CN102206613A - Acquisition and use of tumor-selective replicative adenovirus - thymidine kinase gene construct - Google Patents

Abstract

Belonging to the genetic engineering field in medicine, the invention discloses a construction scheme for artificially modified human adenovirus type 5 (Ad5), and specific application of a tumor-selective replicative adenovirus construct in tumor treatment. With the techniques of PCR (polymerase chain reaction) amplifying fixed point deletion, enzyme cutting, connection, cloning, homologous recombination, transfection, single cloning purification of adenovirus and the like, a recombinant adenovirus construct can be acquired. Acquisition of the construct is characterized in that: 27 bases are deleted in 2(CR2) region of E1A conserved sequence of Ad5 genome; 28530-29360nt in 6.7k/gp19k gene in E3 region are deleted, and meanwhile a fragment of full-length cDNA(1131bp) of HSV-TK (herpes simplex virus thymidine kinase) is inserted into the deletion region. The construct of the invention is a novel oncolytic adenovirus carrier with high tumor-selective replication capability. With the suicide gene of HSV-TK and dissolubility of tumor cells by oncolytic viruses, the dual killing effects have unique utility value in tumor biotherapy.

Description

The acquisition and the purposes of tumor-selective replication type adenovirus-thymidine kinase gene construct

One, technical field

The present invention relates to a kind of human 5 type adenovirus (Human adenovirus type 5, Ad5) constructing plan of recombinant chou, its technical characterictic is: the genomic 920-946nt of directed disappearance Ad5, be GAT CTT ACC TGC CAC GAG GCTGGC TTT, this sequence encoding E1A protein 12 1 to 129 amino acids, on this basis, further lack Ad5E3 district 28530-29360nt, introduce a ClaI restriction enzyme site in above-mentioned disappearance zone; Insert the complete encoding sequence (1131bp of HSV-TK gene simultaneously in this disappearance district, concrete sequence is seen the nucleotide sequence table), thus the tumor-selective replication type adenovirus construct Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK that tumour is had therapeutic action obtained.Summary of the invention belongs to the cma gene field of engineering technology.

Two, background technology

Current, malignant tumour is one of the most serious public health problem of facing of the whole world, and China is one of occurred frequently, the high dead area of tumor disease especially.According to data analyses such as the report of American Cancer Society, international cancer research association (IARC) issue and health ministry statistical communiques: 2006, global new cases 1,230 ten thousand people, dead 7,600,000 people; China's new cases 2,380,000 people, dead 1,780,000 people constitute 26% of the cause of the death, have become the primary cause of the death of China resident, and therefore, tumor disease has had a strong impact on the health of China labor force population, and has restricted the Sustainable development of China's economy to a great extent.Scholarly forecast, morbidity in two, 30 years from now on and mortality ratio will continue to keep ascendant trend.

Through years development, tumor therapeuticing method mainly contains four kinds of operation, chemotherapy, radiotherapy and biotherapies etc. at present, and has formed more sophisticated treatment system, can reach the effect of clinical cure to the part tumor disease.Yet, all there is limitation separately in these four kinds of methods, can only improve the life quality of tumour patient to a certain extent and improve survival rate, development has been in plateau, and attempt by improve treatment plan, further to improve the potentiality of curative effect very limited, result of treatment is not fully up to expectations on the whole.Therefore, curative effect and even radical cure malignant tumour be further improve, new efficient methods of treatment and medicine demanded finding urgently.

The development pole the earth of modern molecular medicine has promoted the conceptual renewal of oncotherapy.We can say, following tumour effectively be controlled at the development and perfection that fundamentally depend on molecular therapy.As the gene therapy of one of main means of molecular therapy, begun to be widely used in the clinical trial treatment of kinds of tumors disease in recent years.As a kind of brand-new treatment pattern, gene therapy becomes effective treatment means that the tool of to more in the past " incurable disease " is wished gradually, demonstrates good potential applicability in clinical practice.In all gene therapy clinical trial protocols of implementing at present, oncotherapy accounts for 2/3, has become the most noticeable in the oncotherapy, progress and has studied frontier the most rapidly, has represented the developing direction of 21 century oncotherapy.Expert analyze that in 10 years from now on, gene therapy will develop into a sophisticated treatment approach, therefore, the application prospect of gene therapy antitumor drug and market potential will be very huge.

One of carrier that reorganization Ad5 carrier is most widely used as therapy of tumor, its Clinical feasibility and security have obtained generally acknowledged, and adenoviral gene treatment carrier has following outstanding biology advantage and clinical application advantage: pattern of infection is wide; Unconformability is gone into host genome, no mutagenesis, carcinogenic danger; Clinical application safety, side effect is slight; Clinical application is convenient; Adenovirus continuous expression time in cell paste is short, especially is fit to oncotherapy; The virus of clinical grade quantity, quality is easy to produce, purifying.

