CN1074459C - Adenovirus-thymidine kinase gene configuration, and its acquiring process and application - Google Patents
Adenovirus-thymidine kinase gene configuration, and its acquiring process and applicationInfo
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- CN1074459C CN1074459C CN98124960A CN98124960A CN1074459C CN 1074459 C CN1074459 C CN 1074459C CN 98124960 A CN98124960 A CN 98124960A CN 98124960 A CN98124960 A CN 98124960A CN 1074459 C CN1074459 C CN 1074459C
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- thymidine kinase
- adenovirus
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- recombinant adenovirus
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Abstract
The present invention relates to a configuration of an adenovirus-thymidine kinase gene and an obtaining method and the application thereof. A full member comprises an adenovirus vector, a thymidine kinase fragment and an RSV-LTP promoter, wherein the ribonucleotide length of the full member is 43Kb, the length of thymidine kinase is 2.8Kb, a starting fragment is 18 bp, and the sequence of the starting fragment is ATGGCTTCGTACCCCTGC. The configuration of an adenovirus-thymidine kinase gene carries out transfection by tumors of a human body, particularly local entity tumors, can kill tumor cells by aiming at specificity under ganciclovir participation, can transfect various solid tumors of human bodies, and particularly has a high antitumoral effect on Chinese people.
Description
The present invention relates to the preparation method and the purposes of a kind of recombinant adenovirus-thymidine kinase construct.
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 (Moolten etc. from doctor Moolten in 1986 in chance opportunity, Cancer Res1986, since 46:5276) thymidine kinase of hsv (TK) gene transfection tumour cell can make chemosensitivity increase, 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 herpes simplex virus thymidine kinase that made transfection also claims the suicide effect.
Obviously, how to obtain recombinant adenovirus-thymidine kinase construct and make its in clinical application, have high transfection efficiency, high expression level and preferably stability be the problem of biologist, physician's common concern.
The object of the invention provides a kind of recombinant adenovirus-thymidine kinase construct of optimization, obtains the method for this system and utilizes the resulting product of above-mentioned vector gene.
For achieving the above object, the present invention by the following technical solutions: a kind of recombinant adenovirus-thymidine kinase construct is characterized in that: the complete long 43Kb of construct comprises: the full gene group DNA of 5 type adenovirus except that E1a, E1b, E3 zone; Thymidine kinase gene is expressed segment, is about 2.8kb, comprising the RSV-LTR promotor, and thymidine kinase cDNA, long 2.3kb (its sequence is seen Fig. 3) opens beginning segment 18bp, its sequence A TGGCTTCGTACCCCTGC.A kind of method that obtains the said gene construct, it is characterized in that: be carrier with the recombinant adenovirus, cut, connect through continuous enzyme, to comprise RSV-LTR promotor and target gene thymidine kinase cDNA total length segment inserts in the recombinant adenoviral vector, through rotaring redyeing 293 cell and cultivate and to be increased, again product is screened with purifying to obtain this construct.
The invention will be further described below in conjunction with example.
Fig. 1: recombinant adenovirus-thymidine kinase construct
Fig. 2: recombinant adenovirus-thymidine kinase construct forms synoptic diagram
Fig. 3: thymidine kinase gene nucleotide sequence
1. the acquisition of recombinant adenovirus (ADV) carrier: the ADV slight human body adenovirus of promptly causing a disease, the adenovirus that this project adopts is the 5th type ADV, it is to be packaged in the pJM17 helper plasmid at first.The pJM17 plasmid is provided by doctor Graham that (Ontario, Canada), the pJM17 plasmid includes 5 type ADV full-length cDNAs and removes E1a in the genome, E1b, E3 coding region through the DNA recombinant technology.After a series of processing, become a kind of replication defective virus vector.Adopt ADV to have following tangible biology advantage as Vectors in Gene Therapy: 1. carefully act as a travelling agent and have pattern of infection widely except that lymphocyte, bone marrow matrix, 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. thymidine kinase gene full-length cDNA coded slices: (asking for an interview accompanying drawing 3)
3. recombinant adenovirus-thymidine kinase construct forming process (asking for an interview accompanying drawing 2):
(1) obtains and contain Bgl II/BamH I (purchase of U.S. GIBCO company) thymidine kinase (HSV-TK) gene segment; It is 18bp that thymidine kinase gene opens the beginning segment, and its sequence A TGGCTTCGTACCCCTGC, transcription termination signal are successive AAA tail band.
(2) utilize Xba I (purchase of U.S. GIBCO company) and Cla I (purchase of U.S. GIBCO company) site to cut pXCJL.1 plasmid (purchase of U.S. Clontch company), obtain the pXCJL.1 segment, in order to linking with the RSV-LTR promotor subsequently.
(3) adopt Xba I and Cla I restriction enzyme site that the pBR322 plasmid (available from U.S. Promega company) that includes RSV-LTR is handled equally, obtain the RSV-LTR segment.
(4) RSV-LTR is connected with pXCJL.1, produces the pXCJL.1/RSV novel plasmid, again purifying after BamH I enzyme is cut.
(5) thymidine kinase gene segment (Bgl II/BamH I) is connected with pXCJL.1/RSV, generation comprises RSV-LTR promotor and the pulsating transfection carrier of target gene thymidine kinase cDNA total length.
(6) the pJM17 plasmid includes 5 type ADV full-length cDNAs and removes E1a in the genome through the DNA recombinant technology, E1b, the E3 coding region, in the binding of the two ends of ADV full-length cDNA inversion terminal tumor-necrosis factor glycoproteins (invert terminal repeat squence is arranged, ITRs), and insert betwixt to duplicate and open the beginning site, plasmid can be increased (referring to Virology1988,163:614).
