CN101173299A - Construction and application of tumour targeting gonad correlation viral vectors - Google Patents
Construction and application of tumour targeting gonad correlation viral vectors Download PDFInfo
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Abstract
The invention discloses a construction method of a tumor targeting adeno-associated virus vector system rAAV-hTERT-gene: a tumor specific hTERT is used to start the foreign genes carried by a sub-control-gland-associated virus to express to construct an adeno-associated virus vector system rAAV-hTERT-gene which has tumor targeting and can be inserted into a therapeutic gene with anticancer effect at will. The invention also discloses the tumor targeting adeno-associated virus rAAV-hTERT-hTRAIL constructed by the above method: the virus is an anti-tumor active hTRAIL gene with wide selectivity carried by a tumor targeting adeno-associated virus vector system rAAV-hTERT-gene. The invention has the advantages of constructing safe, high efficient tumor targeting adeno-associated viruses.
Description
Technical field
The invention belongs to biotechnology and field of gene, specifically, relate to targeting gonad correlation viral vectors rAAV-hTERT-hTRAIL and construction process and application that a specific specificity is expressed in tumour cell.
Background technology
Cancer for most of kinds, what still adopt at present is traditional remedies such as surgical operation, radiotherapy, chemotherapy etc., and in the face of a lot of advanced malignant tumors, it is at a loss what to do that traditional remedies seems, shows mainly that curative ratio is low, recurrence rate and mortality ratio height, prognosis be relatively poor.Gene therapy is a kind of biological high-technology scheme for the treatment of disease by modifying gene, and people lodge at high enthusiasm to genetic treatment of tumor, and 66% gene therapy clinical trial all is used for oncotherapy.Up to the present, genetic treatment of tumor research has obtained impressive progress; For malignant tumour, gene therapy can utilize the difference of molecular level between tumour cell and normal cell, significantly widens " the treatment window " of tumour, can be efficiently, optionally killing tumor cell or the transfer that stops growth of tumour cell and prevent tumour.
Yet through more than ten years more than 600 clinical trial protocol find that although gene therapy is very safe, its anti-tumour effect is very low, have in addition do not have any therapeutic action, this mainly is the problem of present stage gene therapy vector.Present carrier system is all tumour cells of transfection specifically in human body, therapeutic gene is efficiently expressed in tumour, and tumour is to comprise the rapid process of multistep that several genes changes, and the modification by term single gene is difficult to utterly destroy tumour.These a series of problems seriously hamper the clinical efficacy of therapy of tumor.Therefore development carrier system efficient, target transfection tumor cell is most important to present genetic treatment of tumor.
In the gene therapy vector that is widely used at present, virus vector comprises adenovirus (adenovirus, Ad), adeno-associated virus (adeno-associated virus, AAV), retrovirus (retrovirus) and slow virus (lentivirus) etc. be the most commonly used.AAV is noticeable owing to possess following characteristic.The first, the immune response that AAV causes is slight, and pathogenic is low, and report is not arranged so far, and it causes human any disease; The second, as a kind of defective virus, when non-auxiliary virus infects, can only be incorporated in the host cell DNA, be " hiding " Infection Status; The 3rd, adjustable point is integrated, and AAV uniquely a kind ofly is incorporated into eucaryon virus on No. 19 karyomit(e)s of people in the locus specificity mode, thereby avoids random integration and cause the potentially dangerous of mutant host cell; The 4th, host range is wide, comprise division stage cell and non-division stage cell; The 5th, the foreign gene that carries can be for a long time, stably express.Therefore, AAV becomes one of ideal carrier of present transgenosis.Yet, because the AAV2 shortage of host range and tissue or cell-specific widely, make its as carrier be used for clinical before and still have the low drawback of security, target and transfection efficiency during the clinical treatment test.Therefore, successfully be used for the research of oncotherapy,, must overcome these problems as possible, seek the best target strategy of the carrier mediated oncotherapy of AAV2 in order to reach maximum tumor-killing efficient and minimum cytotoxicity at AAV2.