Clinical tumor most carriers on probation are the replication defective sexual gland virus that has lacked E1 and E3 district at present, after entering tumor focus, the killing effect of tumour is depended on the input of external high density treatment virus, the killing effect of the focus far away apart from injection point a little less than.For this reason, in recent years, occurred from the new development trend of replication defective sexual gland virus carrier to condition replicability adenovirus carrier (conditionally replicative adenovirus) conversion, so-called conditionality is duplicated adenovirus carrier and is meant that carrier can optionally duplicate propagation in tumour cell, with the place of tumour cell as production virus, finally reach cracking and kill the therapeutic purpose of tumour cell, after the tumour cell cracking discharges the virus of high density, can further infect the focus tumour cell far away apart from injection point, form the infection ripple of a new round, going round and beginning again forms a kind of positive regeeration, reaches maximum treatment effect.Examples of such carriers another very the ideal advantage be that they can not be finished in normal cell and duplicate, therefore normal cell is not had any lethal effect.In the 21st U.S.'s tumor research annual meeting, a research report from Houston Anderson tumor center shows: behind the local injection, the distribution of replication defective sexual gland virus in human malignant glioma focus be limitation, influenced the performance of curative effect to a certain extent, form a sharp contrast therewith, the adenovirus ONYX-015 that can duplicate at specifically inside tumor cell is by the propagation at tumor by local, its distribution spreads all over full knurl kitchen range, obtains significant curative effect.ONYX-015 be U.S. ONYX Pharma Biotech develop in recent years can be in tumour cell the adenovirus of copy choice, its structure characteristics are on the genome of wt-Ad5, the encoding sequence of excalation E1B 55kDa, and add the translation termination signal, but keep the encoding sequence of 19kDa E1B.

Since entering clinical trial in 1996, obtained very challenging clinical efficacy as the ONYX-015 of condition replicability adenovirus carrier representative products.At present, Pfizer (pifzer) pharmaceutical companies has been bought from ONYX biotech company and has been used patent, and carried out clinical phase test in kinds of tumors, wherein, to recurrent, the treatment of intractable tumor of head and neck is just being carried out the clinical III phase and is being tested, and has obtained very satisfied curative effect, and the clinical I-II phase of other tumours such as transitivity colorectal carcinoma, lung cancer, carcinoma of the pancreas, oral leukoplakia (oral leukoplakia) and glioblastoma is tested well afoot.The patent application of domestic existing 4 examples and ONYX-015 similar principles, wherein patent application (a kind of defective virus and construction process thereof) 99124030.8 is positioned at the 2809-3329bp encoding sequence by disappearance E1B 55kDa, and obtains the construct of E1B 55kDa excalation in the method for disappearance position insertion stop code.Patent application (structure of disappearance apoptosis suppressor virus and the application in therapy of tumor) 96013494.7 is by excalation E1B 55kDa encoding sequence, and at this disappearance position insertion marker gene LacZ, obtain E1B 55kDa excalation and carry the virus formulation body of marker gene simultaneously, another characteristics of this construct are disappearances that the E3 district is arranged.Other two applications for research group's submission of applicant leader, the patent No. 01144628.5 " a kind of acquisition and purposes that lacks the recombination adenovirus construction body of E1A encoding sequence ", disappearance E1A 382-1630nt sequence, filter out the recombination adenovirus construction body that to express the E1A functional protein, the patent No. 01144629.3 " a kind of structure and purposes of uniting disappearance 19kDa, 55kDa E1B encoding sequence recombination adenovirus construction body " filters out the recombination adenovirus construction body that can not express the E1B functional protein.

Domestic and international existing adenoviral gene treatment carrier is compared with the carrier in early stage, no matter treating on the principle, still all has tangible improvement on the actual therapeutic effect, and makes a breakthrough.Yet, still there is following shortcoming in they:＜1〉though possess the treatment advantage of tumour condition replicability based on the carrier of adenovirus Basic of Biology design, because all deficiencies in the design, the effectiveness of the adenovirus of obtaining duplicating in tumour cell, cracking tumour cell is weaker than wild-type adenovirus far away;＜2〉present adenoviral gene is treated in vivo active unstable or lower of carrier-bound exogenous promotor.

Three, summary of the invention

Based on above technical background and great medical requirement, the present invention will disclose a kind of constructing plan of new Ad5 gene therapy vector, the construct that obtains by this scheme has that tumour-specific duplicates, height tumour-specific mass expressing external treatment albumen, powerful remarkable advantages such as tumour-specific bystander effect, and possess the height therapeutic index, be suitable for intravenous administration, can solve crucial weak point in present oncotherapy technology and the practice.

