AU2002301561B2 - Signal Sequence Trapping Method - Google Patents

Description

S&F Ref: 509748D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicants: Chugai Seiyaku Kabushiki Kaisha 5-1, Ukima 5-chome, Kita-ku, Tokyo 115-8543 Japan Toshio Kitamura 6-16-20-406, Shirokane Minato-ku Tokyo 108-0072 Japan Actual Inventor(s): Address for Service: Invention Title: Toshio Kitamura, Tetsuo Kojima Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Signal Sequence Trapping Method The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c 1

SPECIFICATION

SIGNAL SEQUENCE TRAPPING METHOD r Technical Field The present invention belongs to the field of gene engineering and relates to a method for detecting and isolating a DNA encoding a peptide that has secretory ability.

Background Art So far, genes encoding a hormone or growth factor have been isolated and utilized to produce many recombinant proteins that are commercialized as medicines. Most of them are secretory proteins. Therefore, isolation of a gene encoding a novel secretory protein is an extremely important step.in developing a novel medicine. Accordingly, methods for isolating a gene encoding a secretory protein have been developed. For example, Honjo et al. developed a method (unexamined published Japanese patent application No. Hei 6-315380) by utilizing the feature that secretory proteins have a signal sequence that allows intracellularly expressed proteins to translocate to the cell surface. In this method, the signal sequence of the a chain of human IL-2 receptor, a secretory protein, is replaced with a short cDNA fragment corresponding to the 5 '-end sequence of mRNA from a target cell or tissue to construct a library, which is then introduced into cells. Among the clones, IL-2 receptor is expressed on the cell surface of clones with a signal sequence, but not those without a signal sequence. The presence of the signal sequence can thus be detected by the anti-IL-2 receptor antibody.

Genetics Institute, Inc., (Cambridge, MA) developed a more sophisticated system utilizing a yeast metabolic enzyme S. Patent 5,536,637). Invertase, a yeast metabolic enzyme, is a secretory enzyme that cleaves sucrose in the culture medium to glucose and fructose to transfer energy. A mutant strain that does not secrete this enzyme can not grow in a medium containing sucrose as the sole carbon source without glucose. In this method, which utilizes the phenomenon, invertase gene is ligated with cDNA to construct a library, which is then introduced into a mutant yeast strain lacking the invertase gene. Clones containing the signal peptide are isolated by selecting clones capable of growing in a medium containing only sucrose as a carbon source.

However, the method of Honjo et al. is disadvantageous in that laborious steps are required in selecting positive clones due to the use of an antibody. Furthermore, the detection sensitivity is very low. The method of Genetics Institute, Inc., also has a problem in that a clone with poor secretion efficiency in yeast cannot be isolated. In 1o addition, these methods detect only short DNA because of the potential loss of antigenicity or enzymatic activity when the reporter protein is fused with a large protein.

Moreover, the methods fail to detect the type II membrane proteins having their Nterminus within the cell and C-terminus outside the cell.

Disclosure of the Invention According to a first embodiment of the invention, there is provided a method for examining whether a peptide encoded by a cDNA to be tested has secretory ability, the method comprising: preparing a vector comprising a cDNA to be tested, wherein said cDNA is ligated to the 5' upstream of a DNA encoding a peptide capable of inducing cell proliferation through dimerization on a cell surface and lacking secretory ability; introducing the vector prepared in into a cell; culturing the transformant prepared in and detecting the cell's proliferation ability.

According to a second embodiment of the invention, there is provided a method for isolating a cDNA encoding a peptide with secretory ability, the method comprising: preparing a vector comprising a cDNA to be tested, wherein said cDNA is ligated to the 5' upstream of a DNA encoding a peptide capable of inducing cell proliferation through dimerization on a cell surface and lacking secretory ability; introducing the vector prepared in into a cell; culturing the transformant prepared in and detecting the cell's proliferation ability; and [R:\LIBFF] 8407spec.doc:gcc 2a selecting a positive cell judged to have cell proliferation ability from the cells prepared in and isolating the cDNA from said cell.

