostic imaging
Label any cancer cells in the breast to be detected with a detectable marker.
Contacting the identified anti-breast specific protein antibody
Include. In this method, the labeled antibody is
The reaction is performed under conditions that bind to the peptide. specific
In some examples, the antibody interacts with the breast (eg, breast cancer cells).
Interact and fluoresce when contacted,
As a result, breast imaging and visibility are amplified,
Allows for the determination of diseased or non-diseased states. The present invention will be further described with reference to the following examples.
However, the invention is limited to such examples.
It should be understood that not. All parts also
The amounts are by weight unless otherwise specified. To facilitate understanding of the following examples,
Certain methods and / or terms that appear frequently are described.
You. "Plasmids" are preceded by lowercase p and
And / or following capital letters and / or numbers
It is specified by doing. Starting plasmids herein
Is commercially available and publicly available without restriction
Or the plasmid available according to published procedures
Can be built from In addition, the described plasmid
Equivalent plasmids are known in the art and
It will be clear to those skilled in the art. "Digestion" of DNA refers to specific arrangements in the DNA.
Catalytically catalyzes the DNA with a restriction enzyme that acts only on the sequence
Refers to cutting. Various controls used in this specification
Restriction enzymes are commercially available and their reaction conditions,
Cofactors and other requirements are those known to those skilled in the art.
Was used. For analytical purposes, typically 1 μg
Rasmid or DNA fragment contains about 2 units of enzyme
Together with about 20 μl of buffer solution. Plasmi
For the purpose of isolating DNA fragments for constructing
For this purpose, typically 5 to 50 μg of DNA are
Digested in a larger volume with 50 units of enzyme
You. Appropriate buffers and substrate for specific restriction enzymes
Is specified by the manufacturer. About 1 hour at 37 ° C
The incubation time is usually used, but this depends on the supplier
May vary according to instructions. After digestion, the reaction is
Direct electrophoresis on a acrylamide gel to obtain the desired fragment
Isolate. The size separation of the cleaved fragments was performed using Sam.
Brook et al., "Molecular Clonin
g: A Laboratory Manual] C
oldSpring Laboratory Pres
s, (1989) 1% TAE agarose gel
Is done using "Oligonucleotide" is a single-stranded polynucleotide.
Oxynucleotide or two complementary polydeoxy
Any of the nucleotide chains, which are chemically combined
Can be achieved. Such synthetic oligonucleotides are
Does not have a 5 'phosphate, and therefore has the ATP
If no phosphoric acid is added with the
Do not connect with Synthetic oligonucleotides are dephosphorylated
Ligate to unfragmented fragments. "Ligation" refers to two double-stranded nucleic acid fragments.
Process to form a phosphodiester bond between
(Maniatis, T et al., Supra, p. 146). other
If not provided, ligation uses known buffers and conditions
And an approximately equimolar amount of the DNA fragment to be ligated.
10 units of T4 DNA ligase per 0.5 μg
("Ligases"). Unless otherwise stated, Graham, F. et al.
And Van der Eb, A. et al. , Virolo
gy, 52: 456-457 (1973).
Transformations were performed as described. EXAMPLES Example 1 Measurement of Transcription of Breast-Specific Gene
Constant) Whether there is active transcription of breast-specific gene RNA
About 6 ml of venous blood was
Standard venipuncture technique using phosphorus-treated tubes
Obtained using Equivalent volume of phosphate buffered saline for whole blood
And then mix it with a 15-ml polystyrene tube.
8 ml of Ficoll (Pharmacia,
(Uppsala, Sweden). this
The gradient is centrifuged at 1800 × g for 20 minutes at 5 ° C.
Carefully aspirate the lymphocyte and granulocyte layer (about 5 ml)
And release the phosphate to 50 ml in a 50 ml tube.
Re-dilute in opiated saline and 1800 × at 5 ° C.
Centrifuge again at g for 20 minutes. Discard the supernatant, and
Pellet containing nuclear cells, as described by the manufacturer
For RNA extraction using RNazole B method
(Tel-Test Inc., Friendsw
wood, TX). [0188] The amount of mRNA from the gene of interest is determined.
1 to 20 (SEQ ID NOS: 1 to 2)
MR transcribed from a human gene containing the DNA sequence of 0)
Make sure that at least a part of the NA sequence has identity.
Design the probe. Mix this probe with the extracted RNA
And mix the mixed DNA and RNA
Precipitate in ethanol (-70 ° C for 15 minutes). This pere
Resuspend in hybridization buffer, and
And dissolve. Transfer the tube containing this mixture to the DNA
10 to 15 minutes in a 72 ° C water bath to denature
Cubate. Transfer this tube to the desired hybrid
Transfer into water bath quickly at the temperature of the zation. Hybrid
The ligation temperature depends on the content of G + C in DNA.
Exist. Hybridization is performed for 3 hours.
Add 0.3 ml of nuclease-S1 buffer and add
And mix well. 50 μl of 4.0 M ammonium acetate
And the reaction was stopped by adding 0.1 M EDTA
You. This mixture is extracted with phenol / chloroform,
Then add 20 μg of carrier tRNA, and etc.
Precipitate with a volume of isopropanol. 40μ precipitate
dissolved in 1 TE (pH 7.4) and alkaline
Run on agarose gel. After electrophoresis, distribute a small amount of RNA.
