CN1254759A - Adenovirus-thymidine kinase gene configuration, and its acquiring process and application - Google Patents
Adenovirus-thymidine kinase gene configuration, and its acquiring process and application Download PDFInfo
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- CN1254759A CN1254759A CN 98124960 CN98124960A CN1254759A CN 1254759 A CN1254759 A CN 1254759A CN 98124960 CN98124960 CN 98124960 CN 98124960 A CN98124960 A CN 98124960A CN 1254759 A CN1254759 A CN 1254759A
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- 108020004440 Thymidine kinase Proteins 0.000 claims abstract description 17
- 102000006601 Thymidine Kinase Human genes 0.000 claims abstract description 14
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Abstract
The present invention relates to an adenovirus-thymidine kinase gene configuration,and its obtaining method and application. Whole configuration is composed of adenovirus carrier, thymidine kinase fragment and RSV-LTP promotor. The length is 43 Kb for the ribonucleotide of whole configuration, 2.8 Kb for the thymidine kinase, and 18 bp for initial fragment. Its sequence is ATGGCTTCGTACCCCTGC. Through human tumor transfection, especially the transfection to local actual tumor and participation of ganciclovir, it can specifically kill tumor cells, especially for Chinese.
Description
The present invention relates to a kind of gene configuration, particularly a kind of adenovirus-thymidine kinase gene configuration and preparation method and application.
As everyone knows, malignant tumour is the primary disease of harm humans health and life at present, and annual China dies from more than about 1,300,000 people of patient of malignant tumour.The four big methods that are used for the treatment of tumour at present are: operation, chemotherapy, radiotherapy and immune composite treatment, though they can partly improve patient's prognosis, but do not obtain substantial progress as yet, and gene therapy is just occurring with huge vitality and brand-new looks, and the developing direction of next century radical cure tumour has been represented in its development.
Find (people such as Moolten in chance opportunity from doctor Moolten in 1986, Cancer Res1986, since 46:5276) thymidine kinase of hsv (TK) gene transfection tumour cell can make it that chemosensitivity is increased, people study the antitumous effect of TK gene, the ultimate principle of recognizing its treatment tumour is that thymidine kinase can be with nontoxic ganciclovir metabolism is the cytotoxicity product to mammalian cell, the tumour cell spontaneous death in the presence of ganciclovir of simple sore exanthema virus thymidine kinase that made transfection also claims the suicide effect.
Obviously, how to obtain adenovirus-thymidine kinase gene and make that it has high transfection efficiency, high expression level in clinical application, stability is the problem of biologist, physician's common concern preferably.
The object of the invention provides a kind of adenovirus-thymidine kinase gene configuration of optimization, the product that obtains the method for this configuration and utilize the said gene configuration to obtain.
For achieving the above object the present invention by the following technical solutions: a kind of adenovirus-thymidine kinase gene configuration, it is characterized in that: full member comprises adenovirus carrier, thymidine kinase segment and RSV-LTP promotor, the complete long 43Kb of member Nucleotide, the long 2.8Kb of thymidine kinase, open the long 18bp of beginning segment, its sequence A TGGCTTCGTACCCCTGC.
A kind of method that obtains the said gene configuration, it is characterized in that it comprises: (1) is that ATGGCTCGTCCCTGC opens the beginning segment and induces down in sequence, at external and ADV carrier thymidine kinase gene cDNA, through enzyme cut, binding, cotransfection make the RSV-LTP promotor and the thymidine kinase segment is inserted in the adenovirus carrier, (2) the elementary member in (1) is combined once more with helper plasmid PJM17, make member can in 293 cells, cultivate amplification after the binding moulding.
The invention will be further described below in conjunction with embodiment.