According to a third embodiment of the invention, there is provided a DNA encoding a peptide capable of inducing cell proliferation through dimerization on a cell surface and lacking secretory ability, when used for the methods in accordance with the first and second embodiments of the present invention.

According to a fourth embodiment of the invention, there is provided a vector comprising the DNA in accordance with the third embodiment of the present invention, and at the 5' upstream of a DNA, a cloning site for a cDNA to be tested, when used for the methods in accordance with the first and second embodiments of the present invention.

According to a fifth embodiment of the invention, there is provided a cell carrying the vector in accordance with the fourth embodiment of the present invention, when used for the methods in accordance with the first and second embodiments of the present invention.

According to a sixth embodiment of the invention, there is provided the use of the DNA in accordance with the third embodiment of the present invention, for the methods in accordance with the first and second embodiments of the present invention.

According to a seventh embodiment of the invention, there is provided the use of the vector in accordance with the fourth embodiment of the present invention, for the methods in accordance with the first and second embodiments of the present invention.

According to an eighth embodiment of the invention, there is provided the use of the cell in accordance with the fifth embodiment of the present invention for the methods in accordance with the first and second embodiments of the present invention.

The present invention provides a method for examining whether a tested cDNA encodes a peptide with the secretory ability or not. It also provides a method for isolating a cDNA encoding a peptide with the secretory activity, which permits the use of a cDNA encoding a long peptide coding region.

Proteins such as cytokine receptors translocate to the cell surface, dimerize upon binding their ligands and induce cell proliferation. The translocation ability (secretory ability) to the cell surface of the proteins is known to depend on the presence of the signal sequence. The present inventors thought it possible to examine whether a desired peptide [R:\LIBFF]I 8407spec.doc:gcc In 2b

O

o has the secretory activity by removing the signal sequence (or with additional Sextracellular region) from the proteins and replacing it with a fusion protein containing a desired peptide, expressing the fusion protein in cells, and examining the proliferation o ability of the cells. If the peptide has the

IO

rI [R:\LIBFF] 8407spec.doc:gcc 3 secretory ability, the fusion protein translocates to the cell surface, dimerizes, and induces cell proliferation. In contrast, if the peptide does not contain the secretory activity, the fusion protein cannot translocate to the cell surface and induce cell proliferation. Thus, the secretory ability of the fused peptide can be tested by simply examining cell proliferation as an index. Moreover, the inventors thought it possible to perform positive screening for a peptide with the secretory ability by selecting cells that proliferate. Thus, the present inventors used mpl (thrombopoietin receptor) as a protein that triggers cell proliferation through translocation to the cell surface and dimerization, and developed a method for detecting and isolating a peptide possessing the secretory ability.

Specifically, we prepared a DNA encoding human mpl without the secretory ability by removing the secretion signal and most of the extracellular domain from a constitutively active form of mpl, which was found by the present inventors (the mpl is altered to be able to confer autonomous proliferation ability to an IL-3 dependent cell line by the transducing signal in the absence of ligand; Blood 88:1399-1406 (1996)). The DNA was then ligated with cDNA to be tested, a DNA encoding a known secretory protein, or a DNA encoding the secretory peptide from which the secretory signal region was removed. The resulting chimeric genes were expressed in cells, and the proliferation ability of the cells was examined. The results show that the DNA encoding a known secretory protein used as a test cDNA induced cell proliferation whereas no cell proliferation was detected for the DNA encoding the secretory protein from which the secretory signal region was removed.

In this way, the inventors found that the system thus developed can be used to easily detect and isolate a DNA encoding a peptide with secretory activity and containing a long peptide coding region using cell proliferation as an index. Indeed, they performed a screening and succeeded in detecting and isolating DNAs encoding secretory proteins including type I membrane 4 proteins and type II membrane proteins.