Theoretical basis of the present invention is based on the deep understanding of existing research to the Ad5 viral biology.After Ad5 enters cell in 1 hour, Ad5 will give expression to the E1A functional protein, its biological effect mainly comprises two aspects, at first, E1A protein binding retinoblastoma mutator gene (Rb) albumen, host cell endogenous transcription factor E2F is discharged from the mixture of Rb-E2F, meanwhile, E1A albumen will activate Ad5 and give expression to the E1B functional protein, drive kinase whose inhibition with this deactivation host cell endogenous transcription factor P53 cell cycle, at E1A, under the common driving of E1B and E2F, host cell enters the DNA synthesis phase, for virus genomic self-replacation provides precondition; Secondly, Ad5 relies on the early gene (E1B that E1A albumen activates Ad5, E2, E3, E4) and the expression of late gene (L1-6), for the packing of virus genomic self-replacation, virion, the immune clearance of escaping host cell, lysing cell, releasing virus particle, complete life cycle such as infection provides the permission environment again.This shows that it is synthetic that Ad5 relies on other proteic sequential property of E1A protein promoter, finish the life cycle of virus, final cracking host cell.

The encoding sequence of functional protein E1A is positioned at Ad5 genome 560-1112nt, and 1229-1545nt comprises three important sequences, is respectively 677-799nt, and this sequence is E1A conserved sequence 1 (CR1), is used in conjunction with transcribing cofactor P ₃₀₀, 917-979 nt is E1A conserved sequence 2 (CR2), is used in conjunction with retinoblastoma mutator gene (Rb), 1007-1115nt is conserved sequence 3 (CR3), is the genetic transcription active region.Because all there is the defective of Rb regulation and control pathway in nearly all tumour, if the disappearance transformation different in addition to the CR2 district of E1A917-979nt, the effect that keeps the E1A transcripting activating characteristic when reaching the proteic RB binding characteristic of deactivation E1A as far as possible, the adenovirus recombinant chou that obtains thus might effectively duplicate in tumour cell, finally kill tumour cell, on the other hand, the Rb regulation and control pathway that normal cell has, can effectively check the life cycle of its sequential property of adenovirus recombinant chou dependence E1A protein promoter, finally only form abortive infection.Above-mentioned theory is confirmed by numerous institutes.

The new A d5 recombinant chou that we make up, its technical characterictic is: the genomic 920-946nt sequence of directed disappearance Ad5, and further lack Ad5 E3 district 28530-29360nt, introduce a ClaI restriction enzyme site in this disappearance zone; In the ClaI restriction enzyme site, insert the complete encoding sequence (1131bp) of HSV-TK gene, thereby obtain the tumor-selective replication type adenovirus construct Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK that tumour is had therapeutic action.

This carrier has been represented the up-to-date direction of domestic and international adenoviral gene treatment study on the carrier, has following biological characteristics and treatment advantage:＜1〉selectivity expressed of tumour cell copy choice and therapeutic gene efficiently;＜2〉therapeutic gene can obtain instant expression behind the virus infection tumour cell, at the producible viral therapy circle of tumor by local, unites with the advantage of treatment target spot after the tumour cell cracking in addition, can form onlooker's effect on potent side;＜3〉a large amount of treatment target genes are transcribed out in the efficient amplification of tumour cell inner virus simultaneously, allow tumour cell become treatment albumen synthetic " source mill ";＜4〉anticancer spectrum is wide;＜5〉the body inner model to animal model for tumour studies confirm that, by locally injected into tumor or intravenous route of administration, all animal models for tumour equal tool significant therapeutic action of this tumor-selective replication type adenovirus construct to being tried, and can effectively suppress metastases, and it is xicity related that the treatment animal is not had obvious treatment.

Four, description of drawings

Accompanying drawing 1: tumor-selective replication type adenovirus construct Ad5/ Δ E1/ Δ 6.7k/gp19k/HSV-TK E3 plot structure synoptic diagram, 7 kinds of albumen of this regional code, the zone that lacks is 28530-29360bp, be the E36.7k/gp19k gene, insert the complete encoding sequence (1131bp) of HSV-TK gene in this disappearance zone.

Accompanying drawing 2: tumor-selective replication type adenovirus construct Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK structural representation.

Five, specific embodiment

In the following example, except that indicating especially, used enzyme and PCR primer are all available from U.S. Gibco company in the techniqueflow.

The structure of example 1, pXC1 series mutation body (Δ 920-946pXC1)

PXC1 purchase in Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number (Cat.No.): PD-01-03), this plasmid comprises human 5 type adenovirus (Ad5) 22-5790nt sequences.

Adopt 3 PCR methods disappearance 920-946nt, the obtaining of fragment 1: primer 1=5 '-CG GGA TCCGGG CCC CCATTT CC-3 ' is equivalent to 9883-9902nt, and underscore partly is the BamHI restriction enzyme site; Primer 2=5 '- GTC ACT GGGTGG ATC GAT CAC CTCCGG TAC-3 ' is equivalent to 922-905nt, and underscore partly is and primer 3 complementary portions; With pXC1 is template, the performing PCR reaction, and the reaction system cumulative volume is that 100 μ l comprise:

Contain MgCl ₂10 * PCR damping fluid, 10 μ l

2mM?dNTP 10μl

10 μ M primers, 11 μ l

10 μ M primer 2s, 1 μ l

pXC1?10ng/μl 1μl

Pfu high-fidelity Taq enzyme 2.5u

Add water to 100 μ l;

Reaction conditions is: 95 ℃ 30 seconds; 95 ℃ 45 seconds, 60 ℃ 1 minute, 72 ℃ totally 28 circulations in 2 minutes; 72 ℃ were extended 10 minutes.The long 940bp of PCR product forms fragment 1, and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.