Sequence to confirm nucleotide sequence. (More detailed
For a review, see Fabaloro, J. et al. Mot
hods Enzymol. , 65: 718 (198
0)). The two oligonucleotide primers
Used to amplify sequences isolated in the manner described above.
The 5 'primer is 20 nucleotides in length, and
The 3 'primer is complementary to the 3' end of the isolated mRNA
Arrangement. Primers to isolated mRNA
Follow custom design. Reverse transcription reaction and PCR amplification
To Perkin Elmer 9600 PCR machine
(Emeryville, CA)
Continuously. 20 μl diethyl pyrocarbonate treatment
400 ng of total RNA in the
For 1 minute and then immediately on ice just before the addition of PCR reagents.
Cool on top. A total PCR volume of 50-μl is 2.5 units
Taq polymerase (Perkin-Elmer),
2 units of chicken myeloblastosis virus reverse transcriptase (Bo
ehringer Mannheim, Indian
apolis, IN); 200 μM of each dCT
P, dATP, dGTP and dTTP (Perk
in Elmer); 18 pM of each primer,
10 mM Tris-HCl; 50 mM KC
l; and 2 mM MgCl ₂ (Perkin El
mer). The PCR conditions are as follows:
Cycle 1 at 42 ° C. for 15 minutes, then at 97 ° C. for 15 seconds
Cycle 2 at 95 ° C. for 1 minute, 60
° C for 1 minute and 72 ° C for 30 seconds (15 cycles
Cycle 3 is 95 ° C for 1 minute, 60 ° C for 1 minute
For 1 minute at 72 ° C. (10 cycles); cycle
4 at 95 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C.
For 2 minutes (8 cycles); cycle 5 is 15 minutes at 72 ° C.
Minutes (1 cycle); and the last cycle is
At 4 ° C. until removed from the machine. 50
-Concentrate the μl PCR product to 10 μl by vacuum centrifugation.
And then remove the sample, ethidium bromide
1.2% Tris-borate-EDTA containing
Run on agarose gel. The band of expected size is
Indicates that this gene is present in the assayed tissue
You. The amount of RNA in the pellet can be quantified in a number of ways.
obtain. For example, it may weigh. Confirmation of the nucleotide sequence of the PCR product
Performed by microsequencing. PCR products were
n PCR Product Purificatio
nKit (Qiagen, Chatsworth,
Using CA) to purify as described by the manufacturer.
You. 1 μg of the PCR product was added to Tag Dye Deoxy.
Terminator Cycle sequencing kit
Using a Perkin-Elmer 9600 PC
Applied Biosystem on R machine
ms (Foster, CA)
Next, it is subjected to PCR sequencing. The sequencing product was
Tri-Sep column (Princeton Sepa)
ratios, Adelphia, NJ)
And purified as described by this company. Next
This product is then transferred to the Macintosh IIci
ABI model 373A DNA with integrated
Sequencing system (Applied Biosystem)
(ms). Example 2 Bacterial Expression of BSG Protein
And purification and use for monoclonal antibody preparation.
DNA sequence encoding the polypeptide of the present invention (for
In the embodiment, BSG1 (ATCC Accession No. 971)
No. 75)), the 5 ′ sequence of the protein and the protein
PCR oligonucleotide corresponding to vector sequence 3 '
Amplification was first performed using a reotide primer. D
Additional nucleotides corresponding to the NA sequence were added 5 ′ and
And 3 'sequences. 5 'oligonucleotid
Primer has the sequence 5 ′ GCCACATGGGATG
Having TTTTCAAG 3 '(SEQ ID NO: 21);
NcoI restriction enzyme site followed by processed protein
15 nucleotides starting from the first amino acid of the protein
It may include a coding sequence. 3 'sequence 5' GCCGCAGA
TCTGTCTCCCCCACTCTGGGC 3 '
(SEQ ID NO: 22) is complementary to the BglII restriction enzyme site
Sequence and also subsequently encodes the protein.
18 nucleotide nucleotide sequence. Restriction enzyme section
The position was determined using a bacterial expression vector, pQE-60 (Qiage).
n, Inc. Chatsworth, CA)
Corresponds to the restriction enzyme site. pQE-60 is antibiotic resistant
(Amp ^r ), Bacterial origin of replication (ori), IPTG system
Possible promoter operator (P / O), Riboso
Binding site (RBS), 6-His tag, and restriction enzyme
Code the prime site. Then, pQE-60 was converted to Nco
Digest with I and BglII. The proliferating sequence was replaced with pQE-6.
0 and the histidine tag and RBS
The sequence to be inserted in frame. Then
Using the ligation mixture, E. coli strain M15 / re
p4 (Qiagen) is described in Sambrook, J .; Et al.,
Molecular Cloning: A Labo
rattro Manual, Cold Spring
g Laboratory Press, (198
Transform according to the procedure described in 9). M15 / rep
4 contains multiple copies of plasmid pREP4,
It expresses the lacI repressor and
Shin resistance (Kan ^r ) Is also given. Transformants are LB
Identified by their ability to grow on rate, and
Select ampicillin / kanamycin resistant colonies.
Isolate plasmid DNA and use restriction enzyme analysis
Confirm. The clone containing the desired construct was
(100 μg / ml) and Kan (25 μg / ml)
Overnight in liquid culture in LB medium supplemented with both (O /
N) Proliferate. 1: 100- using O / N culture
Inoculate a large culture at a ratio of 1: 250. Cells, 6
00 optical density (OD ⁶⁰⁰ ) Is 0.4 and 0.6
And grown to between Then, IPTG ("iso
Propyl-BD-thiogalactopyranoside ").
