CN102191245B - Method and reagent for detecting tumor cell in circulatory blood by applying tumor peculiar promoter to express reporter gene - Google Patents

Abstract

The invention provides a method and a reagent for detecting a tumor cell in circulatory blood sample and marrow sample. According to the invention, a tumor peculiar promoter is applied to drive an expression of a reporter gene so as to detect the tumor cell in the circulatory blood sample and the marrow sample. The tumor peculiar promoter like a human TERT promoter has a pretty high activity in most of tumor cells, but has no activity in normal cells. A gene carrier like an adenovirus carrier, which is contained in a reporter expression box driven by the tumor peculiar promoter, is used to transfect a karyote in the circulatory blood sample or the marrow sample; and then whether a cell with high expression by a reporter gene exists or not in the sample is detected so as to confirm whether a tumor cell exists or not in the sample. A diagnostic kit containing the promoter, the reporter gene and the gene carrier is provided in the invention and can be used for early diagnosis of a cancer patent and prediction of treatment reaction of the patient.

Description

The application tumor-specific promoters is expressed reporter gene and is detected method and the reagent of circulating tumor cells

Most of cancer patientss die from metastases, shift and refer to that tumour cell is diffused into away from position from original site by the recycle system, and breeding grows into new metastatic tumor (Ecclesand Welch, 2007) in new organ.According to estimates, every gram tumor tissues approximately has 106 cells to enter blood (Chang et al., 2000 every day; Butler and Gullino, 1975), but this major part wherein can be removed (Butler and Gullino, 1975 very soon; Luzzi et al., 1998).But well-known, the intrusion of tumour cell and transfer will occur in early days, and even a lot of years before cancer is made a definite diagnosis will occur.Therefore, the circulating tumor cell that detects in blood is effective ways (Husemannet al., 2008 of early diagnosis of cancer; Gray, 2003; Kohn and Liotta, 1995).In addition, a nearest research shows that there is dependency in total survival time of preoperative circulating tumor cell (CTC) level and cancer patients, and preoperative and postoperative circulating tumor cell quantity may be relatively indicator (Scher et al., 2009 of therapeutic response; De Bono et al., 2008).Therefore, effectively detecting circulating tumor cells not only is of value to the early diagnosis of cancer, and helpful to prognosis and therapeutic response.

The method that detects at present circulating tumor cells has three kinds: 1) detect the immunoassay of epithelial cell specific proteins; 2) based on the detection tumour of molecular assay or the PCR method of epithelial cell specific mrna; 3) detect and the chromosome aberration of related to cancer or the fluorescence in situ hybridization of gene amplification.

Immunoassay are to catch the cell of expressing some tissue, organ or tumour specific antigen by the antibody that use is fixed on magnetic-particle.Due to the cell of most of solid tumors from the epithelial cell source, therefore in epithelial cell specific expressed albumen (as Keratin sulfate cytokeratins (19,8,18and 20) and EpCam (Litvinov et al., 1994)) be used as distinguishing the biomarker of the sick and nucleated blood cell of epithelial cell.But this method may cause some false positives or false negative result.The first, because the tumour cell of invasion changes to mesenchymal cell because of epithelial cell, and lose original epithelium antigen, this will cause false negative result (Willipinski-Stapelfeldt et al., 2005; Went et al., 2004).The second, non-tumour epithelial cell may be present in blood, and perhaps antibody may non-specifically be combined with hemocyte, and this will cause false positive results.According to estimates, in normal control, approximately the Keratin sulfate cytokeratin positive cell of 0%-20% is white corpuscle (Goeminne etal., 2000).Use simultaneously the specific antibody of hemocyte (as CD45) may reduce false positive to dying.The antibody of anti-organ specificity mark; as prostate specific antigen (PSA); carcinomebryonic antigen (CEA); mucin-1 (MUC-1), EGF-R ELISA (EGFR), oncofetal protein (AFP); HER-2; also be used to detect circulating tumor cell, but because not every tumour cell is all expressed those biomarkers, so often can produce false negative result.But the advantage of this method is to use method for distinguishing, measures the circulating tumour cell that comes the concentration and analysis magnetic-particle to catch as morphology, mRNA or gene amplification.The circulating tumour cell also can pass through its physical property (as size and density) enrichment.The size of most of blood leukocytes (lymphocyte and neutrophil leucocyte) is 8～11um, and in patient with breast cancer's blood, the mean diameter of tumour cell is 29～35um (Meng et al., 2004).

Use fluorescence in situ hybridization (FISH), the genome Epidemiological Analysis such as comparative genome hybridization (CGH) and mutation analysis can prove further that cancer is (Swennenhuis et al., 2009 of existing of the chromosome aberration of being correlated with, gene amplification, sudden change; Leversha et al., 2009).But these method ordinary expenditure are expensive, and consuming time.Reverse transcription PCR (RT-PCR), especially Real Time RT-PCR, be a kind of sensitivity, actual, be used for detecting the specific method of mRNAs (Kitago et al., 2009 of some tissue or organ (epithelial cell and organ specificity albumen as mentioned above); Iakovlevet al., 2008).This method can be combined with immunoassay.For example, can be further analyzed their tissue or the content of tumour-specific mRNAs with the circulating tumour cell of immunomagnetic beads enrichment.Although PCR method is highly sensitive aspect detection signal, need to take strict control, avoid the pollution that may produce in process.

Owing to lacking high tumor-specific molecule, the application of immunoassay and quantitative reverse transcription PCR method is restricted, and therefore measures the existence of circulating tumour cell and need to analyze the different kinds of molecules mark.

We develop the method for tumour cell in a kind of new detection blood specimen and bone marrow prepare.This method is used by tissue or tumor-specific promoters and is ordered about reporter gene expression, reaches the testing goal of tumour cell.The expression cassette of the tumor-specific promoters that is ordered about by reporter gene is transfected in karyocyte in blood specimen and bone marrow prepare.Then culturing cell 12-48 hour.Use subsequently the expression of diverse ways and reagent examining report gene.Expression cassette delivery carrier used can be in tumour cell, rather than amplification selectively or express in nucleated blood cell, so detected signal is higher to the specificity of tumour cell.Based on method used be used for the strength of signal of examining report gene and can measure circulating tumor cell in blood specimen and bone marrow prepare.This method is reliable, and is simple, and specificity is high aspect the circulating tumor cell in detecting blood specimen and bone marrow prepare, and to cancer patients's early diagnosis, prediction and the prognosis thereof of therapeutic response are highly profitable.

The reporter gene that can be used for this purpose comprises the gene of the various fluorescins of coding (as green, red, blue, yellow, purple fluorescence albumen), firefly luciferase gene, the renilla luciferase gene, bacteria beta-galactosidase (LacZ) gene etc.The expression of above-mentioned reporter gene can be detected by diverse ways.The expression of fluorescin can be detected by fluorescent microscope and fluorescence-activation flow cytometry method (FACS).The cell of observing under fluorescent microscope can be analyzed the size and shape of cell, and this may be of value to further differentiation normal cell and tumour cell.Fluorescence-activation flow cytometry method can the enrichment fluorescencepositive cell, and further adopts the traditional method checking,, as with immunoassay and PCR assay method, further confirms cell.

Bioluminescence assay can detect firefly luciferase gene, renilla luciferase gene, the expression of bacteria beta-galactosidase (LacZ) gene.Promotor is the cis sequence that regulating cell is expressed.No matter its types of organization of some promotor, biological species, the etap, have activity in most cells.This promotor is called ubiquitous promotor (ubiquitous promoter), as cytomegalovirus early promoter (CMV), Rous sarcoma virus long terminal repeat (RSV), SV40 viral promotors, PGK and EF1 α promotor.Some promotor only has activity in the cell of some type.For example, albumin promoter mainly has activity in liver cell, and probasin and Prostate Specific Antigen Promoter have activity in prostata tissue.Some gene is only expressed in tumour cell, its transcription regulating nucleotide sequence is tumour-specific.But, although may these promotors the specific activity in tumour cell active higher in normal cell, most of promotors are that tissue or cellular type are specific, rather than tumour-specific.They only have activity in some tumour cells.As the alpha-fetoprotein α of liver cancer, the prostate specific antigen of prostate cancer (PSA), the carcinomebryonic antigen of cancer of the stomach (CEA) is exactly such example.The tyrosine oxidase promotor has activity in melanoma, the mucin-1 promotor has activity in mucomembranous cell.On the contrary, people survivin gene, telomere body reverse transcriptase gene (TERT), the midkine gene has activity in a variety of cancers.

Tissue or tumor-specific promoters have been used to the specific gene expression in some cells or tumour cell.For example, these promotors have been used to the tumor specific expression of therapeutic gene in cancer gene therapy.Sometimes these promotors also are used for transgenic animal, control genetically modified expression in some particular organization.

Telomerase is a kind of special ThermoScript II, and it is responsible for (Morin, 1989 of copying of end of chromosome or telomere; Blackburn, 1992).Its activity in immortalized cell line and 85% human cancer cell is very high, but is reticent (Kim et al., 1994 in the normal human cell of differentiation; Counter etal., 1992; Shay and Bacchetti, 1997).The increasing fact shows that telomere body ThermoScript II catalytic subunit gene (TERT) playing an important role aspect the activation Telomerase, and the gene expression dose of telomere body reverse transcriptase gene in primary tumor cell, multiple cancerous cell line and Telomerase positive cell very high (Nakamura et al., 1997; Nakayama et al., 1998; Meyerson et al., 1997).Therefore, people's telomere body reverse transcriptase gene promotor can be used as extensively applicable tumor-specific promoters.In vitro study shows people's telomere body reverse transcriptase gene promotor activity very high (Gu et al., 2000 in the cancer cells of the lung cancer of people and mouse, liver cancer, colorectal carcinoma, mammary cancer, ovarian cancer, the cancer of the brain; Gu et al., 2002a; Gu et al., 2002b; Huang et al., 2002; Lin et al., 2002a; Lin etal., 2002b), (Gu et al. in normal people's fibroblast, 2000), normal people's tracheae (Gu et al., 2000), breast (Lin et al., 2002b), in the epithelial cell of ovary (Huang et al., 2002), (Lin et al., 2002a), normal people CD34 in normal people's primary hepatocyte ⁺There is no activity in (Gu et al., 2002a), normal mice fibroblast (Gu et al., 2002a) in progenitor cell.Telomerase promoter also is used to control condition duplicating virus (comprising oncolytic adenovirus) required viral gene expression (Daviset al., 2006; Huang et al., 2004; Irving et al., 2004).Telomerase specificity oncolytic adenovirus can copy in tumour cell,, so can increase copy number or the expression level of reporter gene, increase detection signal.