Recently, become the focus of scientific research, and obtained new breakthrough with the viral therapy malignant tumour.Several viruses that clinical experiment is recently used, as adenovirus ONYX-015, CV706, hsv G207 and G1716 and retrovirus and adeno-associated virus etc., result of treatment is encouraging.What these viruses had can optionally duplicate in tumour cell, breed, the kill tumor cell, the progeny virus that discharges is diffused into the tumour cell that closes on, and causes a kind of chain reaction in tumor tissues, the whole tumours of final elimination, and in normal cell, do not duplicate; Have enter in the body after can make that the therapeutic gene of mediation is specific expresses in tumour cell, and in normal cell, do not express; The viral oneself protein that also has has the tumour cell cycle of prevention to be carried out, suppress tumor cell proliferation and prevents tumorigenic effect.
Implement the targeting gene therapy of tumour, at first will make up and to mediate specific in tumour cell, the expression of therapeutic gene and the harmless virus of normal cell.Therefore, need transform, utilize the entrained therapeutic gene of tumor-specific promoters control virus, thereby therapeutic gene is only expressed at specifically inside tumor cell gland relevant viral vector, and harmless to normal cell; Thereby improve the target and the curative effect of gene therapy, make gland relevant viral vector more effective and safe be applied to clinical.
Utilizing the entrained therapeutic gene of tumor-specific promoters control virus is one of method that makes up the tumor-targeting adeno-associated virus, it can make therapeutic gene express in tumour cell, and in normal cell, do not express, thereby greatly reduce the side reaction of therapy of tumor and strengthened its target and curative effect.In recent years studies show that Telomerase may be find so far one the new mark of tumour the most widely, plays an important role in cell immortalityization and tumor development process.Under physiological condition, normal cell does not mostly have telomerase activation; And the tumour cell Telomerase Activity height about 85-90%.And its active height is relevant with the grade malignancy of tumour, and the prompting Telomerase can be used as the good target spot of oncotherapy.Reverse transcriptase of telomere, promptly hTERT (human telomerase reverse transcriptase) is the nucleus of Telomerase mixture, its expression status and telomerase activation significant correlation are the key links of cell regulate and control telomerase activation.The hTERT expression of gene mainly is from the transcriptional level adjusted, transcribes rise in the tumour cell about 90%, and does not transcribe in the quiescent stage cell.Therefore, the hTERT promotor is used to control expression of exogenous gene, is expected to improve the target of therapy of tumor.
(human TNF-related apoptosis-inducing ligand hTRAIL) is tumour necrosis factor (Tumor necrosis factor, the TNF) family member who finds recently to the relevant apoptosis induction ligand of human tnf.TRAIL plays a role by the specific receptors approach, can optionally induce the kinds of tumor cells apoptosis, but does not influence the Growth and Differentiation of normal somatic cell.Its receptor expression and distribution characteristics have determined it that tumour cell is had selective killing effect.Compare with other members of TNF family, TRAIL only exists with film mating type in vivo.Experiment in vitro shows, film mating type TRAIL, the cell generation apoptosis of the special acceptor of abduction delivering TRAIL rapidly.Many experimentation on animalies show, TRAIL has the early stage incidence of the tumour of minimizing, suppresses tumor growth and improves the survival rate effect tumor models such as lymphoma, mammary cancer, liver cancer and colorectal carcinomas, and with traditional radiotherapy, chemotherapy synergy is arranged, might become a kind of new type antineoplastic medicine.
Summary of the invention
The technical problem to be solved in the present invention provides the construction process and the application of a kind of tumour targeting gonad correlation viral vectors system, adopts this method can construct safety, tumor-targeting adeno-associated virus efficiently.
In order to solve the problems of the technologies described above, the invention provides the construction process of a kind of tumour targeting gonad correlation viral vectors rAAV-hTERT-gene of system: usefulness tumour-specific hTERT promotor is controlled the entrained exogenous gene expression of adeno-associated virus, is built into to have tumor-targeting and can insert a kind of rAAV-hTERT-gene of gland relevant viral vector system with antitumous effect therapeutic gene arbitrarily.