To a final concentration of 1 mM. IPTG is lacI replay
Gene expression by inactivating
To release the P / O for 3 to more cells
Grow for 4 hours. The cells are then recovered by centrifugation
I do. The cell pellet is treated with the chaotropic agent 6M guar.
Solubilize with Niidine HCl. After clearing, solubilized tan
Proteins are made more rigid by proteins containing 6-His tags
Chromatography on nickel chelate columns under binding conditions
Purify from this solution by chromatography (Hochu
li, E. J. et al. Chromatography
411: 177-184 (1984)). BGS1
The protein (> 90% pure) was converted to 6M guanidine HCl
Elution from the column at pH 5.0 and the purpose of the regeneration
For 3M guanidine HCl, 100 mM sodium phosphate
Thorium, 10 mM glutathione (reduced), and 2
Adjust to mM glutathione (oxidized form). In this solution
After a 12 hour incubation, the protein was
Dialyze against mM sodium phosphate. [0193] The protein thus purified was obtained from
Produce monoclonal antibodies specific for such proteins
Can be used as an epitope. Isolated
Monochromes produced against proteinaceous polypeptides
Null antibodies can be used to inject animals directly with the polypeptide.
Or by administering the polypeptide to the animal
Can be obtained by Then it was obtained in this way
Antibodies bind to the protein itself. Then this
Such an antibody would elicit the protein from its polypeptide
From the resulting tissue, for example, using an ELISA assay,
Can be used to isolate. Example 3 cDNA Libraries from Breast Tissue
Preparation of library) Total cellular RNA was guanidinium
According to the Enol method, the previous description (P. Chomczyn)
ski and N.I. Sacchi, Anal. Bi
ochem. , 162: 156-159 (198
7)), RNAzol (Cinna-Biote)
Prepared from tissue using cx). Further RNA
Include ethanol precipitation. Poly A mRNA is converted to oligo
dT-coated latex beads (Qiagen)
Used to isolate from total RNA. A sufficient amount of total RNA
If available, make a poly A selection twice and select
Ensure better separation from nitrated material. The mRNA selected with oligo dT was ligated with Go
bbler and Hoffman (Gobbler,
U. And B. J. Hoffman, 1983,
Gene, 25: 263).
Used for A synthesis. First-strand synthesis was performed by Moloney.
Mouse sarcoma virus reverse transcriptase (Stratagen)
e) or Superscript II (Molo
RNase H containing no mouse reverse transcriptase,
Gibco-BRL). First chain
Synthesis was performed using primers / reagents containing an Xho I restriction site.
Start with an anchor. Nucleotides used in the synthesis
Mixture prevents restriction enzyme digestion within the cDNA sequence
To include methylated dCTP. Second strand synthesis
For E. coli polymerase Klenow Hula
Using the mentment, and [ ³² P] -dATP,
Incorporate as a tracer for leotide incorporation. Following the second strand synthesis, the cDNA was converted to T4
DNA polymerase or Klenow fragment
Use either one to make blunt ends. Eco RI Ada
Is added to the cDNA and the cDNA is added to Xho.
Digest with I. cDNA is separated by Sephacryl
l Size with S-500 column (Pharmacia)
Fractionation, excess linker and less than about 500 base pairs of c.
Remove the DNA. The cDNA was unidirectionally transformed into pBluesc
ript II phagemid or λ Uni-zap
Any Eco of XR (Stratagene)
Clone into the RI-Xho I site. pBlues
When cloning into script II, use plasmid
To E. E. coli SURE competent cells (St
electroporation into the rat. c
When cloning DNA into Uni-Zap XR
Is a Gigipack II packaging extract (S
Packaging using tratagene). Pa
Infecting SURE cells using the packaging phage,
Amplify. pBluescript containing cDNA insert
t phagemid was isolated from λZap phage
Use Exassist (Stratagene)
And resect. The recovered phagemid was subjected to SOLR
E. FIG. E. coli cells (Stratagene)
You. (Preparation of Template for Sequencing)
Template DNA for 1) boiling method, or 2) PCR amplification
Prepared by The boiling method is described in Holmes and Quigl.
ey (Holmes, DS and M. Quig)
ley, 1981, Anal. Biochem. ,
114: 193). Bluesc
Rip II, or recovered Bluescript
Any of the cDNAs cloned into the t phagemid
The resulting colonies are grown overnight in concentrated bacterial media. 4
00 μl of cells are centrifuged and lysozyme (8
0 μg / ml) and RNase A (4 μg / ml)
Containing STET (0.1 M NaCl, 10 mM T
RIS pH 8.0, 1.0 mM EDTA and 5
% Triton X-100). cell
Boil for 40 seconds and centrifuge for 10 minutes. Up
Remove the supernatant and precipitate the DNA with PEG / NaCl.
Settle and wash with 70% ethanol (2x).
Resuspend the template in water at about 250 ng / μl. The preparation of the template by PCR was performed using Rosent
hal et al. (Rosenthal et al., Nucleic A
cids Res. , 1993, 21: 173-1.