Thus, the present invention relates to: a peptide capable of inducing cell proliferation through dimerization on the cell surface and lacking the secretory ability; the peptide as described in wherein the peptide is derived from a cytokine receptor; the peptide as described in wherein the peptide is derived from mpl; the peptide as described in or wherein the peptide is ligand-independent; the peptide as described in wherein the peptide comprises the amino acid sequence of SEQ ID NO: 4; a DNA encoding the peptide as described in any of (1) to a vector containing the DNA as described in and a cloning site for cDNA at the 5'-upstream region of the DNA; the vector as described in wherein the vector is derived from a retrovirus; the vector described in or wherein a cDNA is inserted into the 5'-upstream of the DNA of a cell carrying the vector as described in (11) a cell as described in wherein the cell is a mammalian cell; (12) a method for examining whether a peptide encoded by a cDNA to be tested contains the secretory ability, the method comprising ligating the test cDNA with the vector of introducing the vector prepared in into a cell, and culturing the transformant prepared in and detecting the cell proliferation ability; (13) a method for isolating a cDNA encoding a peptide with the secretory ability, the method comprising ligating a cDNA library with the vector of introducing the vector prepared in into a cell, culturing the transformant prepared in and detecting the cell proliferation ability, and selecting a positive cell that is judged to have cell proliferation ability in and isolating the cDNA from said cell; (14) a method as described in (12) or wherein the vector is derived from a retrovirus and the cell to be introduced with the vector is a mammalian cell; a cDNA encoding a peptide with the secretory ability, 1) which is isolated by the method of and (16) a peptide encoded by the cDNA as described in The present invention relates to a method for detecting a DNA encoding a peptide having the secretory ability. The detection method features the use of a DNA encoding a peptide capable of inducing cell proliferation through dimerization on the cell surface and lacking the secretory ability for detecting a peptide with the secretory ability. Here, "the peptide capable of inducing cell proliferation through dimerization on the cell surface" includes mpl (Proc. Natl.

Acad. Sci. USA, 89:5640-5644 (1992)), the alpha chain or beta chain of GM-CSF (Blood 83:2802 (1994)), erythropoietin receptor (Nature 348:647 (1990)), c-kit receptor (Blood 85:790 (1995)), and neu (Nature 339:230 (1989)), but are not limited thereto. In the method of the present invention, a cDNA is constructed to encode the above peptides whose secretory ability is eliminated. The secretory ability is usually removed by deleting a region containing the signal sequence.

For example, the signal sequence of the human mpl is the region corresponding to 1 to 25 positions in the amino acid sequence oftheprotein (Proc. Natl. Acad. Sci. USA, 89:5640-5644 (1992)), and that of the beta chain of the human GM-CSF is the region corresponding to 1 to 48 positions (Proc. Natl. Acad. Sci. USA, 87:9655-9659 (1990)). Preferably, the extracellulardomain is also deleted from the peptide.

The peptide encoded by a constructed cDNA is preferably ligand-independent (if the peptide is ligand-dependent, it may 6 lose the ligand-binding ability and become inactive after creating a fusion protein). A method for creating a ligand-independent peptide is to introduce a mutation into the peptide, for example. In case of mpl, the substitution of Ser 498 to Asn can abolish the dependency to the ligand, thrombopoietin (Blood 88:1399-1406 (1996)). The mpl used in the present method preferably comprises the amino acid sequence as described in SEQ ID NO: 4.

The DNA prepared as described above is inserted into an appropriate expression vector. The expression vector is not limited, and preferably is a retrovirus vector, which can be introduced into a variety of cells with high efficiency through virus infection, and stably expresses the DNA inserted into the vector in the cells. Examples of a retrovirus vector include that engineered for CDNA library construction, such as pBabeX (Proc. Natl. Acad. Sci. USA 92:9146-9150 (1995)) or pMX (Exp. Hematol. 24:324-329 (1996)). Also, virus vectors such as adenovirus, EB virus, and papilloma virus, or plasmid vectors such as pEF-BOS (Nucleic Acid Res. 18 and pcD SRa296 (Mol. Cell. Biol. Jan. 466-472 (1988)) can be used. The expression vectors should have a cloning site for a cDNA to be tested for its secretory ability at the 5'-upstream of the above DNA insert to express a fusion protein. The method for creating a cloning site for a cDNA is known to one skilled in the art.