Usually the activity of organizing specific promotor a little less than.In order to improve the transcriptional activation of promotor, keep simultaneously its cell-specific, the investigator can design promotor by the sequence of combination different promoters, thereby forms the promotor of mixed type or mosaic type.The most widely used is the CAG promotor, and it is the enhanser that combines the cytomegalovirus early gene, chicken actin gene promotor and rabbit globulin gene splice acceptor sequence.The promotor of this mixed type, by some viruses or non-virus carrier, has been ordered about the high expression level of many genes in various tissues.Another utilization of cytomegalovirus enhanser is to combine the organizing specific promotor to strengthen the tissue-specific expression of goal gene.For example, the cytomegalovirus enhanser is joined in the reverse transcriptase of telomere promotor, will strengthen the active of reverse transcriptase of telomere promotor and keep its specificity.The engineered promotor that we use comprises reverse transcriptase of telomere promoter sequence and cytomegalovirus enhanser, is used for ordering about the expression of reporter gene.Or even any tissue and tumor-specific promoters, or their mosaic type promotor can be used to replace reverse transcriptase of telomere/cytomegalovirus chimeric promoters.

The activity of tissue or tumor-specific promoters also can be strengthened by the binary expression system, weak tumor-specific promoters wherein is used to express strong interactive transcriptional factor, GAL4-VP16 fusion rotein for example, this transcription factor then starts second promotor orders about destination gene expression.This method can not reduce in the specific situation of promotor, increases weak tumor-specific promoters active, sometimes can surpass more than 100 times (Koch et al 2001).The GAL4-VP16 system has been successfully used to strengthen the activity of carcinomebryonic antigen and reverse transcriptase of telomere promotor.These binary systems also will improve the expression of reporter gene in tumour cell, help to check circulating tumor cells.

Promotor, gene and polyadenylic acid signal sequence, these three key elements are that reporter gene and other any target genes are expressed successful necessary factor in mammalian cell.This three parts arranged sequentially being called " expression cassette " in the unique DNA fragment.The polyadenylic acid signal sequence is normally nonspecific to histocyte.Therefore, in theory, the polyadenylic acid signal sequence that comes from any gene all can be used in expression cassette.Normally used polyadenylic acid signal sequence comes from the Polisac gene, simian virus 40 large T antigen gene, globulin gene and some virogenes.

A common meeting of expression cassette and a carrier DNA connect together, and the transfection target cell is used for genetic expression, for example expresses in the circulating tumour cell.One can be used to transfectional cell, is used for the vector nucleic acid molecule of express nucleic acid sequence, is referred to as carrier.Carrier comprises plasmid, clay (cosmid), virus (phage, animal virus, plant virus) and artificial chromosome, for example yeast artificial chromosome.Expression cassette can be cloned into carrier by the recombinant technology of standard.Plasmid can be transfected into mammiferous cell by diverse ways, for example, and the electroporation of physical property, and the lipoid of chemical and transfection reagent.Plasmid, clay and artificial chromosome belong to non-virus carrier.Some virus is by receptor mediated endocytosis transfection or enter cell effectively, and expresses reporter gene, and this makes them become and shifts the common carrier that external nucleic acid enters cell (for example mammiferous cell).Being used for the virus that transfection reporter gene or other any genes enter cell is called virus vector.Normally used virus vector is from adenovirus, adeno-associated virus (AAV), retrovirus, the retrovirus of slow virus (particular type), vaccinia virus, cytomegalovirus and hsv etc.

Some virus vector can only copy in packing (preparation) cell, can not copy in other target cell.These viruses are called replication-defective vector.Most of retroviral vectors, lentiviral vectors, gland relevant viral vector, and the E1 deficient adenoviral vector all belongs to replication-defective vector is arranged.Some virus vector, can copy in the target cell of some kind, but can not copy in the cell of other kinds.They are called as the carrier of conditional replication ability.For instance, oncolytic virus can copy in tumour cell, but can not copy in other normal cells.Why oncolytic virus has this cell-specific replication, be because some virogenes are undergone mutation, or tumor-specific promoters has replaced viral promotors, thereby some virus replication indispensable genes can only be expressed in tumour cell.Replace the adenovirus E 1 promotor with the reverse transcriptase of telomere promotor, can cause oncolytic adenovirus to copy (Davis et al., 2006 in the cancer cells with high reverse transcriptase of telomere activity; Huang et al., 2004; Irving et al., 2004).Replication defect type and condition duplicating virus carrier, as long as they, with a reporter gene that is ordered about by tumor-specific promoters, just can be used to the check of circulating tumour cell.

Some virus vector is further revised, and has changed its surperficial albumen, makes it have targeting and some specific cell of transfection.Change virocyte surface protein, can strengthen their infectivity in some tumour cells.For example, arginine-glycine-aspartic acid sequence can combine with adenoviral fiber protein, and therefore, adenovirus can not infect cell (Wickham 2000) by adenovirus receptor.These carriers that were modified can be checked the cell of circulating tumour cell and low adenovirus receptor level effectively.

For the analysis of the expression by to reporter gene, check the circulating tumour cell, reporter gene should only be expressed in tumour cell, perhaps at the cells that is verified, and can not express in normal hemocyte.For example: can not, at neutrophil leucocyte, express in lymphocytes and platelets.Two kinds of methods can be used for realizing the expression of this selectivity reporter gene, 1) direction-sense gene transfection and 2) expression of selected gene.Direction-sense gene transfection is to realize by carrier and the transfection method of selecting particular type, so carrier can only enter tumour cell, and epithelial cell and the cell that is verified.Some virus vector just have this type of cell-specific transfection ability.For instance, adenovirus can not infect white cell usually, and therefore, reporter gene can not enter white cell.When analysis report genetic expression, it is negative that these cells can keep.Selected gene is expressed can be by realizing with tumor promotor or particular cell types promotor.Therefore, even reporter gene is transfected in normal hemocyte, it can not be expressed yet, and it is negative that normal plasma cell can keep.Tumour cell, epithelial specific promotor just can be used for reporter gene expresses in tumour cell or epithelial cell, and does not express in hemocyte.For example: the reverse transcriptase of telomere promotor, the survivin promotor, the probasin promotor, the MUC1 promotor, carcinoembryonic antigen promoter, afp promoter, the tyrosine oxidase promotor, and the promotor of its mosaic all can be used for this purpose.

The expression of the reporter gene that drives by tumour and tissue-specific promoter, check the method for circulating tumour cell to comprise following steps, and each step should be taked sfgd. to prevent sample contamination and keep sample aseptic.

1., with anti-coagulant sample of blood, drawn (5 milliliters-10 milliliters), put into sterile tube.Normally used anti-coagulant is ethylenediamine tetraacetic acid (EDTA) and heparin.Any anti-coagulant, as long as can prevent can using of blood coagulation, as long as they can not have side effects to later cell cultures.

2. blood sample is processed, removed red corpuscle.This can pass through gradient centrifugation, and the scheme of passing ficoll solves, or with 75-200 mmole ammonium chloride, carrys out lysed erythrocyte etc., comprises with other medicaments and reduces harm to karyocyte.But the process of redissolve is dissolved fully to reach erythrocytic.Should use phosphate buffered saline (PBS) (PBS) or substratum to clean 2-3 time with the karyocyte that these two kinds of methods are separated.

3., with the expression vector transfectional cell of the reporter gene that carries tumour or tissue-specific promoter's driving, then cell cultures is cultivated in substratum.Incubation time depends on expression vector used and reporter gene expression and detection method used.Sometimes, gene transfection and cell cultures can be carried out simultaneously.

4. any substratum for cultivating mammalian cell all can be used for this purposes.For example: RPMI-1640, MEM, the substratum such as DMEM.Substratum usually can be added with 5%-20% foetal calf serum or calf serum.Substratum can also supplement with microbiotic, as penbritin (50-200 units per ml) and/or Streptomycin sulphate (50-200 ug/ml).Substratum can directly be added in the solution that contains expression vector.Cell is placed in overnight incubation in cell culture incubator, or 24-48 hour.

5. express the cell of reporter gene in the analyzing and testing cell sample.The character that depends on reporter gene, detect the cell of expressing reporter gene with different analytical procedures.For fluorescin, fluorescent microscope, the fluorescence-activation stream type cell analyzer can be used for detecting the positive cell of express fluorescent protein.Luciferase and beta-galactosidase enzymes are detected, can use noclilucence and enzyme assay.

It is essential, before normal value was set up, control group should be included in mensuration.Positive control sample and feminine gender all should just be used sample when test starts.

Particularly, the invention provides following technical proposal:

In one aspect, the invention provides a kind of tumor-specific promoters, it is high that its activity in normal cell of its specific activity in tumour cell is wanted.In an embodiment of first aspect, described tumor-specific promoters comprises human telomerase reverse transcriptase's promotor, survivin promotor, medkine promotor, carcinoembryonic antigen promoter, MUC-1 promotor and probasin gene promoter.In an embodiment of first aspect, described reverse transcriptase of telomere promoter sequence is as shown in SEQ ID NO:1.In an embodiment aspect first, described tumor-specific promoters can be the former promotor of tissue or cellular type specific gene, can be also the combination promotor through transformation.In an embodiment aspect first, described combination promotor is the mosaic type promotor that contains the sequence that is derived from tissue or cellular type specificity promoter and be derived from the enhanser of other promotors.In an embodiment of first aspect, described mosaic type promotor is the TC chimeric promoters, and it comprises human body reverse transcriptase of telomere promotor and cytomegalovirus enhanser.In an embodiment aspect first, wherein said chimeric promoters sequence is as shown in SEQ IDNO:8.