74) is a modification of the method. pBluescript
II or recovered pBluescript phage
Colonies containing the cDNA cloned into the mid,
96 well tissue culture plates in LB containing ampicillin
Incubate overnight at rate. 2 μl of this culture
With the Tricine buffer system (Ponce and Mic)
ol. , Nucleic Acids Res. ,
1992, 20: 1992) and 200 μM dN.
Using TP as a template in a PCR reaction
(Saiki, RK, et al., Science, 23.
9: 487-493, 1988; and Saiki,
RK, et al., Science, 230: 1350-1.
354, 1985). Selected ply for template amplification
The merset is the plasmid that was selected for template sequencing.
Outside the Immerset. The primers used were
5'-ATGCTTCCGGCTCGTATG-3 '
(SEQ ID NO: 23) (this is in pBluescript
5 'of the M13 reverse sequence of
TTTCCCAGTCACGAC-3 ′ (SEQ ID NO: 2
4) (This is the M13 sequence in pBluescript
3 'of Limer). M13 forward and reverse array
Any primer corresponding to the sequence adjacent to
You. Perkin-Elmer 9600 Thermosai
Amplify the template using the following cycler conditions
Do: 94 ° C. for 5 minutes (1 cycle);
At 20 ° C.); 55 ° C. for 20 seconds (1 minute at 72 ° C.) (30 cycles
); 7 min at 72 ° C (1 cycle). Following amplification
The PCR template was precipitated with PEG / NaCl.
And wash three times with 70% ethanol. Mold
Resuspend in water. Example 4 Selection of Selected Clones from Breast Tissue
Separation) Prepared from human breast tissue using two approaches
Specific clones from the cloned cDNA library
Let go. First, clones were cloned into oligonucleotides
Screen libraries directly with probes
To isolate. To isolate a specific clone
For example, a specific oligonucleotide having 30-40 nucleotides
Creotide is applied to Applied Biosystems
Using a DNA synthesizer, one of the partial sequences described in the present application was
And combine them. The oligonucleotide is called T4
Using polynucleotide kinase ³² P- -ATP
And standard protocols (Maniatis
Et al., Molecular Cloning: A La
boratoryManual, Cold Spri
ng Harbor Press, Cold Spr
ing, NY, 1982). λcD
NA library on 1.5% agar plate, density 2
0000-50,000pfu / 150mm play
And sow the seeds. These plates are coated with Nylon membrane
Uses standard phage screening protocol
(Stratagene, 1993)
To synchronize. Specifically, denatured and immobilized fur
Nylon membrane with diDNA was applied to 6 × SSC, 20 m
M NaH ₂ PO ₄ , 0.4% SDS, denatured 5
5 x Denhardt, 00 μg / ml, sonicated
Salmon sperm DNA and 6 × SSC, 0.1%
Prehybridize in SDS. 1 hour pre-high
After hybridization, the membrane is hybridized
Buffer (6 × SSC, 20 mM NaH ₂ PO ₄ , 0.
4% SDS, 500 μg / ml denatured, sonicated
Salmon sperm DNA). ⁶ cpm / m
l ³² Hybridize overnight at 42 ° C with P-probe
You. The membrane was washed at 45-50 ° C. with wash buffer (6 × SSC,
(0.1% SDS) for 20-30 minutes,
And expose to Kodak X-ray film overnight. Positive
Isolate clones and perform secondary and tertiary screening
To purify. Identical to the subsequence described in this application
Sequencing of the purified clones to verify
I do. A cDNA library prepared from human breast tissue
Another approach to screening the blurry is to distribute everything
The purpose is to prepare DNA probes corresponding to the columns. Step
Both ends of the reported subsequence to prepare the lobe
Two oligonucleotides of 17-20 nucleotides of origin
The tide primer is synthesized and purified. These two
Using two oligonucleotides, the cDNA
The probe is amplified using the library template. DNA casting
From the phage lysate of the cDNA library,
Semi-phage DNA preparation protocol (Maniatis
Prepared according to Polymerase chain reaction
Using μg of the above cDNA template, 25 μl of the reaction mix
Perform in compound. The reaction mixture is 1.5-5 mM MgC
l ₂ , 0.01% (w / v) gelatin, 20 μM each d
ATP, dCTP, dGTP, dTTP, 25 pmol
Primers, and a 0.25 Unit Taq
Limerase. 35 cycles of PCR (1 at 94 ° C)
Denaturation for 1 minute; annealing at 55 ° C. for 1 minute;
Extension for 1 minute) was analyzed using a Perkin-Elmer Cet.
us using an automated thermal cycler. Amplified
The product is analyzed by agarose gel electrophoresis and
Excising the DNA band with the measured molecular weight, and
Purify. PCR products, DNA products subclonin
Probe by sequencing and sequencing
Make sure that Connect the probe to Multiprime D
NA Labeling System (Amer
sham), specific activity <1 × 10 ⁹ smell dmp / μg
Label. Using this probe, Stratage
λ cDNA library according to the
Clean. Hybridization was performed at 5 × T
EN 920XTEN (0.3M Tris-HCl
pH 8.0, 0.02M EDTA and 3M NaC
l) 5 × Denharddt's, 0.5% pyrroline
Sodium acid, 0.1% SDS, 0.2 mg / ml
Heat-denatured salmon sperm DNA and 1 × 10 ⁶ cpm / m
l [ ³² P]-55 ° C using labeled probe
For 12 hours. Filter 0.5X TE
N for 20-30 minutes at room temperature, then at 55 ° C.