Next, the prepared vector is ligated with a cDNA to be tested. The test cDNA is ligated into the 5'-upstream of the "DNA encoding a peptide capable of inducing cell proliferation through dimerization on the cell surface," which is inserted into the vector. The test cDNA can be any cDNA encoding a peptide whose secretory ability is to be tested. The test cDNA can be ligated with a vector according to the standard methods.

For example, the ligation method using T4 DNA ligase via an adapter -linker (Maniatis Molecular Cloning).

The prepared vector is then introduced into a cell.

Cells into which the vector is introduced are not limited and 7 include cytokine-dependent proliferating cell such as Ba/F3, OTT-1, FDCP-1, and TF-1 cells. Vectors can be introduced into cells by using standard methods including lipofection, calcium phosphate method, DEAE-dextran method, and electroporation. In retrovirus infection-mediated introduction, the vector is introduced into the packaging cells and integrated into the virus particles. The vector can be introduced by using standard methods such as the calcium phosphate method and lipofection. For example, cells such as BOSC23, Bing (Proc. Natl. Acad. Sci. USA 90:8392-8396 (1993)), NX-E, and NX-A cells (Nolan G.P. Immunity 8:461-471 (1998)) can be used as the packaging cell.

Next, the thus-prepared transformants are cultured and examined for their proliferation ability. When a protein encoded bycDNA inserted into the vector is expressed as a fusion protein with a ligand-independent aetive cytokine receptor, the transformant is cultured in the medium lacking the cytokine (ligand) on which the cell depends. If a significant cell proliferation is detected, the test cDNA is judged to be a "positive clone" encoding a peptide that contains the secretory ability. Alternatively, if no significant cell proliferation is detected, the cDNA is judged to be a "negative clone" that encodes a peptide lacking the secretory ability. When a protein encoded by the inserted cDNA is expressed as a fusion protein with a ligand-dependent cytokine receptor, the transformant is cultured in the presence of the ligand. If a significant cell proliferation is detected, after comparison with a negative control in the absence of the ligand, if necessary, the test cDNA is judged to be a "positive clone." Other conditions for culturing transformants can be appropriately selected by one skilled in the art depending on the types of cells into which the vector is inserted and the nature of the fusion protein to be expressed.

The present invention also relates to a method for isolating a cDNA encoding a peptide that contains the secretory ability. In the method, a cDNA library is ligated into the 8 vector instead of the above test cDNA that is used for detecting cDNA encoding a peptide containing the secretory ability. In one specific embodiment of the invention, cDNAs prepared by using a random primer are ligated with the BstXI adapter and inserted between the two BstXI sites; one is of the vector and the other is inserted into the extracellular cleavage site of the active mpl. The source of the cDNA library is not limited to any specific one, but can be a cell or tissue from which a desired peptide containing the secretory ability is to be isolated. Many standard methods can be used to construct a cDNA library. In the present method, cells judged to be capable of proliferation are selected from the cDNA library-introduced cells. The cDNAs contained in the selected cells are supposed to encode a peptide having the secretory ability. cDNA can be isolated from the cells whose proliferation has been detected by, for example, extracting the genomic DNA or RNA, amplifying the cDNA of interest by PCR using primers designed to encompass the cloning sites (in case of RNA, after converting it into DNA using reverse transcriptase), and recovering the products.

Whether the recovered cDNA is full length or a fragment, or whether it is a cDNA encoding a novel secretory peptide, can be analyzed by comparing the cDNA sequence with those of the known proteins in the database. If the cDNA is not full length, it is used to screen a secondary cDNA library to isolate a full-length cDNA. The secondary cDNA library can be constructed by a method known to one skilled in the art, such as those described in the literature (Molecular Cloning, A Laboratory Manual, 2nd edition. Sambrook J. et al., (1989) Cold Spring Harbor Laboratory Press, New York).

3 A cDNA encoding a peptide with the secretory ability isolated by the method of the present invention can be utilized to produce a recombinant protein that is useful as a medicine or in gene therapy of related diseases. A recombinant protein from the isolated cDNA can be produced by known methods in the art. For example, the cDNA is inserted into an appropriate vector such as pED (Kaufman et al. Nucleic Acids Res.