Fig. 1: adenovirus-thymidine kinase gene configuration figure
Fig. 2: DNA connects and the vector construction synoptic diagram
Fig. 3: TK gene nucleotide series (before should contain a plurality of A and tail band 2.8Kb altogether)
1, the promptly pathogenic slight human body adenovirus of the acquisition of ADV adenovirus carrier: ADV, the ADV that this project adopts removes Ela in the genome through the DNA recombinant technology, E1b, the E3 coding region, total length 7.5Kb becomes a kind of replication defective virus vector.Adopt ADV to have following tangible biology advantage as Vectors in Gene Therapy: (1) has pattern of infection widely except that lymphocyte, marrow stromal cell, its infection need not be in division stage by cell; (2) ADV changes that the form with the genome exosome exists behind the cell over to, does not have the genome of insertion and causes sudden change, carcinogenic danger; (3) virus can be at replication in vitro; (4) the gene continuous expression can reach 2-3 month etc.
2, TK full length gene cDNA coded slices: (asking for an interview accompanying drawing 3)
3, DNA connects and vector construction:
(1) be that ATGGCTTCGTACCCCTGC opens pulsating inducing down of beginning in sequence, at external and PBR322 carrier, above-mentioned TK gene cDNA, cut through enzyme, used enzyme is 456 * bal, Bg111/BamHI, BamHI links, cotransfection makes the RSV promotor and thymidine kinase gene inserts in the adenovirus PBR322 carrier (seeing Fig. 1, Fig. 2).
(2) because above-mentioned member still lacks in replication in vitro amplification ability,, make member cultivate amplification after the binding moulding at 293 cells (a kind of HEKC) so elementary member is combined once more with helper plasmid (PFM17).Select for use 293 cells (ATCC-CRL1573) as virus replication clone, this clone is the transforming gene of V-type ADV, is international breeding isolated viral and the standard cell lines of carrying out ADV titer determination system, and this clone is introduced from the U.S..
(3) short in size might occur owing to member or inverted sequence forms no function member, so need screen member, screening process is carried out on agaropectin, cuts by enzyme subsequently, and RT-PCR detects dna sequencing, filters out the configuration that adheres to specification.
(4) the qualified member that filters out be inoculated in 293 cells and cultivate (37 ℃, CO
2), make its a large amount of amplifications, extract culture supernatant, 80, the 000g ultracentrifugation is isolated and be can be used for treating the adenovirus-thymidine kinase product.
(5) product is carried out C
sCl cesium chloride purifying, the test of application plaque is measured PFU and is tired, 80 ℃ of preservations.
Can utilize the supernatant liquor after the adenovirus-thymidine kinase gene member increases to produce gene prod, show in the preliminary experiment before preliminary clinical according to this adenovirus-thymidine kinase gene constructing system, no matter in tumor cell in vitro system or animal model, this gene prod has clearly, efficient resisting human tumor activity, security is good, it is convenient to use, in wherein the preclinical phase of ovarian cancer being used, efficiently surpass 70%, especially for multiple host's tumor by local direct injection, this product can not excise or excise incomplete local lesion to operation original result of treatment.Because carrier is a kind of human body associated virus of defective, no host gene inserts the danger of sudden change, therefore for being applied to the clinical good development prospect that has.The effective good effect of Chinese's solid tumor particularly.
Mechanism: is that carrier changes over to and makes it in the tumour cell to express with thymidine kinase by adenovirus, ganciclovir is used in adding of continuing, under it participates in, it is nucleoside triphosphate that thymidine kinase transforms single phosphoric acid nucleoside, and with tumor neogenetic DNA chain combination, disturb the synthetic of DNA and the kill tumor cell, and thymidine kinase gene antitumor cell effect can also be passed through another principle " bystander effect ", even only there is 10% swollen sick cell to change thymidine kinase over to, after adding ganciclovir, all the other tumour cells of 90% make its anti-tumour effect reach the amplification synergism because the connection of intercellular slit also will be killed.