And wash for 15 minutes. Dry the filter and-
Using Kodak XAR-5 film at 70 ° C
To autoradiograph. Positive clones are
And purified by a third screening. Isolated
The sequence of the clone is confirmed by DNA sequencing. From the partial sequences described herein, the complete sequence
The general procedure for obtaining the sequence is summarized as follows:
(Procedure 1) A partial sequence clone (a sequence to obtain a partial sequence)
Selected humans from the sequenced cDNA clones)
For example, the DNA is subjected to endonuclease using Eco-RI.
Aase digestion, gel electrophoresis, and low melting point agarose
Purification by isolation of the clone by removal from the
You. The isolated insert DNA is, for example, ³² By P sign
Nick translation or run
Radiolabel with dam primer labeling. Sign
Phage cDNA using the inserted insert as a probe.
Library or plasmid cDNA library
Screen. Clones related to probe cDNA
Colonies containing chromosomes are identified and identified by known purification methods.
And purify. Insert the end of the newly purified clone
Reotide sequencing and identifying the full-length sequence. Then all
Complete sequencing of long clones was performed using Exoncleas.
e by III digestion or primer walking
Do. Fewer deposited clones for which partial sequences were obtained
MRs derived from various tissues
A Northern blot of NA was performed as needed,
Adjust the size of mRNA against what is considered DNA
I can get it. The following procedures 2 and 3 were performed with the deposited
Clones containing the full-length sequence
If not, the full length gene or the full length coding portion of the gene
Can be used to obtain Human breast tissue derived or deposited
Libraries from the clone mixtures obtained are also provided by the present invention.
From a source other than the mixture deposited using the subsequence of
Applicable to obtain full-length sequences from the resulting clones.
is there. (Procedure 2) RACE protocol for recovery of full-length gene A partial cDNA clone was prepared according to the method described by Frohman, M .;
A. Dush, M .; K. , And Martin,
G. FIG. R. (1988) Proc. Nat'l. Ac
ad. Sci. USA, 85: 8998-900.
Rapid Amplification (RACE) Hand of cDNA End as described in 2
By using a sequence, it can be full length. 5 'or
CDNA clones lacking any of the 3 'ends are
Extends to each of the translation start or translation stop codon,
Can be reconstructed to include open base pairs. Most
In some cases, the cDNA lacks the start of its translation. Less than
Briefly describes a modification of this original 5'RACE procedure
I do. Poly A + or total RNA is
pt II (Gibco / BRL) and cDNA
Reverse with column specific antisense or complementary primers
Transcribe. Primers are used with Microcon Conc
Removed from reactants with an extractor (Amicon)
I do. Then, dATP and the end of the first strand cDNA were added.
Terminal deoxynucleotide transferase (Gib
(co / BRL). Thus, necessary for PCR amplification
Generate a simple anchor sequence. Second strand in PCR buffer
DA-tail, Taq DNA polymerase (Per
Kin-Elmer Cetus), three adjacent systems
Restriction sites (XhoI, SalI and ClaI)
Oligo-dT primers containing at the 5 'end, and these
Synthesized from primers containing only restriction sites. These two
Heavy chain cDNA is used with the same primers for 40 cycles.
Nested cDNA-specific
PCR amplification with antisense primer. PCR products
With an ethidium bromide-agarose gel
Release and prediction of DNA encoding the defective protein
The region of the gel containing the cDNA product of the desired size
You. cDNA can be used in Magic PCR Prep Kit
(Promega) and purified from agarose using X
restriction digested with hoI or SalI, and
pBluescript SKII (Stratage
ne) into plasmids such as ShoI and EcoR.
Binds at the V site. Transform this DNA into bacteria
And clone the plasmid clone into the correct protein
Sequence to identify code insert. Right 5 '
The termini are homologues that have putatively identified this sequence and
By comparing the overlap with the partial cDNA clone,
Confirm. Several quality control kits are commercially available.
You. Reagents and methods similar to those described above are available from Gibco
/ BRL in kit form. The second kit
Is available from Clontech, which is
A series of technologies, Dumas et al. (Dumas, JB,
Edwards, M.E. , Delort, J.A. You
And Mallet, Jr. , 1991, Nucl
eic Acids Res. , 19: 5227-52.
32) developed by SLIC (for single-stranded cDNA)
(Single-stranded binding). The main difference in the procedure is the reversal
That the RNA is alkali-hydrolyzed after transcription, and
Anchor primer in which RNA ligase contains a restriction enzyme site
Used to bind DNA to first-strand cDNA
It is. This is a poly that is difficult to sequence beyond.
The necessity of dA-tailing reaction to generate T stretch
To eliminate. Production of 5 'cDNA from RNA
Alternatively, use cDNA library double-stranded DNA
May be. Asymmetric PCR amplified antisense cD
The NA chain is ligated with an antisense cDNA-specific primer and
And a plasmid (plasmid-ancho)
red) Synthesize with a primer. These primers
The symmetric PCR reaction was removed and the shortened cDNA-
Connect specific antisense primers and plasmid
Perform with IDA primer. (Procedure 3) In order to produce a 5 'terminal sequence to obtain a full-length gene,
RNA Ligase Protocol Once the gene of interest has been identified, several methods
Of genes that cannot be present in the original deposited clone,
It can be used to identify 5 'or 3' portions. these
The method includes similar and identical 5 'and 3' RACE.