9 19:4484-4490 (1991)), pEF-BOS (Mizushima et al. Nucleic Acids Res. 18:5322 (1990)), pXM, pJ13, and pJL4 (Gough et al. EMBO J. 4:645-653 (1985)), the vector is introduced into a host cell, the resulting transformant is cultured to allow it to express a recombinant protein, and the recombinant protein is purified.

Brief Description of the Drawings Figure 1 schematically shows the peptides used for 1) detecting secretory ability and the result of detecting cytokine-independent proliferation ability of BAF/03 cells through the expression of the peptides.

est Mode for Carrying out the Invention The present invention is illustrated in detail below with reference to examples, but is not to be construed as being limited thereto.

Example 1. Vector construction In mouse myeloproliferative leukemia virus, env is ligated to the mouse mpl comprising the extracellular domain consisting of 56 amino acids from the transmembrane domain toward the N-terminus, the transmembrane domain, and the intracellular domain. PCR was performed to obtain a cDNA encoding the corresponding region of the human mpl, which is from Leu (449) to the stop codon (636), having the NotI site immediately before the Leu(449) (a single nucleotide insertion) and the Sall site immediately after the stop codon so as to be in the frame of the GM-CSF cDNA shown below. The "pBabeX MPL"" (Blood 88:1399-1406. (1996)), in which active human mpl cDNA is cloned, was used as a template. Primers used are listed in Table 1.

Table 1 Not v-mpl (SEQ ID NO: 1)

(TGCGGCCGCCCTGGAGCTGCGCCCGCGATCCTGCTACCGTTTA)

NotI the sequence of mpl MPL Sal (SEQ ID NO: 2)

(GTATGTCGACTCAAGGCTGCTGCCAATAG)

SalI PCR was performed in a reaction mixture containing jg/ml template DNA, 1 IM each primer, 50 U/ml KOD DNA polymerase (TOYOBO), 1 mM MgCl,, 0.2 mM dNTPs, 120 mM Tris-HCl (pH mM KC1, 6 mM (NH,) 2 SO, 0.1% Triton X-100, and 10 pg/ml BSA by using the GeneAmpPCR System (Perkin Elmer) under the following conditions: denaturation at 98°C for 60 sec, followed by cycles of 98°C for 15 sec, 60°C for 10 sec, and 74°C for 30 sec.

The PCR products were analyzed by electrophoresis on an agarose gel, and a gel piece containing a 0.6 kb fragment of interest was excised to extract DNA. The DNA was then phosphorylated at its 5'-termini with T4 polynucleotide kinase (TOYOBO), and ligated by using T4 DNA ligase (TOYOBO) with the pBluescript vector (Stratagene) that was pretreatedwith SmaI (TaKaRa Shuzo) and Bacterial Alkaline Phosphatase (BAP; TaKaRa Shuzo).

The nucleotide sequence of the active mpl cDNA inserted in the resulting plasmid was verified with the ABI PRISM 310 Genetic Analyzer (Perkin Elmer). The plasmid was digested with NotI (TaKaRa Shuzo) and SalI (TaKaRa Shuzo), and separated by electrophoresis on an agarose gel to isolate a 0.6 kb fragment.

The fragment was ligated with the pMX (Proc. Natl. Acad. Sci.

USA 92:9146-9150. (1995)), which was also digested with NotI and Sall, treated with BAP, and purified by agarose gel electrophoresis, using T4 DNA ligase to obtain pMX v-mpl". The plasmid pMX v-mpl H contains a cDNA encoding an active mpl lacking the secretory ability. The nucleotide sequence of the cDNA 11 insert and the amino acid sequence of the peptide encoded by the cDNA are shown in SEQ ID NO: 3 and NO: 4, respectively.

Next, to obtain a human GM-CSF cDNA in which the stop codon is replaced with a NotI site, PCR was performed by using the pcDSRc 298 hGM-CSF (Proc. Natl. Acad. Sci. USA 82:4360-4364 (1985)) containing the human GM-CSF cDNA as a template.

Primers used are shown in Table 2.