Advantage of the present invention: the present invention selects nontoxic adenovirus vector by optimal design, selected corresponding function Genetic fragment length etc. make gene vector system have specificity, but the multiple human body solid tumor of transfection, for example: Oophoroma, cervical carcinoma, prostate cancer, gastrointestinal cancer cutaneous melanoma and soft tissue neoplasm, and have High antitumaous effect during the preclinical phase of oophoroma used, efficiently surpasses 70%, the expression rate of this product Reach more than 95%.
Claims (4)
1, a kind of adenovirus-thymidine kinase gene configuration, be applicable to the human tumor localized transfection, it is characterized in that: full member comprises adenovirus carrier, thymidine kinase segment and RSV-LTP promotor, the complete long 43Kb of member Nucleotide, the long 2.8Kb of thymidine kinase, opening the beginning segment is 18bp, its sequence A TGGCTTCGTACCCCTGC.
2, a kind of method that obtains the described gene configuration of claim 1, it is characterized in that it comprises: (1) is that ATGGCTCGTCCCTGC opens pulsating inducing down of beginning in sequence, at external and ADV carrier, TK gene cDNA, through enzyme cut, binding, cotransfection make the RSV-LTP promotor and the thymidine kinase segment is inserted in the adenovirus carrier, (2) the elementary member in (1) is combined once more with helper plasmid PJM17, make member can in 293 cells, cultivate amplification after the binding moulding.
3, the method for acquisition gene configuration according to claim 2 is characterized in that: described member is through screening process, and screening ties up on the agaropectin to be carried out, and obtains required correct gene configuration.
4, a kind of preparation that utilizes adenovirus-thymidine kinase gene to make solid carcinomas such as treatment ovarian cancer, cervical cancer, prostate cancer, gastrointestinal cancer, cutaneous melanoma and soft tissue neoplasm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98124960A CN1074459C (en) | 1998-11-25 | 1998-11-25 | Adenovirus-thymidine kinase gene configuration, and its acquiring process and application |
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|---|---|---|---|
| CN98124960A CN1074459C (en) | 1998-11-25 | 1998-11-25 | Adenovirus-thymidine kinase gene configuration, and its acquiring process and application |
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| CN1254759A true CN1254759A (en) | 2000-05-31 |
| CN1074459C CN1074459C (en) | 2001-11-07 |
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Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2712603B1 (en) * | 1993-11-18 | 1996-02-09 | Centre Nat Rech Scient | Recombinant viruses, preparation and use in gene therapy. |
| US6780406B1 (en) * | 1994-03-21 | 2004-08-24 | The Regents Of The University Of Michigan | Inhibition of vascular smooth muscle cell proliferation administering a thymidine kinase gene |
| AU711269B2 (en) * | 1994-08-16 | 1999-10-07 | Crucell Holland B.V. | Recombinant vectors derived from adenovirus for use in gene therapy |
| FR2735789B1 (en) * | 1995-06-23 | 1997-07-25 | Centre Nat Rech Scient | RECOMBINANT ADENOVIRUSES, THEIR USE FOR PREPARING AVA, COMPLEMENTARY CELL LINE AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
| US5772993A (en) * | 1997-01-21 | 1998-06-30 | The University Of Virginia Patent Foundation | Osteocalcin promoter-based toxic gene therapy for the treatment of calcified tumors and tissues |
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1998
- 1998-11-25 CN CN98124960A patent/CN1074459C/en not_active Expired - Lifetime
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Owner name: SHENZHEN TIANDAKANG GENE ENGINEERING CO., LTD Free format text: FORMER OWNER: MA DING Effective date: 20020118 |
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Effective date of registration: 20020118 Address after: 518057, B, building 710, building R3, hi tech Industrial Village, southern high tech Zone, Shenzhen Patentee after: Tiandakang Gene Engineering Co., Ltd., Shenzhen City Address before: 430030 Jiefang Avenue, Hubei, Wuhan 1095 Patentee before: Ma Ding |
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Granted publication date: 20011107 |