Filter probes using specific probes and protocols
Roving (filter probing), claw
Enrichment, but these
It is not limited to. The full-length gene is in the library
And can be identified by probing, while 5 ′
A useful way to generate the ends is to use native subsequences.
Of existing sequence information to produce missing information
It is to use. A similar method for 5'RACE is desired
To produce the missing 5 'end of the full-length gene of
Available. (This method is based on the Front-Ra
Cine et al., Nucleic Acids Re
s. , 21 (7): 1683-1684 (199
3)). Briefly, specific RNA oligonucleotides
Nucleotides are predicted to contain full-length gene RNA transcripts.
RNA and linked RNA oligonucleotides
Of a primer set population containing primers specific to
Ligation at the 5 'end. The known sequence of the gene of interest (E
Using the primers specific to ST), the desired full-length gene
The 5 'portion of the offspring is PCR amplified. This is followed by sequencing
And can be used to produce full-length genes.
You. This method is based on total RNA isolated from a desired source.
And poly A RNA can be used, but this procedure involves
Not necessary. The RNA preparation is then degraded or damaged.
The 5 'phosphate group of the damaged RNA (these are
If it is necessary to eliminate (which may interfere with the gauze process)
It can be treated with phosphatase. Then phosphatase
Inactivate (if used) and
The cap structure at the 5 'end of the RNA.
To remove RNA from tobacco acid pyrophosphatase
To process. This reaction is based on capped RNA.
Leaving a 5 'phosphate group at the 5' end of the T4
Using RNA ligase, RNA oligonucleotide
To This modified RNA preparation is then
First-strand cDN using gene-specific oligonucleotides
Can be used as a template for A synthesis. Then the first chain combination
Specific reactions are performed for the bound RNA oligonucleotide
Primers and known sequences of the gene of interest (ES
Using primers specific to T), the desired 5 ′
Can be used as a template for PCR amplification. Then get
The resulting product was sequenced and analyzed and the 5 '
Check that the sequence belongs to the subsequence. Example 5 Using Baculovirus Expression System
Cloning and expression of BSG1) Full length BSG
DNA sequence encoding one protein (ATCC accession number
No. 97175), the 5 ′ sequence of this gene and
PCR oligonucleotide primer corresponding to 3 'sequence
The 5 ′ primer was sequence 5′-A
AAGGATCCCCCCGCCATCATGGATGT
Having TTTCAAGAAG 3 ′ (SEQ ID NO: 25);
And the BamHI restriction enzyme site (bold) followed by
BSG1 gene (the translation start codon "ATG" is underlined.
Efficient) to initiate translation in eukaryotic cells
Eight nucleotides similar to the signal (Kozak,
M. , J. et al. Mol. Biol. , 196: 9
47-950 (1987)). The 3 'primer had the sequence 5' AAATC
TAGACTAGTCTCCCCCACTCTG 3 '
(SEQ ID NO: 26) and has a restriction endonucleus
XbaI cleavage site and 3 ′ of this BSG1 gene
Contains 21 nucleotides complementary to the sequence. Amplified sequence
Using a commercially available kit (“Geneclean” BIO1)
01 Inc. , La Jolla, Ca. )
And isolated from a 1% agarose gel. Then this
Fragments were digested with the endonucleases BamHI and X
digest with baI, then purify again on 1% agarose gel
did. This fragment is named F2. The vector pA2 (of the pVL941 vector)
Variant, described below) using Baculovirus expression system B
Used for SG1 protein expression (for review
Is described in Summers, M .; D. And Smith,
G. FIG. E. FIG. 1987, Manual of met
hods for baculovirus vector
ors and insect cell culture
re procedures, Texas Agri
cultural Experimental Sta
Tion Bulletin No. See 1555
That). This expression vector is Autographa
californica nuclear polyhedrosis virus (AcM
NPV) strong polyhedrin promoter, followed by
Of the restriction endonucleases BamHI and XbaI
Includes recognition sites. Simian virus (SV) 40 poly
Adenylation site used for efficient polyadenylation
I have. For easy selection of recombinant viruses, E. coli was used.
E. coli-derived β-galactosidase gene
Phosphorous promoter followed by polyhedrin gene
Insert in the same direction as the polyadenylation signal. Polyhed
Phosphorus sequence co-transfected wild-type virus D
Both sequences are included in the viral sequence for cell-mediated homologous recombination of NA.
Adjacent at the edge. Many other baculovirus vectors
Can be used instead of pA2. For example, pRG
1, pAc373, pVL941, and pAcIM1
(Luckow, VA and Summer)
s, M .; D. , Virology, 170: 31-3.
9). The plasmid was replaced with the restriction enzymes BamHI and X
digested with baI and calf intestine by procedures known in the art.
Dephosphorylated using phosphatase. Then, DN
A using a commercially available kit ("Geneclean" BIO
101 Inc. , LaJolla, Ca. )
And isolated from a 1% agarose gel. This vector
The DNA is called V2. The fragment F2 and the dephosphorylated
Rasmid pA2 is ligated using T4 DNA ligase
did. Then, E. E. coli HB101 cells
And using the enzymes BamHI and XbaI
Plasmid containing the BSG1 gene (pBacBS
Bacteria containing G1) were identified. Cloned fragment
Was confirmed by DNA sequencing. 5 μg of plasmid pBacBSG1 was
Lipofection method (Felgner et al., Proc.