Table 2 EcoGMss (SEQ ID NO: 5) (CGAATTCAAAGTTCTCTGGAGGATG) EcoRI EcoGM (SEQ ID NO: 6) (CGAATTCGCCGCCACCATGGCACCCGCCCGCTGCCC) EcoRI GM Not (SEQ ID NO: 7) (AG-CGGCCGCCTCCTGGACTGGCTCCCA) NotI EcoGM was designed to have the translation initiation codon ATG in place of the Ser and, as in EcoGMss and EcoGM, the EcoRI site and the Kozak consensus sequence Cell Biol.

108:29. (1989)) immediately before theATGcodon. Primer pairs of EcoGMss and GM Not were used in PCR to amplify GM-CSF containing the signal sequence, and EcoGM and GM Not were used to amplify GM-CSF lacking the signal sequence. PCR was performed as described above except for using 55 0 C for the annealing temperature, and the products were cloned into the pBluescript The nucleotide sequence of the DNA inserts was verified by using the ABI PRISM 310 Genetic Analyzer (Perkin Elmer). The plasmids were then digested with EcoRI (TaKaRa) and NotI and inserted into the EcoRI-NotI site of the pMX v-mpl

M

as described above, and "pMX GM(+)v-mpl and "pMX GM(-)v-mpl"" were obtained. The "pMX GM(+)v-mpl"" and "pMX GM(-)v-mpl"" encode a fusion protein between the C-terminal part of the active mpl starting from Leu (449) and the entire GM-CSF with or without the signal sequence, respectively. The nucleotide sequences of their cDNA inserts are shown as SEQ ID NO: 8 and 12 NO: 10, and the amino acid sequences of the proteins encoded by the cDNAs are shown as SEQ ID NO: 9 and NO: 11.

Example 2. Viral infection Each of the above plasmids was introduced into packaging cell-line BOSC23 (Proc. Natl. Acad. Sci. USA 90:8392-8396.

(1993)) using LipofectAMINE (Life Technologies). BOSC23 cells were plated into 6-cm dishes (CORNING) with Dulbecco's modified Eagle medium (DMEM; Nissui Pharmaceutical) containing 10% fetal calf serum (FCS; JRH Biosciences). After 6-hr incubation, the cells were washed with OPTI-MEMI reduced serum medium (Life Technologies). Separately, LipofectAMINE (18tl) diluted in 200 4l OPTI-MEM I was mixed with 3 pg samples of each plasmid diluted in 200 ptl OPTI-MEM I. The resulting mixtures were allowed to stand at room temperature for minutes, mixed with 1.6ml OPTI-MEMI, then added to the cells.

After 5 hr, 2 ml of DMEM containing 20% FCS was added to the cells, which were incubated for an additional 19 hr. The medium was then replaced with 3 ml of DMEM containing 10% FCS, and the culture supernatant was recovered after 24 hr. Mouse interleukin-3 (IL-3) and 10 pg/ml polybrene (hexadimethrine bromide, Sigma) were added to the culture supernatant containing the recombinant virus, and Ba/F3 cells were suspended therein for infection. After 24 hr of infection, the cells were washed twice in RPMI1640 (Nissui Pharmaceutical) containing 10% FCS lacking mouse IL-3, and the culture was continued in the same medium.

The cells containing the fusion protein between the entire GM-CSF containing the signal sequence and the active mpl (derived from the pMX GM(+)v-mpl

M

grew in the absence of IL-3 as well as those containing the active mpl with the secretory ability. In contrast, the cells containing the fusion protein between the GM-CSF lacking the signal sequence and the active mpl (pMX GM(-)v-mpl

M

did not grow as well as control Ba/F3 cells into which no fusion protein expression vector was introduced (Figure 1).

13 Example 3. Screening The foll'owing oligonucleotides (Table 3) were synthesized, and their 5'-termini were phosphorylated using T4 polynucleotide kinase. The oligonucleotides were mixed and denatured at 95 0 C, and then annealed by gradually cooling them to 40 0 C to prepare the cassette DNA.