As the improvement of above-mentioned construction process of the present invention, this method may further comprise the steps:
1), design hTERT promotor two ends Auele Specific Primer, use PCR method, be template with the DNA genome of human hepatoma cell strain SMMC-7721, the PCR product of gained be hTERT promotor-378～+ the 78nt sequence; Shown in SEQ IDNO:1;
2), remove, and replace to above-mentioned hTERT promotor, obtain tumour targeting gonad correlation viral vectors pAAV-hTERT-gene with the promotor CMV of restriction enzyme with gland relevant viral vector pAAV-MCS;
3), utilize the adeno-associated virus recombination system, pAAV-hTERT-gene, pAAV-RC and pHelper three plasmid co-transfection AAV293 cells obtain the rAAV-hTERT-gene of tumour targeting gonad correlation viral vectors system.
The present invention also provides the tumor-targeting adeno-associated virus rAAV-hTERT-hTRAIL that makes up according to the method described above, and this virus is that the rAAV-hTERT-gene of tumour targeting gonad correlation viral vectors system carries the hTRAIL gene (being people's trail dna) with extensive selectivity anti-tumor activity.
The present invention also provides the construction process of above-mentioned tumor-targeting adeno-associated virus rAAV-hTERT-hTRAIL, may further comprise the steps:
1), design hTRAIL gene two ends Auele Specific Primer, design special restriction endonuclease sites thereon and be convenient to the clone, the method for using RT-PCR clones total length people hTRAIL gene from healthy human peripheral blood;
2) obtain containing the carrier pAAV-hTERT-hTRAIL of hTRAIL gene, the hTRAIL gene clone is gone among the pAAV-hTERT-gene;
3), utilize the adeno-associated virus recombination system, pAAV-hTERT-hTRAIL, pAAV-RC and pHelper three plasmid co-transfection AAV293 cells obtain tumor-targeting Recombinant rAAV-hTERT-hTRAIL.
Because the hTERT promotor is a tumor-specific promoters widely, and expression of exogenous gene is limited in the tumour cell, therefore in the present invention, the hTERT promotor is set at the outstanding candidate's promotor that makes up the tumour-specific adeno-associated virus.The present invention adopts the method for gene clone to transform 2 type gland relevant viral vectors (AAV2), replace the CMV promotor with the hTERT promotor, made up a new rAAV-hTERT-gene of tumour targeting gonad correlation viral vectors system, this system can mediate therapeutic gene specific efficient in tumour cell and express, and in normal cell, not expressing, thereby killing tumor cell.
Utilization of the present invention has people's trail dna of selectivity antitumor action as therapeutic gene, and under the carrying of cancer target carrier system rAAV-hTERT-gene, comprehensively both advantages have made up Recombinant rAAV-hTERT-hTRAIL.Confirm that after deliberation this method can largely improve the target of adeno-associated virus, thereby improve the security of gland relevant viral vector and gene therapy, lay the first stone for using safely clinically.
The objective of the invention is to the advantage of the high efficiency of the target of tumour-specific hTERT promotor and adeno-associated virus is combined, regulation and control therapeutic gene expression, thereby provide a kind of safer, the tumour targeting gonad correlation viral vectors system is the rAAV-hTERT-gene system efficiently.Another object of the present invention is the high efficiency advantage of the target of tumour-specific hTERT promotor and hTRAIL oncotherapy is combined in, thus provide a kind of safer, tumor-targeting adeno-associated virus rAAV-hTERT-hTRAIL is used for the treatment of the purposes of tumour efficiently.
Therapeutic gene can carry in rAAV-hTERT-gene of the present invention system, to reach safer, to treat the purpose of tumour efficiently, and therefore can be as a kind of more effective safer gene therapy vector platform; Tumor-targeting adeno-associated virus rAAV-hTERT-hTRAIL of the present invention has tumor-targeting and oncotherapy effect, can be used to develop the PTS of effective treatment tumour as a kind of tumor-targeting gene therapy reagent.