In aspect second, the invention provides a kind of carrier, it is to comprise that the tumor-specific promoters by first aspect orders about the reporter gene expression vector of expression.In an embodiment aspect second, the reporter gene of wherein mentioning is a kind of gene usually used as the promoter activity indicator.In an embodiment aspect second, wherein said reporter gene comprises fluorescin (green, redness, yellow and blue fluorescent protein) gene, firefly luciferase gene, renilla luciferase gene, bacteria beta-galactosidase (LacZ) gene.In an embodiment aspect second, wherein said expression vector refers to any carrier for gene transfection.In an embodiment aspect second, wherein said expression vector comprises plasmid, plasmid/lipid complex and virus vector.In an embodiment aspect second, the virus vector of wherein mentioning refers to replication-defective virus carrier and replication competent type virus vector, adenovirus carrier for example, replication competent type adenovirus carrier, herpesvirus vector, vaccinia virus vector etc.In an embodiment of second aspect, described adenovirus carrier is the 5th type adenovirus hominis.The adenovirus carrier that the present invention mentions is to prepare in the damping fluid of stablizing the adenovirus carrier activity.Damping fluid contains Tris, NaCl, EDTA, ethanol, Histidine, sucrose.

In an embodiment aspect second, wherein said virus vector is after viral fiber improves.In an embodiment aspect second, it is to be inserted into by arginine-R-Glyceric acid-aspartic acid (RGD) sequence of encoding the adenoviral gene group that is arranged in map unit 86-91 that wherein said viral fiber improves, thereby make viral scleroproein contain the RGD sequence, change its receptor-specific, the carrier after improvement can enter target cell through integrin albumen.In an embodiment aspect second, wherein the activity of tissue or tumor-specific promoters is strengthened by the binary expression system.In an embodiment aspect second, described binary is expressed system applies green fluorescent protein/TRAIL fusion gene.In an embodiment aspect second, described green fluorescent protein/TRAIL fusion gene is as shown in SEQ ID NO:5.In an embodiment aspect second, the expression vector of wherein mentioning comprises replication competent type adenovirus carrier Ad/E1-GFP and Ad/E1-GFP-RGD, replication-defective adenoviral vector Ad/TERT-GFP, Ad/TERT-LacZ, Ad/GFP-TRAIL and Ad/TERT-Luciferase.

In aspect the 3rd, the invention provides a kind of test kit, it is used for cancer patients's early diagnosis, the prediction of therapeutic response and prognosis thereof, and it comprises the tumor-specific promoters of first aspect and/or the carrier of second aspect.In an embodiment in aspect the 3rd, described test kit is for detection of the tumour cell in blood specimen and bone marrow prepare.

In aspect the 4th, the invention provides a kind of diagnostic reagent, it is used for cancer patients's early diagnosis, and the prediction of therapeutic response and prognosis thereof, comprise the tumor-specific promoters of first aspect and the carrier of second aspect.In an embodiment aspect the 4th, described diagnostic reagent is for detection of the tumour cell in blood specimen and bone marrow prepare.

In aspect the 5th, the tumor-specific promoters that the invention provides first aspect is for the preparation of the application of the diagnostic reagent of the test kit of the carrier of second aspect and the third aspect and fourth aspect.

Description of drawings:

Fig. 1, compare the LacZ gene at normal cell (NHBE, NHLF, CD34+), the expression in healthy tissues (spleen and liver) and cancer cells (H1299, A549, UV-2237M).Cell or the mouse adenovirus carrier (Ad/CMV-GFP, Ad/CMV-LacZ, Ad/hTERT-LacZ) of same dose) transfection.Detect LacZ gene (beta-galactosidase enzymes) activity with the X-gal staining.Positive cell is blue.The cell title is listed in left side.Top is used carrier, right-hand prompting cell (tissue) attribute, normal or cancer cells.

Fig. 2, cancer cells (H1299, A549, LOVO, DLD1) and normal cell (CD34+, NHFB, NHBE) with the adenovirus carrier (Ad/CMV-GFP of same dose, Ad/CMV-LadZ, Ad/hTERT-LacZ) after transfection, with luminescence method, analyze betagalactosidase activity.Betagalactosidase activity represents with relative light intensity (relative light units (RLU)/μ g albumen).Institute's indicating value is three average+standard deviations (SD) of analyzing.

Fig. 3. by the specifically expressing of GFP-Bax fusion gene in tumour of adenovirus carrier Ad/hTERT-gBax mediation.Left side, tumour cell (H1299, A549, H322, H358) and normal fibroblast (NHFB) with Ad/CMV-GFP or Ad/TERT-GFP/Bax (2000 virions/cell) transfection of same dose, after 48 hours in the expression of fluorescence microscopy Microscopic observation GFP or GFP/Bax.The GFP that CMV drives expresses in all cells, and the GFP-Bax that TERT drives only expresses in tumour cell, and does not express in NHFB.Tumour and liver GFP and GFP-Bax after treat with Ad/CMV-GFP or Ad/TERT-GFP/Bax on right side, mice with tumor, the expression of Bax.Bax expresses and detects with Immunohistochemical Method.Natural death of cerebral cells detects with the TUNEL method.GFP-Bax and Bax only express and cause to transfer and die in tumour, but do not express in normal liver tissue.

Fig. 4. top, adenovirus carrier Ad/gTRAIL schematic diagram. adenovirus E 1 district (map unit 1.3-9.3) replaces by the expression cassette by GT promoter expression GFP/TRAIL with by the expression cassette of TERT promoter expression Gal4/GV16.Below, Ad/CMV-GFP or the Ad/gTRAIL transfection of same dose of tumour cell (DLD-1) and normal cell (NHFB), expression and the cell state of observation GFP or GFP-TRAIL after 48 hours.GFP-TRAIL is only at DLD1 cells and cell killing.Fig. 5.TERT/CMV (TC) promoter, fusion schematic diagram.This figure contains the TC-GFP expression cassette.The TC promoter sequence is seen SEQ ID NO:8.

Fig. 6, the expression in normal cell (NHFB) and tumour cell (MiaPaca2, H1299, DLD1) of condition replication type adenovirus (Ad/GFP-E1) schematic diagram (top) and E1 gene thereof.Ad/GFP-E1 is by TC promoters driven GFP and E1.The E1 protein expression detects with Western blotting (Western blot).Fig. 7.The expression (left side) of the GFP gene that condition replication type adenovirus (Ad/GFP-E1) carries in tumour cell (H1299) and normal cell (NHFB), the figure top is transfection dosage.Right side, the expression of GFP gene in noumenal tumour that condition replication type adenovirus (Ad/GFP-E1) carries.

Fig. 8, the condition replication type adenovirus (Ad/GFP-E1-RGD) after scleroproein is modified.Top, the virus vector schematic diagram, same as shown in Figure 6, by TC promoters driven GFP and E1.But the scleroproein of virus contains the RGD sequence.Below, compare Ad/CMV-GFP, Ad/GFP-E1, the Ad/GFP-E1-RGD expression in solid tumor.Virus vector is expelled in solid tumor, observes GFP after two weeks and express.Ad/GFP, because not copying in tumour, expresses disappearance with tumor propagation.Ad/GFP-E1, Ad/GFP-E1-RGD all have GFP to express in tumour, and do not express in normal cell around.But it is higher that the GFP of Ad/GFP-E1-RGD expresses.

Fig. 9, normal blood sample, add tumour cell (top) and do not add tumour cell (below), uses respectively Ad/GFP-E1, and the Ad/GFP-E1-RGD transfection, expresses at fluorescence microscopy Microscopic observation GFP after 24 hours.The GFP positive cell is only in adding the blood sample of tumour cell, and not in the blood sample of negative for tumor cells.

Figure 10.The blood sample that compares normal people (normal) and tumour (lung cancer) patient.Respectively get 5 milliliters of blood, separate white corpuscle, and with Ad/GFP-E1 transfection white corpuscle, after 24 hours at the Microscopic observation cell.Left side, fluoroscope (black and white is shone), right side, common mirror, but the visual field that communicates.Without the GFP positive cell, GFP positive cell (tumour cell) is arranged in patients with lung cancer blood in normal blood sample.

Figure 11 is the collection of illustrative plates of Ad/CMV-LacZ.E1 gene (the 456bp-3228bp of the 5th type adenovirus hominis, the 1.3-9.3 of collection of illustrative plates unit) deleted, and the expression cassette that inserts an expression casette (as shown in the figure) .AD/TERT-LacZ in this zone contains the TERT promotor, the poly a-signal sequence (BGHpA) of LacZ gene (LacZ) and Polisac gene.

The collection of illustrative plates unit of the 5th type adenovirus hominis is presented at the top of figure.

Figure 12 is the collection of illustrative plates of Ad/TERT-LacZ.E1 gene (the 456bp-3228bp of the 5th type adenovirus hominis, the 1.3-9.3 of collection of illustrative plates unit) deleted, and the expression cassette that inserts an expression casette (as shown in the figure) .AD/TERT-LacZ in this zone contains the TERT promotor, the poly a-signal sequence (BGHpA) of LacZ gene (LacZ) and Polisac gene.

The collection of illustrative plates unit of the 5th type adenovirus hominis is presented at the top of figure.

Figure 13 is the collection of illustrative plates of Ad/TERT-Bax.E1 gene (the 456bp-3228bp of the 5th type adenovirus hominis, the 1.3-9.3 of collection of illustrative plates unit) deleted, and the double expression boxes that inserts double gene expression box (as shown in the figure) .AD/TERT-Bax in this zone contains the TERT promotor, TetR-GV16 gene (TetR-GV16), the poly a-signal sequence (BGHpA) of Polisac gene, the TRE promotor, GFP/Bax gene (GFP/Bax), and SV40 poly a-signal sequence (sv40polyA).