Table 3 5'-GGCCCCAGCACAGTGGC-3' (SEQ ID NO: 12) (SEQ ID NO: 13) The pMX GM(-)v-mpl", which was digested with NotI (TaKaRa) and treated with BAP, was mixed with the cassette and ligated using T4 DNA ligase. The direction of the cassette in the resulting plasmid was verified by DNA sequencing to be in the order of BstXI and NotI (pMX GM(-)v-mpl m2 Total RNA was prepared from the rat neuroblastic cell line MNS70 using the TRIZOL reagent (GIBCO BRL) and passed through the oligo dT column (Pharmacia) to prepare polyA(+) RNA. Double-stranded cDNA was synthesized with the random hexamer contained in the SuperScript Choice System (GIBCO BRL). The cDNA was bluntended, ligated with the BstXI adapter (Invitrogen), and fractionated by using the SizeSep 400 Spun Column (Pharmacia).

The cDNA was then mixed and ligated with the pMX GM(-)v-mpl" 2 which was digested with BstXI (TaKaRa) and treated with BAP, using T4 DNA ligase. The DNA was introduced into DHIOB E. coli (GIBCO BRL) by electroporation using Gene Pulser (BioRad) to construct a cDNA library.

Plasmids were extracted from the recombinant E. coli containing a cDNA library and purified by using the JETstar column (GENOMED). The library plasmids were introduced into BOSC23 packaging cells by using LipofectAMINE as described above. Mouse IL-3 (10 ng/ml) and 10 -tg/ml polybrene (Hexadimethrine Bromide, Sigma) were added to the culture 14 supernatant containing the recombinant virus, and Ba/F3 cells were suspended therein for infection. After 24-hr infection, the cells were washed twice with phosphate buffer and cultured further in RPMI1640 containing 10% FCS. The genomic DNA was prepared from the clones that grew in the absence of IL-3, and PCR was performed using primers designed to encompass the cDNA insertion site to recover the cDNA fragments.

Table 4 10 5'-GGGGGTGGACCATCCTCTA-3' (SEQ ID NO: 14) 5'-CGCGCAGCTGTAAACGGTAG-3' (SEQ ID NO: PCR was performed in 50 pl of the reaction mixture containing 500 ng genomic DNA, 500 pM each primer, 2.5 U TaKaRa LATaq (TaKaRa), 2.5 mMMgCl,, 0. 3 mMdNTPs, and the accompanying buffer using the GeneAmpPCR System2400 in the following process: denaturation at 989C for 60 sec, followed by 30 cycles of 98°C for 20 sec and 68°C for 120 sec. PCR products were separated by electrophoresis on an agarose gel, the gel pieces containing the amplified fragments were excised, and DNA was purified. The nucleotide sequence of the DNA fragments purified from the resulting 190 clones was determined, and 150 clones were found to be cDNAs encoding a known secrete protein or a membrane protein, or its part. The other 40 clones were found to encode unknown secrete proteins. Some of the thus-obtained known secretory proteins are shown in Table where "length" indicates the length of the ORF of the obtained cDNA fragment by the number of amino acid residues. The average length of the clones encoding a known secrete protein was 273 amino acid residues. "Accession number" indicates the accession number in the GenBank protein database. It should be noted that the background in the-present method such as detecting a cDNA encoding a protein other than a secrete protein or cDNA that was inserted in the opposite direction was 1% or less.

15 Table Length Accession No. Name S 288 350 561 161 176 218 382 286 159 259 254 482 224 105 482 322 1805299 416630 468563 112929 2494287 2507439 118115 3219172 461671 1082724 1777354 205167 1708023 1139548 135818 129731 1172451 1709256 2367641 Amyloid precursor Amyloid-like protein 1 Amyloid precursor-like protein 2 Amyloid A4 protein homologue precursor o-acetyl GD3 ganglioside synthase Syndecan 3 (heparan sulfate proteoglycan core protein) Cyr61 protein (growth factor binding protein) collagen alpha 1(V) collagen alpha 1 type 1 Prostacyclin-stimulating factor SHPS-1,