Natl. Acad. Sci. USA, 84:
7413-7417 (1987)).
μg of commercially available linearized baculovirus (“Bacu
loGold ^TM baculovirus DN
A ", Pharmingen, San Dieg
o, CA. ) And co-transfected. 1 μg of BaculoGold ^TM Will
DNA and 5 μg of plasmid pBacBSG1
With 50 μl of serum-free Grace's medium (Life Te)
channels Inc. , Gaithers
burg, MD) containing microtiter plates
Mix in sterile well. Thereafter, 10 μl of lipofer
Add Kutin and 90 μl Grace medium and mix
And incubated for 15 minutes at room temperature. Next
The transfection mixture without serum.
35 mm tissue culture plate containing 1 ml of Grace's medium
Sf9 insect cells (ATCC CR
L 1711). New plate
The solution was shaken back and forth to mix.
The plate was then incubated for 5 hours at 27 ° C.
Was. After 5 hours, place the transfection solution on a plate
1 ml supplemented with 10% fetal bovine serum
Grace insect medium was added. Incubate plate
Return to beta and continue culturing at 27 ° C. for 4 days. After 4 days, the supernatant was collected and Summe
rs and Smith (supra)
Workup assay was performed. As a modification, stained blue
"Blue Ga", which allows easy isolation of plaques
l ”(Life Technologies In
c. , Gaithersburg)
Sgel was used. (Detailed description of "plaque assay"
Also has Life Technologies In
c. Insect cell culture distributed by Gaithersburg
Nursing and baculovirology user guides (9-10
P.)). After 4 days of serial dilution, the virus was added to the cells.
And plaque stained blue with Eppendorf
Picked up with pet chips. Next, the recombinant virus
Mug agar in an eppen containing 200 μl of Grace's medium
Resuspended in Dorf tube. Agar, centrifuge briefly
Removed by release and contains recombinant baculovirus
The supernatant was added to Sf9 cells seeded on a 35 mm dish.
Used to infect. Four days later, these culture dishes are
The supernatant was collected and then stored at 4 ° C. Sf9 cells were supplemented with 10% heat-inactivated FBS.
Grow in filled Grace's medium. Infect cells
Recombinant baculovirus V-BS with severe (MOI) 2
Infected with G1. After 6 hours, the medium is removed and the
SF900 II medium without thionine and cysteine
Ground (Life Technologies Inc.,
Gaithersburg). 42 hours
Later, 5μCi ³⁵ S-methionine and 5 μCi
³⁵ S-cysteine (Amersham) was added. Fine
The cells are incubated for an additional 16 hours, after which the cells are
Collected by heart separation and labeled protein
Possible by DS-PAGE and autoradiography
Visualized. Example 6 Recombinant BS in COS cells
G1 expression) The expression of the plasmid, BSG1 HA,
Vector pcDNAI / Amp (Invitrogen)
Derived from. The vector pcDNAI / Amp contains:
Contains: 1) SV40 origin of replication, 2) Ampicillin resistance
Sex gene, 3) E. E. coli origin of replication, 4) polyline
Kerr region, SV40 intron, and polyadenylation
CMV promoter followed by a site. The whole precursor and its
Codes HA tag fused in-frame to 3 'end
The DNA fragment to be converted into the polylinker region of the vector.
Region. Therefore, recombinant protein expression
Is governed by the CMV promoter. HA tag is
(I. Wilson, H. Nima
n, R.I. Heighten, A Cherenso
n, M. Connolly, and R.C. Lerne
r, 1984, Cell 37, 767 (198
4)) like influenza hemagglutinin protein
Corresponding to the next epitope. HA to target protein
Using an antibody that recognizes the HA epitope
This allows easy detection of recombinant proteins. The plasmid construction strategy is described below.
: DNA sequence encoding BSG1 (ATCC contract)
No. 97175) using the following two primers:
Constructed by PCR: 5 'primer AAAAG
GATCCCCCGGCCATCATGGATGTTTTT
CAAGAAG 3 '(SEQ ID NO: 27) is a BamHI
BSG1 code starting at the site followed by the start codon
3 'sequence AAATC comprising 18 nucleotides of sequence
TAGACTAAAGCGTAGTCTGGGACGT
CGTATGGGTACTCCTGGGGTCTCCC
CCACTCTGGGC 3 ′ (SEQ ID NO: 28)
baI site, translation stop codon, HA tag, and Bam
The last 18 nucleotides of the HI coding sequence (stop codon
Is not included). Therefore, PCR
The product is a BamHI site, HA fused in frame.
BSG1 coding sequence followed by tag, translation adjacent to HA tag
Includes a translation stop codon, and an XbaI site. PCR
Amplified DNA fragments and vectors (pcDNAI
/ Amp) using BamHI and XbaI restriction enzymes
And digested and ligated. The ligation mixture is c
oli SURE strain (Stratagene Clon
ing Systems, 11099 North
Torrey Pines Road, La Jol
la, available from CA 92037)
And inoculate the transformed culture into ampicillin medium plates
And selected resistant colonies. Plasmid DNA
Was isolated from the transformant and the correct fragment
The presence was checked by restriction enzyme analysis. Recombinant BSG protein
For expression of COS cells, DEAE-DEXTRA
N method (J. Sambrook, E. Fritsc)
h. Maniatis, Molecular C
loning: Alabourory Manua
1, Cold Spring Laboratory
Press, (1989)).