In sum, the present invention has following beneficial effect:
1, the present invention organically combines target and the high efficiency of therapeutic gene hTRAIL and the specificity of gland relevant viral vector of tumor-specific promoters hTERT, further improve target, security and the high efficiency of therapy of tumor, can be used for the treatment of multiple malignant tumour;
2, the invention provides a kind of tumor-targeting adeno-associated virus gene therapy vector system: the rAAV-hTERT-gene system, various therapeutic genes can carry in this system, and are safer, efficient;
3, the invention provides the hTRAIL therapeutic gene is used for the treatment of tumour under the mediation of tumour targeting gonad correlation viral vectors system high efficiency;
4, the tumor-targeting adeno-associated virus rAAV-hTERT-hTRAIL of the present invention's structure has very high target and curative effect, proves optionally kill tumor cell through cell experiment and animal experiment, and normal cell is not almost had toxicity; The tumor growth that can suppress tumor bearing nude mice effectively is for the treatment cancer provides a good new technique platform and candidate PTS, for therapy of tumor is from now on laid a solid foundation.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is the AAV carrier that carries the hTRAIL gene of hTERT promotor control and the structure sketch of contrast virus vector (being rAAV-hTERT-hTRAIL and rAAV-hTERT-EGFP); The wherein expression of hTERT promotor control therapeutic gene;
Fig. 2 is that the SDS-PAGE electrophoresis of tumour targeting gonad correlation viral vectors rAAV-hTERT-hTRAIL of the present invention and control vector rAAV-hTERT-EGFP is identified synoptic diagram; Among the figure: 1 represents rAAV-hTERT-EGFP, and 2 represent rAAV-hTERT-hTRAIL; M represents Protein Marker;
Fig. 3 observes the expression figure of green fluorescence down for fluorescent microscope behind rAAV-hTERT-EGFP the infects different cell strains.Among the figure: a is normal cell strain L02; B, c, d are tumor cell line, and wherein b is SMMC-7721, and c is SGC-7901, and d is A549;
Fig. 4 is the external specificity kill and wounding effect figure to tumour cell of rAAV-hTERT-hTRAIL;
Fig. 5 is that apoptotic flow cytometer detects figure.Hepatoma cell strain SMMC-7721 is by the treatment of equivalent virus rAAV-hTERT-hTRAIL and contrast viral rAAV-hTERT-EGFP infection after three days, and the hypodiploid peak of its apoptosis is detected in PI dyeing back by FACS; Among the figure: a is the PBS contrast, and b is that rAAV-hTERT-EGFP infects, and c is that rAAV-hTERT-hTRAIL infects;
Fig. 6 is the result schematic diagram of tumor-targeting adeno-associated virus rAAV-hTERT-hTRAIL at nude mouse internal therapy liver cancer cell transplanted tumor.
Embodiment
The invention will be further described below in conjunction with specific embodiment.Should be understood that following examples only to be used to the present invention is described and be not used in the scope of the present invention that limits.