BIT

120 kDa sialoglycoprotein (a hepatic lysosomal membrane protein) K-glypican Seizure-related gene product 6 type2 precursor G-protein coupled thrombin receptor Protein Disulfide Isomerase perlecan (basement membrane heparin sulfate proteoglycan) neurocan (proteoglycan core protein precursor) neuropilin-2 (semaphorin III receptor) (cont'd) 16 Table 5 (cont'd) Length Accession No. Name 211 126638 Lysyl oxidase 308 2627143 Neural cadherin 459 3123675 Notch 140 1718156 Vascular endothelial growth factor 534 627989 Endothelin-converting enzyme 89 114393 Sodium/potassium-transporting ATPase beta-1 chain Industrial Applicability The present invention provides a method for detecting and isolating a cDNA encoding a secretory peptide using a peptide capable of triggering cell proliferation through its dimerization on the cell surface but lacking the secretory ability. Since the method utilizes cell proliferation as an index for detection, it is extremely easy and sensitive.

Moreover, compared to the conventional methods that enable detecting a short DNA fragment, this method enables detecting and isolating a cDNA containing a longer peptide coding region, and thus provides more information from the first isolated clones. In addition, the method enables detecting and isolating secretory proteins including type I and type II membrane proteins.

Claims (17)

1. A method for examining whether a peptide encoded by a cDNA to be tested has secretory ability, the method comprising: preparing a vector comprising a cDNA to be tested, wherein said cDNA is ligated to the 5' upstream of a DNA encoding a peptide capable of inducing cell proliferation through dimerization on a cell surface and lacking secretory ability; introducing the vector prepared in into a cell; culturing the transformant prepared in and detecting the cell's proliferation ability.

2. A method for isolating a cDNA encoding a peptide with secretory ability, the method comprising: preparing a vector comprising a cDNA to be tested, wherein said cDNA is ligated to the 5' upstream of a DNA encoding a peptide capable of inducing cell proliferation through dimerization on a cell surface and lacking secretory ability; introducing the vector prepared in into a cell; culturing the transformant prepared in and detecting the cell's proliferation ability; and selecting a positive cell judged to have cell proliferation ability from the cells prepared in and isolating the cDNA from said cell.

3. The method of claim 1 or 2, wherein the vector is derived from a retrovirus and the cell to be introduced with the vector is a mammalian cell.

4. A DNA encoding a peptide capable of inducing cell proliferation through dimerization on a cell surface and lacking secretory ability, when used for the methods of any one of claims 1-3.

5. A vector comprising the DNA of claim 4, and at the 5' upstream of a DNA, a cloning site for a cDNA to be tested, when used for the methods of claims 1-3.

6. The vector of claim 5, wherein the vector is derived from a retrovirus.

7. The vector of claim 5 or 6, wherein the cDNA to be tested is inserted into the upstream of a DNA of claim 4.

8. A cell carrying the vector of claim 7, when used for the methods of any one of claims 1-3.

9. The cell of claim 8, wherein said cell is a mammalian cell.

Use of the DNA of claim 4 for the methods of any one of claims 1-3. [R:\LIBFF] I 8407spec.doc:gcc 18

11. Use of the vector of any one of claims 5-7 for the methods of any one of claims 1-3.

12. Use of the cell of claim 8 or 9 for the methods of any one of claims 1-3.

13. A method for examining whether a peptide encoded by a cDNA to be tested has secretory ability, substantially as hereinbefore described with reference to any one of the examples.

14. A method for isolating a cDNA encoding a peptide with secretory ability, substantially as hereinbefore described with reference to any one of the examples.

A DNA encoding a peptide capable of inducing cell proliferation through dimerization on a cell surface and lacking secretory ability, when used for the methods of claim 13 or 14.

16. A vector comprising the DNA of claim 4, and at the 5' upstream of a DNA, a cloning site for acDNA to be tested, when used for the methods of claim 13 or 14.

17. A cell carrying the vector of claim 7, when used for the methods of claim 16. Dated 31 May, 2005 Chugai Seiyaku Kabushiki Kaisha Toshio Kitamura Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R:\LIBFF1 I 8407spec.doc:gcc