Transfected. Expression of BSG HA protein
Was detected by radiolabeling and immunoprecipitation (E.
Harlow, D.M. Lane, Antibod
ies: A Laboratory Manual,
Cold Spring Harbor Labor
atry Press, (1988)). Cells
Two days after transfection, ³⁵ With S-cysteine
Labeled for 8 hours. The culture medium is then recovered and the cells
With a surfactant (RIPA buffer (150 mM NaC)
1, 1% NP-40, 0.1% SDS, 1% NP
-40, 0.5% DOC, 50 mM Tris (pH
7.5)) (Wilson, I. et al., Ibid.)
37: 767 (1984)). Cell lysates and media
Both of the nutrient media were prepared using HA-specific monoclonal antibodies.
And settled. Precipitated protein was purified by 15% SDS-
Analyzed on PAGE gel. The human breast-specific gene polypeptide and
DNA (RNA) encoding such a polypeptide
And the production of such polypeptides by recombinant technology
A procedure for performing the above is disclosed. Such a polynucleotide
Or polypeptide as a diagnostic marker for breast cancer
And to determine if breast cancer has spread
A method for use as a medicament is also disclosed. Cancer
Can be used to target vesicles and breast cancer
Breast cancer-specific gene polymorphs that can be used as part of
Antibodies specific for the peptides are also disclosed. For polypeptides
Method for screening antagonists against
And therapeutic uses thereof are also disclosed. Numerous modifications and variations of the present invention are described above.
It is possible with reference to the teachings and therefore the scope of the appended claims.
Within the bounds, the invention may be practiced other than as specifically described.
obtain. According to the present invention, a human breast-specific gene
Encoding polypeptides and such polypeptides
DNA (RNA) and such polypeptides
Procedures for producing by recombinant technology, such polynus
Chromotide or polypeptide as diagnostic marker for breast cancer
As and determine if breast cancer has spread
For use as a drug for cancer
Can be used to target
Cancer specific gene polypeptide that can be used as a part
Antibodies specific for
Methods for screening for antagonists
And its therapeutic use.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the full-length c of the breast-specific gene 1 of the present invention.
DNA sequence. FIG. 2 is a partial cDNA sequence of the present invention and its corresponding deduced amino acid sequence of breast-specific gene 2. FIG. 3 shows the partial cDNA sequence of the present invention and the deduced amino acid sequence of breast-specific gene 3. FIG. 4 is a partial cDNA sequence of the present invention and its corresponding deduced amino acid sequence of breast-specific gene 4. FIG. 5 shows part c of the breast-specific gene 5 of the present invention.
DNA sequence. FIG. 6 shows the partial cDNA sequence of the present invention and the deduced amino acid sequence of breast-specific gene 6. FIG. 7 shows part c of the breast-specific gene 7 of the present invention.
DNA sequence. FIG. 8 shows part c of the breast-specific gene 8 of the present invention.
DNA sequence. FIG. 9 shows part c of the breast-specific gene 9 of the present invention.
DNA sequence. FIG. 10 is a partial cDNA sequence of the breast-specific gene 10 of the present invention. FIG. 11 is a partial cDNA sequence of the breast-specific gene 11 of the present invention. FIG. 12 is a partial cDNA sequence of the breast-specific gene 12 of the present invention. FIG. 13 is a partial cDNA sequence of the breast-specific gene 13 of the present invention. FIG. 14 is a partial cDNA sequence of the breast-specific gene 14 of the present invention. FIG. 15 is a partial cDNA sequence of the breast-specific gene 15 of the present invention. FIG. 16 is a partial cDNA sequence of the breast-specific gene 16 of the present invention. FIG. 17 is a partial cDNA sequence of the breast-specific gene 17 of the present invention. FIG. 18 is a partial cDNA sequence of the breast-specific gene 18 of the present invention. FIG. 19 is a partial cDNA sequence of the breast-specific gene 19 of the present invention. FIG. 20 is a partial cDNA sequence of the breast-specific gene 20 of the present invention.

   ────────────────────────────────────────────────── ─── Continuation of front page    (71) Applicant 597018381             9410 Key West Avenue,               Rockville, Marylan             d 20850, United State             s of America (72) Inventor Hongjun Ji             United States Maryland 20874,               German Town, Country Ridge             The drive 13144 (72) Inventor Craig A. Rosen             United States Maryland 20850−             3338, Layton'sville, Rolling             Hill Road 22400 F-term (reference) 4B024 AA01 AA11 BA80 CA04 CA11                       DA02 EA02 EA03 EA04 GA11                       GA18 GA19 HA03 HA11

Claims (1)

Claims 1. An isolated polynucleotide, comprising:
The following: (a) a polynucleotide encoding the same polypeptide as the polynucleotide of FIG. 1; (b) a code comprising a DNA having at least 90% identity to the DNA of one of FIGS. (C) a polynucleotide that hybridizes to the polynucleotide of (a) and has at least 70% identity to the polynucleotide; and (c) a polynucleotide that encodes the same mature polypeptide as the human gene having the portion; d) a polynucleotide selected from the group consisting of a polynucleotide encoding a mature polypeptide identical to a human gene having a coding portion comprising DNA having at least 90% identity to the DNA contained in the deposited clone. An isolated polynucleotide.