The collection of illustrative plates unit of the 5th type adenovirus hominis is presented at the top of figure.

TERT=human telomerase reverse transcriptase gene promoter

TRE=tsiklomitsin response factor promotor

TetR=tsiklomitsin arrestin

Figure 14 is the collection of illustrative plates of Ad/TERT-gTRAIL.E1 gene (the 456bp-3228bp of the 5th type adenovirus hominis, the 1.3-9.3 of collection of illustrative plates unit) deleted, and the double expression boxes that inserts double gene expression box (as shown in the figure) .AD/TERT-gTRAIL in this zone contains the TERT promotor, GAL4-GV16 gene (GAL4-GV16), the poly a-signal sequence (BGHpA) of Polisac gene, the GT promotor, GFP/TRAIL gene (GFP-TRAIL), and SV40 poly a-signal sequence (sv40polyA).

TERT=human telomerase reverse transcriptase gene promoter

The GT=GT promotor, contain the GAL4/TATA sequence

The collection of illustrative plates unit of the 5th type adenovirus hominis is presented at the top of figure.

Figure 15 is the collection of illustrative plates of Ad/GFP-E1.The E1 gene promoter of the 5th type adenovirus hominis (456bp-543bp, the 1.3-1 of collection of illustrative plates unit.5) deleted, and at the expression cassette of this zone insertion one expression casette (as shown in the figure) .AD/GFP-E1, contain the TC promotor, GFP gene (GFP), the poly a-signal sequence (BGHpolyA) of Polisac gene.And be the TC promotor, and by it, control the expression of E1 gene.The TC=TC promotor, contain human telomerase reverse transcriptase's gene promoter and cmv enhancer sequence.The collection of illustrative plates unit of the 5th type adenovirus hominis is presented at the top of figure.

Figure 16 is the collection of illustrative plates of AD/CMV-GFP.E1 gene (the 456bp-3228bp of the 5th type adenovirus hominis, the 1.3-9.3 of collection of illustrative plates unit) deleted, and the expression cassette that inserts an expression casette (as shown in the figure) .AD/CMV-GFP in this zone contains the CMV promotor, the poly a-signal sequence (BGHpA) of green fluorescence protein gene (GFP) and Polisac gene.

The collection of illustrative plates unit of the 5th type adenovirus hominis is presented at the top of figure

Figure 17 is the collection of illustrative plates of Ad/GFP-E1-RGD.The E1 gene promoter of the 5th type adenovirus hominis (456bp-543bp, the 1.3-1 of collection of illustrative plates unit.5) deleted, and at the expression cassette of this zone insertion one expression casette (as shown above) .AD/GFP-E1-RGD, contain the TC promotor, GFP gene (GFP), the poly a-signal sequence (BGHpolyA) of Polisac gene.And be the TC promotor, and by it, control the expression of E1 gene.The viral scleroproein of this carrier is modified, and contains the RGD sequence.

The TC=TC promotor, contain human telomerase reverse transcriptase's gene promoter and cmv enhancer sequence RGD=Arg-Gly-Asp aminoacid sequence, it is inserted into collection of illustrative plates unit approximately in the adenoviral fiber protein gene of 86-91, and this makes the scleroproein of expression contain the CDCRGDCFC aminoacid sequence.

The collection of illustrative plates unit of the 5th type adenovirus hominis is presented at the top of figure.

The present invention will further illustrate by following embodiment, but it should be noted that the present invention is not limited to described embodiment.Scope of the present invention is only limited by accompanying claim.

Embodiment

Materials and methods:

The present invention's concrete adenovirus carrier used is the 5th type adenovirus hominis (human adenovirustype 5), the plasmid that builds adenovirus carrier can be available from Stratagene company (http://www.stratagene.com/lit/vector.aspx) (catalog#240010) or CellBiolabs, and Inc. company (http://www.cellbiolabs.com) (catalog#VPK-250).

adenovirus carrier described in following embodiment (builds the plasmid of adenovirus carrier from Stratagene company, catalog#240010) construction process is identical with bibliographical information, (see document FangB, Eisensmith RC, Li XHC, Finegold MJ, Shedlovsky A, Dove W, Woo SLC.Gene Therapy for Phenylketonuria:phenotypic correction in a geneticallydficient mouse model by adenovirus-mediated hepatic gene transfer.GeneTherapy 1:247-254, 1994).General first with the expression cassette of molecule clone technology with goal gene,, as TERT-GFP, be inserted into (shuttle plasmid) in a shuttle plasmid.Make two ends of this expression cassette contain respectively a bit of DNA sequence dna from recombinant adenovirus.Shuttle plasmid and another are contained the almost plasmid (as pJM17, pBHG10) of whole or very big part recombinant adenoviral vector sequence, cotransfection is in 293 cells again.Cell was cultivated for 1～2 week in incubator.Homologous recombination can occur in shuttle plasmid and pJM17 or pBHG10 in 293 cells, form the adenovirus carrier of restructuring.Whether detect resulting adenovirus with the method for polymerase chain reaction,PCR method or testing goal genetic expression again is needed recombinant adenoviral vector.Finally, the DNA that extracts adenovirus carries out sequential analysis, further verifies the sequence at recombinant adenoviral vector restructuring position.

Embodiment 1. reverse transcriptase of telomere promotors (reverse transcriptase of telomere) drive the expression of tumour-specific reporter gene LacZ.

Fig. 1 shows, the relatively expression of human telomerase reverse reversed transcriptive enzyme promotor and cytomegalovirus promoter driving reporter gene LacZ.

In experiment; (this carrier is to build (Gu et al through recombinant DNA technology with the replication deficient human adenovirus carrier (Ad/TERT-LacZ) that is loaded with the LacZ gene (SEQ ID NO:2) that the long reverse transcriptase of telomere core promoter (SEQID NO:1) of 378bp drives for we; 2000) the carrier collection of illustrative plates that builds is seen Figure 12) infect (concrete infection method is seen Gu J et al, 2000) normally or tumour cell.(its construction process is seen Gu et al will to be loaded with the adenovirus carrier (Ad/CMV-LacZ) of the LacZ gene that CMV promotor (SEQ ID NO:9) drives, 2000, appended carrier collection of illustrative plates Figure 11 is seen in promotor, the arrangement of LacZ gene on carrier) as positive control.Virus infection unit is 1000 particles/cell.The Human Lung Cancer cell strain (H1299 and A549) of cultivating, mouse fibrosarcoma cell (UV-2237), normal human body inoblast (NHLF), matter chief cell between CD34+, normal human body bronchial epithelial cell (NHBE) is (available from American Type CultureCollection or Clonetics Corp (San Diego, CA), with the adenovirus particles cells infected of same dose after 24 hours, analyze galactosidase activity (concrete grammar is seen Gu J etal, 2000) with the X-gal staining.X-gal staining analysis demonstration, in all JEG-3, CMV promotor and reverse transcriptase of telomere promotor can drive stronger beta-galactosidase enzymes and express.And in two normal cell strains, only have the cell that has infected Ad/CMV-LacZ virus to produce high-caliber LacZ genetic expression (nearly 100%) (Fig. 1).Only produced considerably less LacZ-positive cell with the normal cell of the Ad/TERT-LacZ virus infection of same pharmaceutical quantities.In the detection of all cells, in the activity of the reverse transcriptase of telomere promotor in cancer cells, be significantly higher than (P≤0.05) in normal cell.

Same result also appears in experiment to mouse.With 5x10 ¹⁰Adenovirus carrier injects mouse tail vein, two days later mouse is put to death, and gets each organ and carry out the betagalactosidase activity analysis.The mouse of injecting normal saline is as the background reference substance.Detect (concrete grammar is seen Gu J et al, 2000) and arrived high-caliber betagalactosidase activity in the mouse liver of injecting Ad/CMV-LacZ virus and spleen.In other organs of the mouse with the Ad/CMV-LacZ virus treated, the activity of enzyme and background reference substance (being the mouse of injecting normal saline) are consistent.Can find after contrast, at the mouse liver that injects Ad/TERT-LacZ, the activity of the enzyme in the organ of spleen and other mouse, all within the being seen scope of background reference substance.The reporter gene that Ad/TERT-LacZ is described express with through phosphate buffered saline buffer, the mouse that control vector is processed is identical.(the results detailed in Fig. 1)

The comparison of embodiment 2. reverse transcriptase of telomere promotors and cytomegalovirus promoter activity

use phosphate buffered saline buffer, (construction process is seen Gu et al. to control vector (Ad/CMV-GFP), 2000. the carrier collection of illustrative plates is seen accompanying drawing 16), Ad/TERT-LacZ (Figure 12) or Ad/CMV-LacZ. (Figure 11) transfection (namely adding phosphate buffered saline buffer or recombinant adenovirus particle (1000 virions/cell) in cell culture fluid) cancer cells (H1299, A549, Lovo, and DLD1) and normal cell (human body inoblast (NHFB), matter chief cell between CD34+, normal human body bronchial epithelial cell (NHBE)) (available from Clonetics Corp (San Diego, CA)).LacZ expresses (concrete grammar is seen Gu et al, 2000) with the check of betagalactosidase activity analytical method.The betagalactosidase activity that comes from cytomegalovirus or reverse transcriptase of telomere promotor, only differed 2-10 doubly in cancer cells.But in normal cell, they but differ over 500 times.The results detailed in Fig. 2.

Embodiment 3.CMV promotor and reverse transcriptase of telomere promotor are ordered about the expression of green fluorescent fusion protein

Same result also obtains in cytomegalovirus (CMV) promotor and reverse transcriptase of telomere promotor are ordered about the experiment of green fluorescent fusion protein.