Obtaining of fragment 2: primer 3=5 '- GAG GTG ATC GAT CCA CCC AGT GACGAC GAG-3 ' is equivalent to 911-947nt, and underscore partly is and the primer 2 complementary portion; Primer 4=5 '-TGC TCT AGACAC AGG TGATGT CG-3 ' is equivalent to 1344-1325nt, and underscore partly is the XbaI enzyme cutting site;

With pXC1 is template, the performing PCR reaction, and reaction conditions is the same, and the long 400bp of product forms fragment 2, and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.

Obtaining of fragment 3: 50ng/2 μ l fragment 1 is mixed with 25ng/1 μ l fragment 2, and as the reaction of template performing PCR, upstream primer is a primer 1, downstream primer is a primer 4, reaction conditions is the same, and product is about 1400bp, forms fragment 3, with QIAquick8PCR product purification test kit (QIAGEN, German Cat:28142) behind the purifying, uses BamHI, the XbaI double digestion spends the night, and enzyme is cut product recovery endonuclease bamhi after 1% agarose gel electrophoresis separates and is used for the clone.

With pXC1 BamHI, the XbaI double digestion spends the night, and enzyme is cut product and separated back generation 2 bands at 1% agarose gel electrophoresis, is about 1400bp and 8500bp, reclaims the 8500bp endonuclease bamhi and is used for the clone.

Get 8500bp pXC1 endonuclease bamhi and the 90ng fragment 3 of 40ng, do ligation with DNA T4 ligase enzyme, get 1.5 μ l and transform 100 μ l DH5 α competence bacteriums, shop ware incubated overnight, second day single colony clone of picking, extract the plasmid of amplification in it, the dna sequencing evaluation and screening obtains lacking pXC1 plasmid encoding mutant body-Δ 920-946pXC1 of 121-129AA 920-946nt, is used for the clone of recombinant adenovirus.

The structure of example 2, Δ 920-946Ad5 recombinant adenovirus

PBHGE3 purchase in Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number (Cat.No.): PD-01-12), this plasmid comprises the full gene group sequence except that Ad5 packaging signal (194-358nt).

When pBHGE3 obtained from Microbix Biosystem Inc., total amount was 10 μ g, and first electricity changes the competence bacterium over to, and the picking positive colony extracts plasmid, the plasmid CsCl that obtains ₂-EB ultracentrifugation purifying.

Homologous recombination method obtains Δ 920-946Ad5 recombination adenovirus construction body, and method is as follows:

In the 15cm culture dish, plant 7.5 * 10 ⁵293 cells, nutrient solution are 10%FBS DMEM, and by second day, cell should be 1-1.5 * 10 ⁶, about 70% cytogamy; Preceding 3-4 hour of transfection changes fresh medium into.

Preparation cotransfection DNA-calcium phosphate solution: with 2 * HBS (280mM NaCl, 43mMHEPES, 10mM KCl, the 10mM Na of 1600 μ l sterilization ₂HPO ₄.7H ₂O, 2%dextrose, pH 7.05-7.15)/pBHGE3 and each 42 μ g/ of Δ 920-946pXC1 add distilled water to the 2840 μ l mixing of sterilization, slowly add the CaCl of 50 μ l 2.5M ₂, put upside down mixing, at room temperature allow DNA/CaCl ₂Precipitate 45-60 minute, form slightly turbid precipitation.

Add the above-mentioned mixed solution of 500 μ l to 293 cells that contain 5ml 60mm culture dish, in 37 ℃, 5%CO2, hatched 4-6 hour, inhale and remove aforesaid liquid, wash once with PBS.

Handle 1-2 minute to promote transfection efficiency with the glycerine/DMEM that contains 15%, wash once, be changed to complete culture solution with PBS.

Prepare 1.8% LMP agarose, autoclaving is distributed into 5ml, melts in boiling water with preceding, and insulation is at 45 ℃, and the time spent adds equivalent 4%FBS DMEM, and the shop is gone in the culture dish at once.

Nutrient solution is removed in suction, adds the 5ml aforesaid liquid.Every 4-5 days, add the 3ml aforesaid liquid.

14-21 days, plaque occurred, selected 6-12 plaque.Plaque is transferred in the EP pipe of the 1.5ml that contains serum-free DMEM substratum, hatched 24 hours at 37 ℃.

In 24 orifice plate culture dish, plant 1 * 10 ⁵293 cells, nutrient solution are 10%FBS DMEM, and by second day, cell should be 2 * 10 ⁵, about 70% cytogamy is inhaled and is removed liquid, gets 100 μ l from above-mentioned Incubating Solution and (is approximately 10 ³Virus) add, rock liquid gently 3 times, at 37 ℃, 5%CO ₂In hatched 90 minutes.