1, design primer:
hTERT：
sense：5’-TGGCCCCTCCCTCGGGTTAC-3’
antisense(EcoR?I)：5’-CCC
GAATTCCGCGGGGGTGGCCGGGGCCA-3’
hTRAIL：
sense(EcoR?I)：5’-CTC
GAATTC?ATGGCTATGATGGAGGTCCAG-3’
antisense(Hind?III)：5’-CCT
AAGCTTCAGGTC?AGTTAGCCAACTAA-3’
EGFP：
sense(EcoR?I)：5’-CTC
GAATTC?ATGGTGAGCAAGGGCGAGGAG-3’
antisense(Hind?III)：5’-CCT
AAGCTTTTATCTAGATCCGGTGGATC-3’
2, the structure of relevant plasmid:
One, the structure of pAAV-hTERT-hTRAIL:
1), utilize above-mentioned hTERT promotor two ends Auele Specific Primer: 5 '-GCTCCCAGTGGATTCGCG-3 ' (sense) and 5 '-CCC
GAATTCCGCGGGGGTGGCCGGGGCCA-3 ' (antisense contains EcoR I restriction enzyme site) is a template with the total DNA genome that extracts human hepatoma cell strain SMMC-7721, by RT-PCR method amplification hTERT promotor, the PCR condition: 94 ℃ * 1min, 55 ℃ * 2min, 72 ℃ * 1min.The PCR product of gained be hTERT promotor-378～+ the 78nt sequence; This sequence is specifically shown in SEQ ID NO:1;
2), the promotor CMV of gland relevant viral vector pAAV-MCS (this vector plasmid that can select for use this laboratory to preserve) is removed with restriction enzyme SnaB I and EcoR I, simultaneously with above-mentioned 1) resulting hTERT promotor PCR product cuts with EcoR I enzyme, two kinds of products after enzyme cut connect, promptly carrier promotor CMV is replaced to the hTERT promotor, obtain tumour targeting gonad correlation viral vectors pAAV-hTERT-gene;
3), utilize above-mentioned hTRAIL gene two ends Auele Specific Primer: 5 '-CTC
GAATTCATGGCTATGATGGAGGTCCAG-3 ' (sense contains EcoR I site) and 5 '-CCT
AAGCTTCAGGTCAGTTAGCCAACTAA-3 ' (antisense contains Hind III site) is a template with the genome DNA of extracting from healthy human peripheral blood, increase by the RT-PCR method, the PCR condition: 94 ℃ * 1min, 55 ℃ * 1min, 72 ℃ * 1min30s.The product of amplification is the hTRAIL gene;
4), with above-mentioned 2) the tumour targeting gonad correlation viral vectors pAAV-hTERT-gene that obtains is behind EcoR I and HindIII double digestion, with above-mentioned 3) the EcoR I of PCR product hTRAIL gene is connected with Hind III double digestion product, obtains containing the vector plasmid pAAV-hTERT-hTRAIL of hTRAIL gene.
Two, the structure of pAAV-hTERT-EGFP:
1), utilize above-mentioned EGFP gene two ends Auele Specific Primer:
5 '-CTC
GAATTCATGGTGAGCAAGGGCGAGG AG-3 ' (sense contains EcoR I site) and 5 '-CCT
AAGCTTTTATCTA GATCCGGTGGATC-3 ' (antisense contains Hind III site) is a template with plasmid pEGFP-C1 (this plasmid is preserved for this laboratory), increase by PCR method, the PCR condition: 94 ℃ * 1min, 55 ℃ * 1min, 72 ℃ * 1min.The product of amplification is the EGFP gene;
2) the tumour targeting gonad correlation viral vectors pAAV-hTERT-gene that, the front has been made up is behind EcoR I and HindIII double digestion, with be connected with segment behind the Hind III double digestion PCR product EGFP gene through EcoR I, obtain containing the vector plasmid pAAV-hTERT-EGFP of EGFP gene;
3, the structure of tumour targeting gonad correlation viral vectors:
The production of gland relevant viral vector is carried out according to three plasmid co-transfection methods, and is as described below.With above-mentioned constructed plasmid carrier pAAV-hTERT-EGFP (or pAAV-hTERT-hTRAIL) and pAAV-RC and pHelper (these two kinds of plasmids are available from U.S. stratagene company) with 1: 1: 1 ratio, respectively get 10 μ g, be laid on the AAV293 cell of 10cm culture dish with the method transfection of calcium phosphate, change liquid after 6 hours, collecting cell and supernatant after 66-72 hour, the cesium chloride purified virus, the purified virus liquid that obtains is respectively rAAV-hTERT-EGFP and rAAV-hTERT-hTRAIL.Be illustrated in figure 1 as structure iron, Figure 2 shows that the purity evaluation figure of the virus behind the purifying.