With Ad/CMV-green fluorescent protein (Ad/CMV-GFP) (Figure 16) or (Figure 13) transfected with human lung carcinoma cell (H1299 of Ad/TERT-green fluorescent protein/Bax (Ad/TERT-GFP/Bax), A549, H322 and H358), namely add 1000 virions/cell in cell culture fluid (MEM or DMEM).(concrete construction process is seen Gu etal. for Ad/CMV-GFP (Figure 16) and Ad/TERT-GFP/Bax, 2002b. the carrier collection of illustrative plates is seen accompanying drawing 13) be replication-defective adenoviral vector, insert and replace adenovirus E 1 district (seeing being attached to collection of illustrative plates) and build by their expression cassettes (CMV-GFP (the GFP gene order is seen SEQ ID NO:3) or TERT-GFP/Bax (GFP/Bax gene fusion sequence is seen SEQ ID NO:4)) separately, the promotor of expression cassette is seen the appended sequence of gene (TetR and TRE sequence, be numbered SEQ ID NO:11 and 12).Cell after transfection through cultivating (37 ℃, 5%CO ₂, nutrient solution is the DMEM that contains 10% calf serum) and 24-48 hour, the expression of green fluorescent protein and green fluorescent protein-Bax fusion rotein can be detected under fluorescent microscope (Olympus).Fig. 3 demonstration, cytomegalovirus-green fluorescent protein can be expressed in all cancer cells (H1299, A549, H322 and H358) and normal cell (NHFB).But reverse transcriptase of telomere-green fluorescent protein can only be expressed in cancer cell, and can not express in normal NHFB cell.Test in Mice Body has also obtained same result (concrete testing method is seen Gu et al., 2002b).With 5x10 ¹⁰Adenovirus carrier injects the tumour of mouse tail vein or mouse, two days later mouse is put to death, and gets its liver organization or tumor tissues, and fluorescent microscope after freezing microtome section (Olympus) is lower to be observed.CMV-GPF is expressed in tumor tissues and normal liver organization.But TERT-GFP/Bax is only expressed in tumor tissues.These results show that TS reporter gene or destination gene expression obtain by the reverse transcriptase of telomere promotor, result such as Fig. 3.

Embodiment 4. use binary expression systems carry out tumour-specific genetic expression

Application green fluorescent protein/TRAIL fusion gene (its sequence of GFP/TRAIL is seen SEQ IDNO:5) is observed tumor cell specific genetic expression.The binary expression system of mentioning in this explicit declaration book carries out tumour-specific genetic expression.

In this case, Gal4/VP16 fusion transcription factor (its sequence is as shown in SEQ ID NO:6) is used to strengthen tumour-specific genetic expression.The reverse transcriptase of telomere promotor is used to drive Gal4/VP16 and expresses.Its target promotor Gal4/TATA (GT) of the Gal4/VP16 reactivation that is expressed (its sequence is as shown in SEQ ID NO:7) promotor, the latter is used for ordering about the expression of green fluorescent protein-TRAIL.The benefit of this binary expression system is, transgene expression level can significantly strengthen (over 100 times), but the specificity of promotor is retained (Koch et al 2001).

As seen in Figure 4, will insert together the adenovirus E 1 district by the expression cassette of GT promoter expression GFP/TRAIL with by the expression cassette of TERT promoter expression Gal4/GV16, (concrete carrier collection of illustrative plates is seen Fig. 4 and Figure 14 to be built into carrier A d/gTRAIL.These two expression cassettes are connected in mode end to end.From right to left, be followed successively by GT promotor-GFP/TRAIL fusion gene-SV40 poly a-signal-TERT promotor-GAL4/GV16 transcription factor gene-Niu growth hormone gene poly a-signal).(transfection dosage is 500～2000moi) colon cancer cell DLD1 and normal fibroblast (NHFB), cultivates after 24-48 hour and observes expression and the cellular form of green fluorescent protein (GFP or GFP/TRAIL) to distinguish transfection with Ad/gTRAIL and Ad/CMV-GFP (control vector).CMV-GFP all expresses in cancer cells and inoblast, but does not cause necrocytosis.And Ad/gTRAIL is only showed and cell is killed at cancer cells DLD1, does not but show in inoblast.

Embodiment 5. builds the TC chimeric promoters

The TC chimeric promoters comprises human body reverse transcriptase of telomere promotor and cytomegalovirus enhanser (its concrete sequence is as shown in SEQ ID NO:8).This inserted type promotor has the specificity of reverse transcriptase of telomere promotor, and the activity of promotor also is eager to excel a lot than the reverse transcriptase of telomere promotor of wild-type.Chimeric promoters has comprised initiator codon base pair (the small segment enhancer sequence of 456 to-2 (gene pool numberings: M21925)) and cytomegalovirus promoter, i.e.-1017 to-901 sequences of human body cytomegalovirus early protein initiator codon (the gene pool numbering: M21925) before of corresponding reverse transcriptase of telomere.This inserted type promotor is named as TC promotor (SEQ ID NO:8).What show in Fig. 5 is the plasmid structure, and it has comprised orders about the promotor in the TC of egfp expression box.

Embodiment 6. condition replication type adenovirus are expressed reporter gene green fluorescent protein (Ad/GFP-E1).

Green fluorescence protein gene and adenovirus E 1 gene order about by TC mosaic promotor (seeing embodiment 5).

The structure of Ad/GFP-E1 is that (the concrete structure of expression cassette is seen Davis JJet al with the TC-GFP expression cassette, 2006) before being inserted into the adenovirus E 1 district, replace adenovirus E 1 promotor (be about to the TC promoter sequence and be inserted into the upstream of E1 gene, collection of illustrative plates is seen accompanying drawing 15) with the TC promotor.Ad/GFP-E1 transfection inoblast (NHFB) and cancer cells (MiaPaca2 (available from ATCC.ORG) with same dosage (1000moi), DLD1 and H1299, (concrete grammar is seen Davis JJ et al. to detect the expression of E1 albumen with Western blotting (Western blot) after 24-48 hour, 2006), or at fluorescence microscopy Microscopic observation GFP express.As seen the condition replication type adenovirus can be in cancer cells expressing green fluorescent protein and E1A, but can not express these genes in normal cell, for example can not express these genes (seeing Fig. 6 and Fig. 7) normal fibroblast (NHFB).When cell was loaded with virus vector (Ad/CMV-GFP, the expression of the CMV promoters driven green fluorescent protein) infection of CMV-GFP, cancer cells (H1299) and normal cell (NHFB) be expressing green fluorescent protein all.But, when cell is infected by Ad/GFP-E1, only have H1299 cell expressing green fluorescent protein.The NHFB cell is expressing green fluorescent protein (Fig. 7) not.Same,, when Ad/GFP-E1 is injected in tumour, only have tumour energy expressing green fluorescent protein, and organizing normally around tumour can not be expressed (Fig. 7).

The condition replication type adenovirus carrier that embodiment 7. scleroproeins are modified

Condition replication type adenovirus after the virus fiber improves is expressed reporter gene green fluorescent protein (Ad/GFP-E1-RGD) (Figure 17).

Condition replication type adenovirus in Fig. 8 be that (seeing above-described embodiment 6) is the same in Fig. 6, but its surface fiber albumen is modified.Coding arginine-glycine-aspartic acid (RGD) sequence is inserted into the adenoviral gene group that is arranged in map unit 86-91, thereby make viral scleroproein head (knob) contain (RGD) sequence (seeing appended adenoviral fiber protein gene order, as shown in SEQID NO:10).This adenovirus can infect and contain low-level adenovirus receptor or the tumour cell of gland-containing virus acceptor not.Fig. 8 is exactly control vector (Ad/CMV-GFP), the expression of Ad/GFP-E1 and the Ad/GFP-E1-RGD green fluorescent protein in tumour.

First mouse fibrosarcoma cell (UV-2237) is seeded in the mouse shank, after it forms tumour (approximately about a week), with same dose (5x10 ⁹Virion/mouse) Ad/CMV-GFP (control vector), Ad/GFP-E1 and Ad/GFP-E1-RGD are expelled in tumour, observe the expression of GFP in tumour and tumour normal surrounding tissue after two weeks, result shows, in the tumour that is infected by Ad/GFP-E1-RGD, green fluorescent protein is more intense.Annotate, normal surrounding tissue does not have reporter gene to express.

Embodiment 8. checks tumour cell in blood sample.

Can the test adenovirus be used to detect tumour cell by the TC promoter expression green fluorescent protein of inserted type.100 human body tumour cells (the human lung carcinoma cell H1299 of cultivation) are joined in 5 milliliters of normal blood of human body samples.Blood sample is removed red blood cell with hypotonic chloride leach solution.With phosphate buffered saline buffer, white cell is cleaned three times, then use 1x10 ⁴The Ad/GFP-E1 of virion or Ad/GFP-E1-RGD cells infected (namely add 1x10 in the 0.5ml nutrient solution ⁴Virion, directly be added in above-mentioned white corpuscle, mixes), under 37 ℃, the white corpuscle after transfection and tumour cell were cultivated 24 hours in RPMI 1640 substratum.Observation of cell under fluorescent microscope.Not adding the blood sample of tumour cell also cultivates with Ad/GFP-E1 or the Ad/GFP-E1-RGD of same amount.Result shows, only has the blood sample that has added tumour cell green cell (Fig. 9) to occur.Do not have the blood sample of tumour cell any green cell not occur.Such result shows that the tumor characteristic promotor can be used for the reporter gene expression of tumour cell in the check blood sample.

Embodiment 9. blood of patients with cancer pattern detection.

Get 5 milliliters of blood from a routine patients with lung cancer and a routine normal control, after separating karyocyte with Ficoll gradient centrifugation method, add the Ad/GFP-E1 of 10000 virus infection units in white corpuscle.White corpuscle is incubated at the culture plate (available from Fisher Scientific) of one 48 orifice plates, one, every hole sample, cultivate two days later at the fluorescence microscopy Microscopic observation again.Have no the green fluorescent protein positive cell in normal specimens, and in the sample of patients with lung cancer, a visual field just there are several positive cells (Figure 10).Figure 10 left side is under fluorescent microscope; Right side, with the left side same field of view, but under simple microscope.Annotate, fluorescin is the whitening look in black and white is shone.