Add perfect medium to 1ml, cell is positioned over 37 ℃, hatched among the 5%CO2 5-10 days, occur until CPE completely, so-called CPE, i.e. cellulotoxic effect, cell show as and become round, floating, cell based on kernel.If after 10 days, CPE does not occur completely, tiring of then prompting virus is too low, need carry out second amplification of taking turns.

Culture plate is carried out the circulation of freezing/thawing of three-wheel, discharge virus, lysate is collected in the 15ml test tube, maximum velocity centrifugation 10 minutes is collected supernatant liquor, freezes in-80 ℃, and this liquid is called s-generation virus, is approximately 5 * 10 ⁷/ ml virus.

Above virus is increased once more, at 75cm ²Plant 5 * 10 in the culture dish ⁶293 cells, nutrient solution are 10ml10%FBS DMEM, and by second day, cell should be 1 * 10 ⁷, about 70% cytogamy;

Preceding 3-4 hour of transfection changes fresh medium into;

Get 1ml s-generation virus storage liquid and add perfect medium, be used for transfection to 1ml; This MOI is about 5; Remove the liquid of 75cm2 culture dish, add above liquid, shake gently three times; In 37 ℃, 5%CO2, hatched 90 minutes; Add 9ml 2%FBS DMEM, in 37 ℃, 5%CO2, hatched 4-7 days, be used to extract the screening that viral DNA is used for positive-virus.

Because 293 cellular genome comprise complete e1a gene, pollute 293 cell DNAs easily during extraction positive-virus DNA, cause and identify failure, Δ 920-946Ad5 is increased once in tumour cell Hela more for this reason, be used for identifying that step is as follows:

In 6 orifice plate culture dish, plant 1 * 10 ⁵Hela cell, nutrient solution are 10%FBS DMEM, and by second day, cell should be 2 * 10 ⁵, about 70% cytogamy is inhaled and is removed liquid, gets 100 μ l from above-mentioned filtered solution and (is approximately 10 ³Virus) add, rock liquid gently 3 times, at 37 ℃, 5%CO ₂In hatched 90 minutes.

Add perfect medium to 1ml, cell is positioned in 37 ℃, 5%CO2 hatched 5-10 days, occur until CPE completely, cell is scraped, be collected in 1.5ml EP pipe, the centrifugal supernatant of abandoning, add 300 μ l PBS solution, carry out the circulation of freezing/thawing of three-wheel, discharge virus, with lysate maximum velocity centrifugation 10 minutes, collect supernatant liquor, freeze in-80 ℃, with the Qiagen test kit mini DNA isolation kit of company, DNA is extracted in the explanation of reference reagent box.With the viral DNA is template, the performing PCR reaction, upstream primer=5 '-CG GGA TCC GGG CCC CCA TTT CC-3 ', downstream primer=5 '-TGCTCT AGA CAC AGG TGA TGT CG-3 ', the reaction system cumulative volume is that 100 μ l comprise: contain 10 * PCR damping fluid, 10 μ l/2mM dNTP, 10 μ l/10 μ M upstream primers, the 1 μ l of MgCl2,10 μ M downstream primers, 1 μ l/ viral DNA 10ng/pfu high-fidelity Taq enzyme 2.5u/ adds water to 100 μ l.Reaction conditions is: 95 ℃ 30 seconds/95 ℃ 45 seconds, 60 ℃ 1 minute, 72 ℃ 2 minutes totally 28 circulations extended 10 minutes for/72 ℃.The PCR product is 1400bp, and behind the conventional electrophoretic separation purifying, detectable level is used for dna sequencing, and sequencing primer is: 5 '-AGCCGGAGCAGAGAGCCTTG-3 ', choose correct clone and be used to make up Ad5/ Δ E1/ Δ 6.7k/gp19k/HSV-TK recombinant adenovirus.

The structure of the subcloning vector pCDNA3.1-Δ 6.7k/gp19k in example 3, Ad5E3 district

Ad5E3 district full name is an Ad5 early region 3, this zone under the driving of endogenous promotor, the 7 kinds of albumen of encoding, sequence, structure, function be referring to accompanying drawing 1:12.5k, 27858-28179nt, function is not bright; 6.7k 27547-28736nt and RID complex body suppress 1,2 expression at cell surface of TRAIL acceptor together; Gp19k, 28735-29215nt in conjunction with MHC I class antigen, suppresses it to the presenting of cell surface, and escapes the removing of CTL; ADP, 29419-29770nt, lysing cell, releasing virus; RID α, 29784-30057nt forms mixture with RID β, prevents the cracking of TNF, removes FAS antigen; RID β, 30062-30458nt and 14.7k, 30453-30837 suppresses the cracking of TNF.

The purpose of this experiment is to lack 28530-29360nt zone, E3 district, and inserts exogenous therapeutic gene in the E36.7k/gp19k zone of recombinant adenovirus.