The specific expressed analysis of reporter gene EGFP in gland relevant viral vector is carried at tumour cell and normal cell of embodiment 2, the mediation of hTERT promotor:
The survival rate of cell after virus treated detects (Cancer Research, 1989,49 (17): 4785-90) by mtt assay.Step is as follows: normal cell L02 and three kinds of tumour cell SMMC-7721, SGC7901, A549 are laid on respectively in 96 orifice plates with the amount of 5000 cells in every hole, cultivate after 24 hours respectively with 5 * 10
4/ cell adds two kinds of viruses (being rAAV-hTERT-hTRAIL and rAAV-hTERT-EGFP) cells infected, measures cell survival rate with MTT respectively after 1,2,3,4,5 day.Specific as follows, the nutrient solution that will contain virus is removed, and changes the normal nutrient solution that contains 5mg/ml MTT into, cultivates the nutrient solution that will contain MTT after 4 hours and removes, with the DMSO cracking, shake up, and be that reference is measured 595nm place absorbancy with 655nm place absorbancy then.
Cell survival rate (%)=A595 (sample)/A595 (contrast) * 100%.
Fig. 4 shows is the survival rate that mtt assay detected tumour cell and normal cell after different virus is handled 5 days.The result as shown in Figure 4, rAAV-hTERT-hTRAIL can specific killing tumour cell, and very little to normal cytotoxicity, have tumor-selective, and kill rate becomes positive correlation with action time.Contrast viral rAAV-hTERT-EGFP normal cell and tumour cell are not all had influence.
Treat viral rAAV-hTERT-hTRAIL and the viral rAAV-hTERT-EGFP of contrast respectively with 1 * 10
4After the dosage of/cell infected the SMMC-7721 tumour cell, collecting cell, PBS were washed once, and 70% ethanol fixedly is no less than 30min for 4 ℃.The centrifugal ethanol that goes, PBS washes once, with 37 ℃ of digestion of RNaseA 30min of 100 μ l 0.5g/L.(150mmol/LNaCl) effect 30min is with the apoptosis percentage of flow cytometer detection cell for 100 μ g/ml propidium iodides, 1%Triton X-100 to add 400 μ l propidium iodide (PI) dye liquors.
Result such as Fig. 5 show, rAAV-hTERT-hTRAIL infects and causes SMMC-7721 to produce significant apoptosis hypodiploid peak, contrast viral rAAV-hTERT-EGFP and PBS then seldom, illustrate that rAAV-hTERT-hTRAIL can come the kill tumor cell by the specific apoptosis pathway of inducing tumor cell.
With the 4-5 female nude mice subcutaneous vaccination hepatoma cell strain SMMC7721 in age in week, every mouse is 5 * 10
6Cell is worked as tumour and is reached 100～150mm after 12 days
3Carry out the animal grouping.The treatment group gives 5 * 10
10The rAAV-hTERT-hTRAIL of v.g/mice treats, two groups of contrast components, and the 1st group is phosphoric acid buffer (PBS) treatment group, and the 2nd group of rAAV-hTERT-EGFP with same dose treats, and test-results is as shown in Figure 6.
As seen from Figure 6, rAAV-hTERT-hTRAIL can significantly suppress growth of tumor than rAAV-hTERT-EGFP, and prolongs the tumor bearing nude mice life cycle.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ?ID?NO：1
tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60
ctcgctggcg tccctgcacc ctggggggag cgcgagcggc gcgcgggcgg ggaagcgcgg 120
cccagacccc cgggtccgcc cggagcagct gcgctgtcgg ggccaggccg ggctcccagt 180
ggattcgcgg gcacagacgc ccaggaccgc gctccccacg tggcggaggg actggggacc 240
cgggcacccg tcctgcccct tcaccttcca gctccgcctc ctccgcgcgg accccgcccc 300
gtcccgaccc ctcccggtcc ccgcccagcc ccctccggcc ctcccagccc ctccccttcc 360
tttccgcggc cccgccctct cctcgcggcg gcatttcagg cagccgtgcg tcctgctgcc 420
gacgtgggaa gccctggccc cggcaccccc gcgatg 456
Claims (4)
1. the construction process of the rAAV-hTERT-gene of tumour targeting gonad correlation viral vectors system, it is characterized in that: usefulness tumour-specific hTERT promotor is controlled the entrained exogenous gene expression of adeno-associated virus, is built into to have tumor-targeting and can insert a kind of rAAV-hTERT-gene of gland relevant viral vector system with antitumous effect therapeutic gene arbitrarily.