Appendix 1, described promotor or gene order

Human telomerase reverse transcriptase's core promoter sequence (SEQ ID NO:1)

accctgggagcgcgagcggcgcgcgggcggggaagcgcggcccagacccccgggtccgcccggagcag

ctgcgctgtcggggccaggccgggctcccagtggattcgcgggcacagacgcccaggaccgcgctccccac

gtggcggagggactggggacccgggcacccgtcctgccccttcaccttccagctccgcctcctccgcgcgga

ccccgccccgtcccgacccctcccgggtccccggcccagccccctccgggccctcccagcccctccccttcct

ttccgcggccccgccctctcctcgcggcgcgagtttcaggcagcgctgcgtcctgctgcgcacgtgggaagcc

ctggccccggccacccccgcc

LacZ gene order (SEQ ID NO:2)

atgtcgtttactttgaccaacaagaacgtgattttcgttgccggtctgggaggcattggtctggacaccagcaagg

agctgctcaagcgcgatcccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcct

tgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgc

gcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtg

cgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctacac

caacgtaacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcacat

ttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaactcggcgtttcatctgt

ggtgcaacgggcgctgggtcggttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttac

gcgccggagaaaaccgcctcgcggtgatggtgctgcgttggagtgacggcagttatctggaagatcaggatat

gtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatgttg

ccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagttgcgtgacta

cctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaa

ttatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggagcg

ccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcc

tgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcga

ggcgttaaccgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctg

ctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcg

accgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccg

atgatccgcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccga

gtgtgatcatctggtcgctggggaatgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaat

ctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacaccacggccaccgatattatttgcccg

atgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgcta

cctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaa

atactggcaggcgtttcgtcagtatccccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgatt

aaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggcgatacgccgaacgatcgccagtt

ctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaacaccagcagcag

tttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgagct

cctgcactggatggtggcgctggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaa

ggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcg

tagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcg

gaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatttttg

catcgagctgggtaataagcgttggcaatttaaccgccagtcaggctttctttcacagatgtggattggcgataaaa

aacaactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagc

gacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgtt

gttgcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatcagggga

aaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgattaccgttgatgttgaagtg

gcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagagcgggtaaa

ctggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgcc

attgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaatta

tggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatggaaacc

agccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattggtgg

cgacgactcctggagcccgtcagtatcggcggaattacagctgagcgccggtcgctaccattaccagttggtct

ggtgtcaaaaataa

GFP gene order (SEQ ID NO:3)

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaac

ggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatct

gcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcag

ccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgc

accatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtga

accgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaact

acaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgcca

caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccg

tgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatc

acatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtactcagat

ctcgagctcaagcttcgaattctgcagtcgacggtaccgcgggcccgggatccaccggat

GFP/Bax gene fusion sequence (SEQ ID NO:4)

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaac

ggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatct

gcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcag

ccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgc

accatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtga

accgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaact

acaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgcca

caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccg

tgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatc

acatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtactcagat

atggacgggtccggggagcagcccagaggcggggggcccaccagctctgagcagatcatgaagacagggg

cccttttgcttcagggtttcatccaggatcgagcagggcgaatggggggggaggcacccgagctggccctgga

cccggtgcctcaggatgcgtccaccaagaagctgagcgagtgtctcaagcgcatcggggacgaactggacag

taacatggagctgcagaggatgattgccgccgtggacacagactccccccgagaggtctttttccgagtggcag

ctgacatgttttctgacggcaacttcaactggggccgggttgtcgcccttttctactttgccagcaaactggtgctca

aggccctgtgcaccaaggtgccggaactgatcagaaccatcatgggctggacattggacttcctccgggagcg

gctgttgggctggatccaagaccagggtggttgggacggcctcctctcctactttgggacgcccacgtggcaga

ccgtgaccatctttgtggcgggagtgctcaccgcctcgctcaccatctggaagaagatgggctga

GFP/TRAIL gene fusion sequence (SEQ ID NO:5)

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaac

ggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatct

gcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcag

ccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgc

accatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtga

accgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaact

acaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgcca

caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccg

tgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatc

acatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtactcagat

ctcgagctcaagcttcgaattcctgcagcccggcgaaggagggccaccatggccatgatggaggtccagggg

ggacccagcctgggacagacctgcgtgctgatcgtgatcttcacagtgctcctgcagtctctctgtgtggctgtaa

cttacgtgtactttaccaacgagctgaagcagatgcaggacaagtactccaaaagtggcattgcttgtttcttaaaa

gaagatgacagttattgggaccccaatgacgaagagagtatgaacagcccctgctggcaagtcaagtggcaac

tccgtcagctcgttagaaagatgattttgagaacctctgaggaaaccatttctacagttcaagaaaagcaacaaaat

atttctcccctagtgagagaaagaggtcctcagagagtagcagctcacataactgggaccagaggaagaagca

acacattgtcttctccaaactccaagaatgaaaaggctctgggccgcaaaataaactcctgggaatcatcaagga

gtgggcattcattcctgagcaacttgcacttgaggaatggtgaactggtcatccatgaaaaagggttttactacatc

tattcccaaacatactttcgatttcaggaggaaataaaagaaaacacaaagaacgacaaacaaatggtccaatata

tttacaaatacacaagttatcctgaccctatattgttgatgaaaagtgctagaaatagttgttggtctaaagatgcaga

atatggactctattccatctatcaagggggaatatttgagcttaaggaaaatgacagaatttttgtttctgtaacaaat

gagcacttgatagacatggaccatgaagccagttttttcggggcctttttagttggctaag

Gal4/VP16 merges transcription factor sequence (SEQ ID NO:6)

Atgaagctactgtcttctatcgaacaagcatgcgatatttgccgacttaaaaagctcaagtgctccaaagaaaaac

cgaagtgcgccaagtgtctgaagaacaactgggagtgtcgctactctcccaaaaccaaaaggtctccgctgact

agggcacatctgacagaagtggaatcaaggctagaaagactggaacagctatttctactgatttttcctcgagaag

accttgacatgattttgaaaatggattctttacaggatataaaagcattgttaacaggattatttgtacaagataatgtg

aataaagatgccgtcacagatagattggcttcagtggagactgatatgcctctaacattgagacagcatagaataa

gtgcgacatcatcatcggaagagagtagtaacaaaggtcaaagacagttgactgtatcgccggaattcccgggg

atcaccgcccccattaccgacgtcagcctgggagacgaactccgcctggacggcgaggaggtggatatgacg

cccgccgacgccctggacgacttcgacttggagatgctgggggacgtggagtcccccccgggtccgggattta

ccccccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttaccgatgccc

ttggaattgacgagtacggtgggtag

Gal4/TATA (GT) promoter sequence (SEQ ID NO:7)

gtcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctcc

gagcggagtactgtcctccgagcggagactctagagggtatataat

TC chimeric promoters sequence (the line part is from the CMV promotor, and all the other are from the TERT promotor)

(SEQ ID NO：8)

accctgggagcgcgagcggcgcgcgggcggggaagcgcggcccagacccccgggtccgcccggagcag

ctgcgctgtcggggccaggccgggctcccagtggattcgcgggcacagacgcccaggaccgcgctccccac

gtggcggagggactggggacccgggcacccgtcctgccccttcaccttccagctccgcctcctccgcgcgga

ccccgccccgtcccgacccctcccgggtccccggcccagccccctccgggccctcccagcccctccccttcct

ttaccgcggccccgccctctcctcgcggcgcgagtttcaggcagcgctgcgtcctgctgcgcacgtgggaagc

cctggccccggccacccccgcc agatctgatctgtaggcgtgtacggtgggaggcctatataagcagagctcgt

ttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatcc

a

CMV promoter sequence (SEQ ID NO:9)

Gcccggttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaata

atgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgc

ccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcc

tggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccat

ggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccacccc

attgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccatt

gacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcg

cctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccggga

acggtgcattggaacggacctgc

Adenoviral fiber protein sequence (the line part is insertion sequence, contains the RGD sequence) (SEQ ID NO:10)

atgaagcgcgcaagaccgtctgaagataccttcaaccccgtgtatccatatgacacggaaaccggtcctccaact

gtgccttttcttactcctccctttgtatcccccaatgggtttcaagagagtccccctggggtactctctttgcgcctatc

cgaacctctagttacctccaatggcatgcttgcgctcaaaatgggcaacggcctctctctggacgaggccggca

accttacctcccaaaatgtaaccactgtgagcccacctctcaaaaaaaccaagtcaaacataaacctggaaatatc

tgcacccctcacagttacctcagaagccctaactgtggctgccgccgcacctctaatggtcgcgggcaacacac

tcaccatgcaatcacaggccccgctaaccgtgcacgactccaaacttagcattgccacccaaggacccctcaca

gtgtcagaaggaaagctagccctgcaaacatcaggccccctcaccaccaccgatagcagtacccttactatcac

tgcctcaccccctctaactactgccactggtagcttgggcattgacttgaaagagcccatttatacacaaaatggaa

aactaggactaaagtacggggctcctttgcatgtaacagacgacctaaacactttgaccgtagcaactggtccag

gtgtgactattaataatacttccttgcaaactaaagttactggagccttgggttttgattcacaaggcaatatgcaact

taatgtagcaggaggactaaggattgattctcaaaacagacgccttatacttgatgttagttatccgtttgatgctca

aaaccaactaaatctaagactaggacagggccctctttttataaactcagcccacaacttggatattaactacaaca

aaggcctttacttgtttacagcttcaaacaattccaaaaagcttgaggttaacctaagcactgccaaggggttgatg

tttgacgctacagccatagccattaatgcaggagatgggcttgaatttggttcacctaatgcaccaaacacaaatc

ccctcaaaacaaaaattggccatggcctagaatttgattcaaacaaggctatggttcctaaactaggaactggcctt

agttttgacagcacaggtgccattacagtaggaaacaaaaataatgataagctaactttgtggaccacaccagctc

catctcctaactgtagactaaatgcagagaaagatgctaaactcactttggtcttaacaaaatgtggcagtcaaata

cttgctacagtttcagttttggctgttaaaggcagtttggctccaatatctggaacagttcaaagtgctcatcttattat

aagatttgacgaaaatggagtgctactaaacaattccttcctggacccagaatattggaactttagaaatggagatc

ttactgaaggcacagcctatacaaacgctgttggatttatgcctaacctatcagcttatccaaaatctcacggtaaaa

ctgccaaaagtaacattgtcagtcaagtttacttaaacggagacaaaactaaacctgtaacactaaccattacacta

aacggtacacaggaaacaggagacacaact tgtgactgccgcggagactgtttctgcccaagtgcatactctat

gtcattttcatgggactggtctggccacaactacattaatgaaatatttgccacctcgagttacactttttcatacattg

cccaagaataag

TRE sequence(SEQ ID NO：11)：

Tttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtga

aagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagag

aaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcag

tgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagctcggtacc

cgggtcgagtaggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctgga

gacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcct

TetR sequence(SEQ ID NO：12)

atgtctagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtttaac

aacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggcttt

gctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaa

gattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggta

cacggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagag

aatgcattatatgcactcagcgctgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgct

aaagaagaaagggaaacacctactactgatagtatgccgccattattacgacaagctatcgaattatttgatcacc

aaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagt

gggtccgcgtacagccgcgcgcgtacgaaaaacaattacgggtctaccatcgagggcctgctcgatctcccgg

acgacgacgcccccgaagaggcggggctggcggctccgcgcctgtcctttctccccgcgggacacacgcgc

agactgtcgacggcccccccgaccgatgtcagcctgggggacgagctccacttagacggcgaggacgtggc

gatggcgcatgccgacgcgctagacgatttcgatctggacatgttgggggacggggattccccgggtccggga

tttaccccccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttaccgatg

cccttggaattgacgagtacggtgggtag

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IB096878 sequence table .ST25 sequence table

＜110〉east, Zhejiang gene biological Products Co., Ltd

＜120〉the application tumor-specific promoters is expressed reporter gene and is detected method and the reagent of circulating tumor cells

<130>IB096878

<160>12

<170>PatentIn version 3.1

<210>1

<211>378

<212>DNA

＜213〉artificial sequence

<400>1

accctgggag cgcgagcggc gcgcgggcgg ggaagcgcgg cccagacccc cgggtccgcc 60

cggagcagct gcgctgtcgg ggccaggccg ggctcccagt ggattcgcgg gcacagacgc 120

ccaggaccgc gctccccacg tggcggaggg actggggacc cgggcacccg tcctgcccct 180

tcaccttcca gctccgcctc ctccgcgcgg accccgcccc gtcccgaccc ctcccgggtc 240

cccggcccag ccccctccgg gccctcccag cccctcccct tcctttccgc ggccccgccc 300

tctcctcgcg gcgcgagttt caggcagcgc tgcgtcctgc tgcgcacgtg ggaagccctg 360

gccccggcca cccccgcc 378

<210>2

<211>3144

<212>DNA

＜213〉artificial sequence

<400>2

atgtcgttta ctttgaccaa caagaacgtg attttcgttg ccggtctggg aggcattggt 60

ctggacacca gcaaggagct gctcaagcgc gatcccgtcg ttttacaacg tcgtgactgg 120

gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 180

cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 240

gaatggcgct ttgcctggtt tccggcacca gaagcggtgc cggaaagctg gctggagtgc 300

gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat 360

gcgcccatct acaccaacgt aacctatccc attacggtca atccgccgtt tgttcccacg 420

gagaatccga cgggttgtta ctcgctcaca tttaatgttg atgaaagctg gctacaggaa 480

ggccagacgc gaattatttt tgatggcgtt aactcggcgt ttcatctgtg gtgcaacggg 540

cgctgggtcg gttacggcca ggacagtcgt ttgccgtctg aatttgacct gagcgcattt 600

ttacgcgccg gagaaaaccg cctcgcggtg atggtgctgc gttggagtga cggcagttat 660

ctggaagatc aggatatgtg gcggatgagc ggcattttcc gtgacgtctc gttgctgcat 720

aaaccgacta cacaaatcag cgatttccat gttgccactc gctttaatga tgatttcagc 780

cgcgctgtac tggaggctga agttcagatg tgcggcgagt tgcgtgacta cctacgggta 840

acagtttctt tatggcaggg tgaaacgcag gtcgccagcg gcaccgcgcc tttcggcggt 900

gaaattatcg atgagcgtgg tggttatgcc gatcgcgtca cactacgtct gaacgtcgaa 960

aacccgaaac tgtggagcgc cgaaatcccg aatctctatc gtgcggtggt tgaactgcac 1020

accgccgacg gcacgctgat tgaagcagaa gcctgcgatg tcggtttccg cgaggtgcgg 1080

attgaaaatg gtctgctgct gctgaacggc aagccgttgc tgattcgagg cgttaaccgt 1140

cacgagcatc atcctctgca tggtcaggtc atggatgagc agacgatggt gcaggatatc 1200

ctgctgatga agcagaacaa ctttaacgcc gtgcgctgtt cgcattatcc gaaccatccg 1260

ctgtggtaca cgctgtgcga ccgctacggc ctgtatgtgg tggatgaagc caatattgaa 1320

acccacggca tggtgccaat gaatcgtctg accgatgatc cgcgctggct accggcgatg 1380

agcgaacgcg taacgcgaat ggtgcagcgc gatcgtaatc acccgagtgt gatcatctgg 1440

tcgctgggga atgaatcagg ccacggcgct aatcacgacg cgctgtatcg ctggatcaaa 1500

tctgtcgatc cttcccgccc ggtgcagtat gaaggcggcg gagccgacac cacggccacc 1560

gatattattt gcccgatgta cgcgcgcgtg gatgaagacc agcccttccc ggctgtgccg 1620

aaatggtcca tcaaaaaatg gctttcgcta cctggagaga cgcgcccgct gatcctttgc 1680

gaatacgccc acgcgatggg taacagtctt ggcggtttcg ctaaatactg gcaggcgttt 1740

cgtcagtatc cccgtttaca gggcggcttc gtctgggact gggtggatca gtcgctgatt 1800

aaatatgatg aaaacggcaa cccgtggtcg gcttacggcg gtgattttgg cgatacgccg 1860

aacgatcgcc agttctgtat gaacggtctg gtctttgccg accgcacgcc gcatccagcg 1920

ctgacggaag caaaacacca gcagcagttt ttccagttcc gtttatccgg gcaaaccatc 1980

gaagtgacca gcgaatacct gttccgtcat agcgataacg agctcctgca ctggatggtg 2040

gcgctggatg gtaagccgct ggcaagcggt gaagtgcctc tggatgtcgc tccacaaggt 2100

aaacagttga ttgaactgcc tgaactaccg cagccggaga gcgccgggca actctggctc 2160

acagtacgcg tagtgcaacc gaacgcgacc gcatggtcag aagccgggca catcagcgcc 2220

tggcagcagt ggcgtctggc ggaaaacctc agtgtgacgc tccccgccgc gtcccacgcc 2280

atcccgcatc tgaccaccag cgaaatggat ttttgcatcg agctgggtaa taagcgttgg 2340

caatttaacc gccagtcagg ctttctttca cagatgtgga ttggcgataa aaaacaactg 2400

ctgacgccgc tgcgcgatca gttcacccgt gcaccgctgg ataacgacat tggcgtaagt 2460

gaagcgaccc gcattgaccc taacgcctgg gtcgaacgct ggaaggcggc gggccattac 2520

caggccgaag cagcgttgtt gcagtgcacg gcagatacac ttgctgatgc ggtgctgatt 2580

acgaccgctc acgcgtggca gcatcagggg aaaaccttat ttatcagccg gaaaacctac 2640

cggattgatg gtagtggtca aatggcgatt accgttgatg ttgaagtggc gagcgataca 2700

ccgcatccgg cgcggattgg cctgaactgc cagctggcgc aggtagcaga gcgggtaaac 2760

tggctcggat tagggccgca agaaaactat cccgaccgcc ttactgccgc ctgttttgac 2820

cgctgggatc tgccattgtc agacatgtat accccgtacg tcttcccgag cgaaaacggt 2880

ctgcgctgcg ggacgcgcga attgaattat ggcccacacc agtggcgcgg cgacttccag 2940

ttcaacatca gccgctacag tcaacagcaa ctgatggaaa ccagccatcg ccatctgctg 3000

cacgcggaag aaggcacatg gctgaatatc gacggtttcc atatggggat tggtggcgac 3060

gactcctgga gcccgtcagt atcggcggaa ttacagctga gcgccggtcg ctaccattac 3120

cagttggtct ggtgtcaaaa ataa 3144

<210>3

<211>786

<212>DNA

＜213〉artificial sequence

<400>3

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtac 720

tcagatctcg agctcaagct tcgaattctg cagtcgacgg taccgcgggc ccgggatcca 780

ccggat 786

<210>4

<211>1305

<212>DNA

＜213〉artificial sequence

<400>4

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtac 720

tcagatatgg acgggtccgg ggagcagccc agaggcgggg ggcccaccag ctctgagcag 780

atcatgaaga caggggccct tttgcttcag ggtttcatcc aggatcgagc agggcgaatg 840

gggggggagg cacccgagct ggccctggac ccggtgcctc aggatgcgtc caccaagaag 900

ctgagcgagt gtctcaagcg catcggggac gaactggaca gtaacatgga gctgcagagg 960

atgattgccg ccgtggacac agactccccc cgagaggtct ttttccgagt ggcagctgac 1020

atgttttctg acggcaactt caactggggc cgggttgtcg cccttttcta ctttgccagc 1080

aaactggtgc tcaaggccct gtgcaccaag gtgccggaac tgatcagaac catcatgggc 1140

tggacattgg acttcctccg ggagcggctg ttgggctgga tccaagacca gggtggttgg 1200

gacggcctcc tctcctactt tgggacgccc acgtggcaga ccgtgaccat ctttgtggcg 1260

ggagtgctca ccgcctcgct caccatctgg aagaagatgg gctga 1305

<210>5

<211>1621

<212>DNA

＜213〉artificial sequence

<400>5

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtac 720

tcagatctcg agctcaagct tcgaattcct gcagcccggc gaaggagggc caccatggcc 780

atgatggagg tccagggggg acccagcctg ggacagacct gcgtgctgat cgtgatcttc 840

acagtgctcc tgcagtctct ctgtgtggct gtaacttacg tgtactttac caacgagctg 900

aagcagatgc aggacaagta ctccaaaagt ggcattgctt gtttcttaaa agaagatgac 960

agttattggg accccaatga cgaagagagt atgaacagcc cctgctggca agtcaagtgg 1020

caactccgtc agctcgttag aaagatgatt ttgagaacct ctgaggaaac catttctaca 1080

gttcaagaaa agcaacaaaa tatttctccc ctagtgagag aaagaggtcc tcagagagta 1140

gcagctcaca taactgggac cagaggaaga agcaacacat tgtcttctcc aaactccaag 1200

aatgaaaagg ctctgggccg caaaataaac tcctgggaat catcaaggag tgggcattca 1260

ttcctgagca acttgcactt gaggaatggt gaactggtca tccatgaaaa agggttttac 1320

tacatctatt cccaaacata ctttcgattt caggaggaaa taaaagaaaa cacaaagaac 1380

gacaaacaaa tggtccaata tatttacaaa tacacaagtt atcctgaccc tatattgttg 1440

atgaaaagtg ctagaaatag ttgttggtct aaagatgcag aatatggact ctattccatc 1500

tatcaagggg gaatatttga gcttaaggaa aatgacagaa tttttgtttc tgtaacaaat 1560