Adopt PCR method disappearance Ad5E3 district 28530-29360nt 3 times:

Obtaining of fragment 1: primer 1=5 '-ATACGCGCCCACCGAAAC-3 ' is equivalent to 27306-27323nt; Primer 2=5 '- AATCTATGGATATCGATTAGCTGCGCCTTTGGCCTA-3 ' is equivalent to 28511-28529nt, (underscore partly is and primer 3 complementary portions that ATCGAT is a Cla I restriction enzyme site);

With Ad5DNA is template, the performing PCR reaction, and the reaction system cumulative volume is that 100 μ l comprise:

Contain MgCl ₂10 * PCR damping fluid, 10 μ l

2mM?dNTP 10μl

10 μ M primers, 11 μ l

10 μ M primer 2s, 1 μ l

Ad5DNA?200ng/μl 1μl

Pfu high-fidelity Taq enzyme 2.5u

Add water to 100 μ l;

Reaction conditions is:

94 ℃ 30 seconds;

94 ℃ 30 seconds, 46 ℃ 1 minute, 72 ℃ totally 30 circulations in 1 minute;

72 ℃ were extended 10 minutes.

The PCR product is 1234bp (fragment 1), and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.

Obtaining of fragment 2: primer 3=5 '- ATCGACGCGGATCGATACTCTATGTGGGATATGCTC-3 ' (being equivalent to 2961-29380n) (underscore partly is and the primer 2 complementary portion that ATCGAT is a Cla I restriction enzyme site) primer 4=5 '-ATGTCTTTGAGGCTTGGAGG-3 ' (being equivalent to 30137-30118nt); The PCR reaction conditions is the same, and product is 794bp (fragment 2), and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.

Obtaining of fragment 3: with fragment 1 and fragment 2 balanced mix, as the reaction of template performing PCR, upstream primer is a primer 1, and downstream primer is a primer 4, reaction conditions is the same, product is about 2000bp (fragment 3), with QIAquick 8PCR product purification test kit (QIAGEN, German, Cat:28142) behind the purifying, cut with the EcoRI enzyme and to spend the night, enzyme is cut product and is separated the back at 1% agarose gel electrophoresis and reclaim, and endonuclease bamhi is used for follow-up ligation.

(Invitrogen, U.S.A. Cat:V79020) cut with the EcoRI enzyme and spend the night, and enzyme is cut product and separated the back at 1% agarose gel electrophoresis and reclaim endonuclease bamhi, is used for follow-up ligation behind the dephosphorylation with pCDNA3.1.

With PCR reaction product fragment 3 cut with enzyme, pCDNA3.1 is connected behind the dephosphorylation, get 1.5 μ l and change 100 μ l DH5 α competence bacteriums over to, the picking positive colony, plasmid is carried for a short time, the dna sequencing evaluation and screening obtains lacking pCDNA3.1 plasmid encoding mutant body-pCDNA3.1-Δ 6.7k/gp19k of 29477-29714nt, is used to insert the subclone of exogenous therapeutic gene.

Example 4, in the middle insertion HSV-TK full-length cDNA fragment of carrier pCDNA3.1-Δ 6.7k/gp19k

HSV-TK full length gene fragment is amplification from this breadboard first-generation adenovirus ADV-TK: upstream primer is 5 '-CC ATCGATATGGCTTCGTATCCCGGCC-3 ', this fragment is equivalent to the 1-19nt (underscore partly is Cla I restriction enzyme site) of HSV-TK cDNA encoding sequence; Downstream primer is 5 '-CC ATCGATTCAGTGAGCCGCCCCCATCT-3 ', this fragment is equivalent to the 1131-1112nt (underscore partly is Cla I restriction enzyme site) of HSV-TK cDNA encoding sequence, and amplified fragments comprises HSV-TK full-length cDNA encoding sequence, and total length is 1143bp.

The reaction system cumulative volume is that 100 μ l comprise:

Contain MgCl ₂10 * PCR damping fluid, 10 μ l

2mM?dNTP 10μl

10 μ M upstream primers, 1 μ l

10 μ M downstream primers, 1 μ l

ADV-TK?DNA 2μl

Pfu high-fidelity Taq enzyme 2.5u

Add water to 100 μ l;

Reaction conditions is:

94 ℃ 30 seconds;

94 ℃ 30 seconds, 52 ℃ 1 minute, 72 ℃ totally 30 circulations in 1 minute;

72 ℃ were extended 10 minutes.

The PCR product is 1143bp, after Cla I enzyme is cut, and conventional electrophoretic separation purifying, detectable level is used for follow-up reaction.After carrier pCDNA3.1-Δ 6.7k/gp19k uses Cla I enzyme tangent line shapeization, conventional electrophoretic separation purifying, be connected with HSV-TKcDNA behind the dephosphorylation, get 1.5 μ l and change 100 μ l DH5 α competence bacteriums over to, the picking positive colony, plasmid is carried for a short time, and the dna sequencing evaluation and screening obtains inserting the segmental plasmid encoding mutant body of HSV-TK full-length cDNA (1131bp)-pCDNA3.1-Δ 6.7k/gp19k/HSV-TK at Cla I restriction enzyme site, is used for the clone of follow-up recombinant adenovirus.