2. the construction process of the tumour targeting gonad correlation viral vectors rAAV-hTERT-gene of system according to claim 1 is characterized in that may further comprise the steps:
1), design hTERT promotor two ends Auele Specific Primer, use PCR method, be template with the DNA genome of human hepatoma cell strain SMMC-7721, resulting PCR product be hTERT promotor-378～+ the 78nt sequence;
2), remove, and replace to above-mentioned hTERT promotor, obtain tumour targeting gonad correlation viral vectors pAAV-hTERT-gene with the promotor CMV of restriction enzyme with gland relevant viral vector pAAV-MCS;
3), utilize the adeno-associated virus recombination system, pAAV-hTERT-gene, pAAV-RC and pHelper three plasmid co-transfection AAV293 cells obtain the rAAV-hTERT-gene of tumour targeting gonad correlation viral vectors system.
3. according to the tumor-targeting adeno-associated virus rAAV-hTERT-hTRAIL of claim 1 or 2 described methods structures, it is characterized in that: this virus is that the rAAV-hTERT-gene of tumour targeting gonad correlation viral vectors system carries the hTRAIL gene with extensive selectivity anti-tumor activity.
4. the construction process of tumor-targeting adeno-associated virus rAAV-hTERT-hTRAIL as claimed in claim 3 is characterized in that may further comprise the steps:
1), design hTRAIL gene two ends Auele Specific Primer, design special restriction endonuclease sites thereon and be convenient to the clone, the method for using RT-PCR clones total length people hTRAIL gene from healthy human peripheral blood;
2) obtain containing the carrier pAAV-hTERT-hTRAIL of hTRAIL gene, the hTRAIL gene clone is gone among the pAAV-hTERT-gene;
3), utilize the adeno-associated virus recombination system, pAAV-hTERT-hTRAIL, pAAV-RC and pHelper three plasmid co-transfection AAV293 cells obtain tumor-targeting Recombinant rAAV-hTERT-hTRAIL.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103055325A (en) * | 2011-10-20 | 2013-04-24 | 中国科学院上海生命科学研究院 | Specific gene-virus therapeutic drug for colorectal cancer |
| CN103288966A (en) * | 2013-05-17 | 2013-09-11 | 华侨大学 | Fusion receptor and gene medicine thereof for treating colorectal cancer |
| CN108330147A (en) * | 2017-01-20 | 2018-07-27 | 上海吉凯基因化学技术有限公司 | A kind of foundation of recombined glandulae correlation viral vectors production technology |
| CN109182279A (en) * | 2018-08-09 | 2019-01-11 | 湖北科技学院 | A kind of the novel oncolytic virus and its construction method of selectively killing tumor stem cell |
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| CN103055325A (en) * | 2011-10-20 | 2013-04-24 | 中国科学院上海生命科学研究院 | Specific gene-virus therapeutic drug for colorectal cancer |
| CN103288966A (en) * | 2013-05-17 | 2013-09-11 | 华侨大学 | Fusion receptor and gene medicine thereof for treating colorectal cancer |
| CN103288966B (en) * | 2013-05-17 | 2015-01-21 | 华侨大学 | Fusion receptor and gene medicine thereof for treating colorectal cancer |
| CN108330147A (en) * | 2017-01-20 | 2018-07-27 | 上海吉凯基因化学技术有限公司 | A kind of foundation of recombined glandulae correlation viral vectors production technology |
| CN109182279A (en) * | 2018-08-09 | 2019-01-11 | 湖北科技学院 | A kind of the novel oncolytic virus and its construction method of selectively killing tumor stem cell |
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