gagcacttga tagacatgga ccatgaagcc agttttttcg gggccttttt agttggctaa 1620

g 1621

<210>6

<211>744

<212>DNA

＜213〉artificial sequence

<400>6

atgaagctac tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag 60

tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac 120

tctcccaaaa ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg 180

ctagaaagac tggaacagct atttctactg atttttcctc gagaagacct tgacatgatt 240

ttgaaaatgg attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat 300

aatgtgaata aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta 360

acattgagac agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt 420

caaagacagt tgactgtatc gccggaattc ccggggatca ccgcccccat taccgacgtc 480

agcctgggag acgaactccg cctggacggc gaggaggtgg atatgacgcc cgccgacgcc 540

ctggacgact tcgacttgga gatgctgggg gacgtggagt cccccccggg tccgggattt 600

accccccacg actccgcccc ctacggcgct ctggatatgg ccgacttcga gtttgagcag 660

atgtttaccg atgcccttgg aattgacgag tacggtgggt agatgtttac cgatgccctt 720

ggaattgacg agtacggtgg gtag 744

<210>7

<211>121

<212>DNA

＜213〉artificial sequence

<400>7

gtcggagtac tgtcctccga gcggagtact gtcctccgag cggagtactg tcctccgagc 60

ggagtactgt cctccgagcg gagtactgtc ctccgagcgg agactctaga gggtatataa 120

t 121

<210>8

<211>507

<212>DNA

＜213〉artificial sequence

<400>8

accctgggag cgcgagcggc gcgcgggcgg ggaagcgcgg cccagacccc cgggtccgcc 60

aggagcagct gcgctgtcgg ggccaggccg ggctcccagt ggattcgcgg gcacagacgc 120

ccaggaccgc gctccccacg tggcggaggg actggggacc cgggcacccg tcctgcccct 180

tcaccttcca gctccgcctc ctccgcgcgg accccgcccc gtcccgaccc ctcccgggtc 240

cccggcccag ccccctccgg gccctcccag cccctcccct tcctttaccg cggccccgcc 300

ctctcctcgc ggcgcgagtt tcaggcagcg ctgcgtcctg ctgcgcacgt gggaagccct 360

ggccccggcc acccccgcca gatctgatct gtaggcgtgt acggtgggag gcctatataa 420

gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 480

tccatagaag acaccgggac cgatcca 507

<210>9

<211>630

<212>DNA

＜213〉artificial sequence

<400>9

gcccggttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 60

cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 120

gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 180

tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 240

agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 300

ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 360

ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 420

aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 480

gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga 540

gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg 600

gccgggaacg gtgcattgga acggacctgc 630

<210>10

<211>1774

<212>DNA

＜213〉artificial sequence

<400>10

atgaagcgcg caagaccgtc tgaagatacc ttcaaccccg tgtatccata tgacacggaa 60

accggtcctc caactgtgcc ttttcttact cctccctttg tatcccccaa tgggtttcaa 120

gagagtcccc ctggggtact ctctttgcgc ctatccgaac ctctagttac ctccaatggc 180

atgcttgcgc tcaaaatggg caacggcctc tctctggacg aggccggcaa ccttacctcc 240

caaaatgtaa ccactgtgag cccacctctc aaaaaaacca agtcaaacat aaacctggaa 300

atatctgcac ccctcacagt tacctcagaa gccctaactg tggctgccgc cgcacctcta 360

atggtcgcgg gcaacacact caccatgcaa tcacaggccc cgctaaccgt gcacgactcc 420

aaacttagca ttgccaccca aggacccctc acagtgtcag aaggaaagct agccctgcaa 480

acatcaggcc ccctcaccac caccgatagc agtaccctta ctatcactgc ctcaccccct 540

ctaactactg ccactggtag cttgggcatt gacttgaaag agcccattta tacacaaaat 600

ggaaaactag gactaaagta cggggctcct ttgcatgtaa cagacgacct aaacactttg 660

accgtagcaa ctggtccagg tgtgactatt aataatactt ccttgcaaac taaagttact 720

ggagccttgg gttttgattc acaaggcaat atgcaactta atgtagcagg aggactaagg 780

attgattctc aaaacagacg ccttatactt gatgttagtt atccgtttga tgctcaaaac 840

caactaaatc taagactagg acagggccct ctttttataa actcagccca caacttggat 900

attaactaca acaaaggcct ttacttgttt acagcttcaa acaattccaa aaagcttgag 960

gttaacctaa gcactgccaa ggggttgatg tttgacgcta cagccatagc cattaatgca 1020

ggagatgggc ttgaatttgg ttcacctaat gcaccaaaca caaatcccct caaaacaaaa 1080

attggccatg gcctagaatt tgattcaaac aaggctatgg ttcctaaact aggaactggc 1140

cttagttttg acagcacagg tgccattaca gtaggaaaca aaaataatga taagctaact 1200

ttgtggacca caccagctcc atctcctaac tgtagactaa atgcagagaa agatgctaaa 1260

ctcactttgg tcttaacaaa atgtggcagt caaatacttg ctacagtttc agttttggct 1320

gttaaaggca gtttggctcc aatatctgga acagttcaaa gtgctcatct tattataaga 1380

tttgacgaaa atggagtgct actaaacaat tccttcctgg acccagaata ttggaacttt 1440

agaaatggag atcttactga aggcacagcc tatacaaacg ctgttggatt tatgcctaac 1500

ctatcagctt atccaaaatc tcacggtaaa actgccaaaa gtaacattgt cagtcaagtt 1560

tacttaaacg gagacaaaac taaacctgta acactaacca ttacactaaa cggtacacag 1620

gaaacaggag acacaacttg tgactgccgc ggagactgtt tctgcccaag tgcatactct 1680

atgtcatttt catgggactg gtctggccac aactacatta atgaaatatt tgccacctcg 1740

agttacactt tttcatacat tgcccaagaa taag 1774

<210>11

<211>432

<212>DNA

＜213〉artificial sequence

<400>11

tttaccactc cctatcagtg atagagaaaa gtgaaagtcg agtttaccac tccctatcag 60

tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag aaaagtgaaa 120

gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc 180

tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa 240

gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagctcggt 300

acccgggtcg agtaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa 360

ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga 420

ccgatccagc ct 432

<210>12

<211>1008

<212>DNA

＜213〉artificial sequence

<400>12

atgtctagat tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc 60

ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca 120

ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta 180

gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt 240

aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat 300

ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta 360

tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt 420

actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca 480

cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa 540

ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa 600

cttaaatgtg aaagtgggtc cgcgtacagc cgcgcgcgta cgaaaaacaa ttacgggtct 660

accatcgagg gcctgctcga tctcccggac gacgacgccc ccgaagaggc ggggctggcg 720

gctccgcgcc tgtcctttct ccccgcggga cacacgcgca gactgtcgac ggcccccccg 780

accgatgtca gcctggggga cgagctccac ttagacggcg aggacgtggc gatggcgcat 840

gccgacgcgc tagacgattt cgatctggac atgttggggg acggggattc cccgggtccg 900

ggatttaccc cccacgactc cgccccctac ggcgctctgg atatggccga cttcgagttt 960

gagcagatgt ttaccgatgc ccttggaatt gacgagtacg gtgggtag 1008

Claims (7)

1. the application tumor-specific promoters is expressed the purposes of reporter gene in the preparation of the early diagnosis for the preparation of detecting circulating tumor cells, and the sequence of wherein said tumor-specific promoters is as shown in SEQ ID NO:8.

2. the purposes of claim 1, wherein said tumor-specific promoters is the former promotor of tissue or cellular type specific gene, or through the combination promotor of transformation.

3. the purposes of claim 2, wherein said tumor-specific promoters are human telomerase reverse transcriptase's promotors of transformation, the enhancer sequence that it contains human telomerase reverse transcriptase's promoter sequence and is derived from the CMV promotor.

4. the purposes of claim 3, its activity in normal cell of the specific activity of wherein said tumor-specific promoters in tumour cell want high.

5. the purposes of claim 1, wherein said reporter gene is a kind of gene usually used as the promoter activity indicator.

6. the purposes of claim 5, wherein said reporter gene is the gene of fluorescin.

7. the purposes of claim 5, wherein said reporter gene is selected from firefly luciferase gene, renilla luciferase gene, bacteria beta-galactosidase gene.

Images