Example 5, tumor-selective replication type adenovirus Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK obtain

The connection method of setting up according to reference " Adenovirus methods and protocol; edited by William S.M.Wold; construction of mutations in theadenovirus early region 3 (E3) transcription units; 11-24 " (ligation protocol) of obtaining of tumor-selective replication type adenovirus Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK is carried out, and key step comprises the extraction of TP DNA; Be used for the preparation of recombination adenovirus construction E3 district DNA; Ligation; Cotransfection 293 cell homologous recombination; The picking of plaque and evaluation; The amplification of positive-virus and purifying.Detailed method is referring to the document.Obtain recombination adenovirus construction body Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK by above experimental procedure, this adenovirus has lacked Ad5920-946nt sequence (GAT CTT ACC TGC CAC GAG GCT GGC TTT), this sequence encoding E1A protein 12 1 to 129 amino acids, and disappearance Ad5 E3 district 28530-29360nt, in the ClaI restriction enzyme site, insert HSV-TK full length gene cDNA fragment, other genome structures and wt-Ad5 are identical, and its sequence and structure are seen accompanying drawing 2 and appendix 1.

The evaluation of example 6, tumor-selective replication type adenovirus Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK treatment effect

1, the evaluation of the tumor cell in vitro lethal effect of tumor-selective replication type adenovirus

The purpose of this experiment is to identify that Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK tumor-selective replication type adenovirus is to a series of tumours and Normocellular lethal effect, experiment will be with the positive contrast of wt-Ad5, with the negative contrast of replication-defective virus Ad5CMV-GFP.Selecting clone for use is human lung cancer cell line A549, the high metastatic prostate cancer clone of people PC3M-1E8, human osteoblast cell's oncocyte is U-2OS, CCL188 HCT-8, MCF-7 MCF-7, Proliferation of Human Ovarian Cell is SKOV-3, the human hepatoma cell line HepG2, human esophageal carcinoma cell line Eca-109, SGC-7901 MKN-45, human glioma cell line SHG-44, department of human head and neck squamous cell cancerous cell line AGZY-973, human pancreatic cancer cell BxPC-3, people's endometrial carcinoma cell is HEC-1-A, people's rectum cancer cell is that Colo320 and KB cell are CNE-2.The clone of selecting for use has different P53, the RB gene phenotype; Have different tissues-derivedly, so experimental result can be represented different types of tumors.The normal cell of selecting for use is lung tracheole epithelial cell, prostate epithelial cell, the BMNC in the vascular endothelial cell in former generation, former generation, selects for use principle to be: the normal tissue cell that will contact a large amount of adenovirus after the intravenous administration.

In 24 orifice plate culture dish, plant 1 * 10 ⁵Tumour or normal epithelium cell, nutrient solution are 10%FBS DMEM or RIPM1640, to second day about 70% cytogamy, inhale and remove liquid, and adding reaches 70% required MOI and is diluted to 100 μ l adding, rocks liquid gently 3 times, at 37 ℃, 5%CO ₂In hatched 90 minutes.

Add and to contain 10%FBS DMEM or RIPM1640 substratum and add ganciclovir (GCV) simultaneously to 2ml, cell is positioned among 37 ℃ of 5%CO2 hatched 3 days, flow cytometer detects the apoptosis rate of cell.

Experimental result shows, positive control wt-Ad5 non-selectivity ground cracking tumour and normal cell; The lethal effect of replication-defective adenoviral Ad5CMV-GFP pair cell very a little less than, tumor-selective replication type adenovirus construct Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK+GCV is killing tumor cell optionally, its effect significantly is better than the lethal effect of wt-Ad5 pair cell, and this tumor-selective replication type adenovirus construct is to the obviously influence of normal control cells system's nothing simultaneously.The result shows that fully tumor-selective replication type adenovirus construct Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK selectivity efficient kills tumour cell, and concrete data see the following form:

2, the evaluation of duplicating efficiency in the tumour cell of tumor-selective replication type adenovirus

For further carrying out detection by quantitative Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK tumor-selective replication type adenovirus construct duplicating efficiency in tumour cell, carried out following experiment:

Variously in 24 orifice plate culture dish go into 1 * 10 ⁵Various tumour cells or former generation normal cell, nutrient solution was 10%FBSDMEM or RIPM1640, by second day, about 70% cytogamy is inhaled and is removed liquid, adds Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK 10MOI and is diluted to 100 μ l adding, rock liquid gently 3 times, at 37 ℃, 5%CO ₂In hatched 90 minutes.

Add and to contain 2%FBS DMEM substratum, cell is positioned in 37 ℃, 5%CO2 hatched 3 days to 1ml.

Add 300 μ l PBS solution, carry out the circulation of freezing/thawing of three-wheel, discharge virus,, collect supernatant liquor, freeze, use TCID in-80 ℃ with lysate maximum velocity centrifugation 10 minutes ₅₀In 293 cells, detect the titre of adenovirus.

The result shows and duplicates the back by Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK in tumour cell it is tired and is better than initial 1000-1500 doubly, and infectious titer does not have considerable change in normal cell, and the result shows that fully this tumor-selective replication type adenovirus has the characteristic of duplicating at specifically inside tumor cell.