(7) pJM17 and pXCJL.1/RSV/HSV-TK plasmid are added in 293 cells of adherent growth and carry out cotransfection.Selecting for use 293 cells (ATCC-CRL1573) as virus replication clone, 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..Carry 5 type ADV E1 coding regions in 293 cells, duplicating of 5 type ADV provides E1 albumen, when pJM17 and pXCJL.1/RSV/HSV-TK plasmid homologous recombination take place during cotransfection in 293 cells, finally prepares recombinant adenovirus-thymidine kinase construct.
(8) 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.
(9) filter out in the qualified construct rotaring redyeing 293 cell of recombinant adenovirus-thymidine kinase gene, through cultivate (37 ℃, CO2), make its a large amount of amplifications, extract culture supernatant, 80, the 000g ultracentrifugation is isolated and be can be used for treating recombinant adenovirus-thymidine kinase construct product.
(10) product is carried out the gorgeous purifying of CsCl chlorination, the test of application plaque is measured PFU and is tired ,-80 ℃ of preservations.
Can utilize the supernatant liquor after recombinant adenovirus-thymidine kinase construct increases to produce gene prod, according to before preliminary clinical, showing in the preliminary experiment, the product of this recombinant adenovirus-thymidine kinase construct preparation has obvious anticancer purpose, security is good, it is convenient to use, and in wherein the preclinical phase of ovarian cancer being used, efficiently surpasses 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 is inserted the danger of people's sudden change, therefore, is applied to the clinical good development prospect that has, and particularly Chinese's solid tumor is had better effects.
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 realize by another principle " bystander effect ", even only there is 10% tumour cell to change thymidine kinase over to, after adding people's 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 phase Answer promoter and functional gene fragment length etc., make gene construct have specificity, prepared medicine But the multiple human body solid tumor of the purposes transfection of thing, for example: oophoroma, cervical carcinoma, prostate cancer, Gastrointestinal cancer, cutaneous melanoma and soft tissue neoplasm, and have better lethal effect, this product The expression rate of product reaches more than 95%, during the preclinical phase of oophoroma is used, efficiently surpasses 70%.
Claims (3)
1. recombinant adenovirus-thymidine kinase construct is characterized in that: the complete long 43Kb of construct comprises: the full gene group DNA of 5 type adenovirus except that E1a, E1b, E3 zone; Thymidine kinase gene is expressed segment, is about 2.8kb, and comprising the RSV-LTR promotor, long 18bp and sequence are the thymidine kinase cDNA that opens beginning segment and long 2.3Kb and have Fig. 3 sequence of ATGGCTTCGTACCCCTGC.
2. method of producing recombinant adenovirus according to claim 1-thymidine kinase construct, it is characterized in that: be carrier with the recombinant adenovirus, cut, connect through continuous enzyme, to comprise RSV-LTR promotor and target gene thymidine kinase cDNA total length segment inserts in the recombinant adenoviral vector, through rotaring redyeing 293 cell and cultivate and to be increased, again product is screened with purifying to obtain this construct.
3. be used to prepare the purposes of treatment ovarian cancer, cervical cancer, prostate cancer, gastrointestinal cancer, cutaneous melanoma and soft tissue neoplasm medicine according to the recombinant adenovirus-thymidine kinase construct of claim 1.
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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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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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| Publication Number | Publication Date |
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| CN1254759A CN1254759A (en) | 2000-05-31 |
| CN1074459C true CN1074459C (en) | 2001-11-07 |
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| CN98124960A Expired - Lifetime CN1074459C (en) | 1998-11-25 | 1998-11-25 | Adenovirus-thymidine kinase gene configuration, and its acquiring process and application |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995014102A1 (en) * | 1993-11-18 | 1995-05-26 | Rhone-Poulenc Rorer S.A. | Recombinant viruses coding for thymidine kinase in gene therapy |
| WO1995025807A1 (en) * | 1994-03-21 | 1995-09-28 | Regents Of The University Of Michigan | Inhibition of arterial smooth muscle cell proliferation |
| EP0707071A1 (en) * | 1994-08-16 | 1996-04-17 | Introgene B.V. | Recombinant vectors derived from adenovirus for use in gene therapy |
| AU6363996A (en) * | 1995-06-23 | 1997-01-22 | Rhone-Poulenc Rorer S.A. | Recombinant adenoviruses, use thereof for preparing aavs, complementary cell line, and pharmaceutical compositions containing said adenoviruses |
| 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 |
-
1998
- 1998-11-25 CN CN98124960A patent/CN1074459C/en not_active Expired - Lifetime
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995014102A1 (en) * | 1993-11-18 | 1995-05-26 | Rhone-Poulenc Rorer S.A. | Recombinant viruses coding for thymidine kinase in gene therapy |
| WO1995025807A1 (en) * | 1994-03-21 | 1995-09-28 | Regents Of The University Of Michigan | Inhibition of arterial smooth muscle cell proliferation |
| EP0707071A1 (en) * | 1994-08-16 | 1996-04-17 | Introgene B.V. | Recombinant vectors derived from adenovirus for use in gene therapy |
| AU6363996A (en) * | 1995-06-23 | 1997-01-22 | Rhone-Poulenc Rorer S.A. | Recombinant adenoviruses, use thereof for preparing aavs, complementary cell line, and pharmaceutical compositions containing said adenoviruses |
| 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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| Publication number | Publication date |
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| CN1254759A (en) | 2000-05-31 |
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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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