3, the evaluation of tumor-selective replication type adenovirus Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK anti-tumor in vivo effect

The tumour cell animal model: select the BALB/c nude mice in 4～6 ages in week, the stomach cancer cell in the vegetative period of taking the logarithm is the MKN-45 cell, adjusts cell density 1 * 10 ⁶/ 100 μ l, every nude mice right side back subcutaneous vaccination 200 μ l, when treating that tumor average diameter reaches 0.4-0.6cm, be divided into 4 groups at random by the tumour size, promptly medium control group, wt-Ad5 group, Ad5CMV-GFP group and Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK organize every group of 10 nude mices, difference intratumor injection PBS, wt-Ad5, Ad5CMV-GFP and Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK, every 2 * 10 ⁸Pfu/ (only inferior), once a day, continuous 5 days.The 2nd～18 day abdominal injection GCV 125mgkg ^-1D ^-1Observe and measure gross tumor volume weekly 2 times, (100 days or gross tumor volume are greater than 1cm until the experiment terminal point ³).

Experimental result shows that when the experiment terminal point, wt-Ad5 group average tumor inhibiting rate is 36% ± 5%; Ad5CMV-GFP group tumor control rate is-7% ± 3%; Three nude mice tumours of Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK group disappear fully, the average tumor inhibiting rate is 88% ± 5%, and the result shows that fully tumor-selective replication type adenovirus construct Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK has definite anti-tumor in vivo effect.

The vena systemica medication is to the treatment of stomach carcinoma in situ model and to the inhibition of tumour micrometastasis: when the cancer of the stomach Subcutaneous tumor grows to 0.7-0.8cm, select the better tumor animal of overall health of patients, the cervical vertebra dislocation is put to death, aseptic condition takes out the knurl piece down, cut into the knurl piece of 1-2mm with scalpel, be transplanted under the lesser gastric curvature mucous membrane.Animal B ultrasonic monitoring is divided into 4 groups at random when tumor growth during to 3mm, and promptly PBS control group, wt-Ad5 group, Ad5CMV-GFP group and Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK organize, and every group of 10 nude mices are with 2 * 10 ⁸The 2nd～18 day abdominal injection GCV 125mgkg treated in the intravenous injection of pfu virus continuously 5 days ^-1D ^-1Animal B ultrasonic monitoring gross tumor volume.(30 days or gross tumor volume are greater than 1.2cm to testing terminal point ³), take out lung, liver, sky, colon and peritonaeum after, portal vein and hilar lymph node, the pathology detection micrometastasis.Experimental result shows that when the experiment terminal point, the PBS control group all has many organs, tissue tumor to shift, and wt-Ad5 group tumor control rate is 8 many organ metastasis of generation tumour in 21% ± 4%, 10 nude mices; The Ad5CMV-GFP tumor control rate is-7% ± 4%; The incidence of metastases is 100%; 3 nude mice tumours of Δ AD5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK group disappear fully, and tumor control rate is 82% ± 7%, 1 tumour sky, colon implantation metastasis take place.Above result shows that fully tumor-selective replication type adenovirus construct AD5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK intravenous administration has the effect of significant anti-tumor in vivo and inhibition metastases.

Claims (4)

1. tumor-selective replication type adenovirus construct, its technical characterictic is:

This tumor-selective replication type adenovirus construct is called Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK, this construct is to have lacked its 920-946nt GAT CTT ACC TGC CACGAG GCT GGC TTT on the basis of wild-type Ad5 (concrete sequence is seen the nucleic acid table), proteic 121 to 129 amino acids of this sequence encoding Ad5E1A, lack the 28530-29360nt that the E3 district is positioned at the 6.7k/gp19k gene regions simultaneously, introduce a ClaI restriction enzyme site in this disappearance zone; Utilize the complete encoding sequence (1131bp, concrete sequence is seen the nucleotide sequence table) of this restriction enzyme site insertion HSV-TK gene, other genome structure and wild-type Ad5 identical (seeing Figure of description for details).

2. prepare the method for the described tumor-selective replication type adenovirus of claim 1 construct, it is characterized in that:

Lack the coding region 920-946nt that comprises E1A in adenopathy 22nt to the 5790nt sequence pXC1 plasmid respectively with pcr amplification fixed point deletion technology, sequence is GAT CTT ACC TGC CAC GAG GCT GGC TTT, form new carrier Δ 920-946pXC1, Δ 920-946pXC1 plasmid and shuttle vectors pBHGE3 cotransfection 293 cells filter out the recombination adenovirus construction body Δ 920-946Ad5 that expresses E1A mutant functional protein; Adopt pcr amplification fixed point deletion techno-absence Ad5 E3 district 28530-29360nt, and at ClaI restriction enzyme site of above-mentioned disappearance zone introducing, form new carrier pCDNA3.1-Δ 6.7k/gp19k, insert complete encoding sequence (1131bp) nt of HSV-TK gene at ClaI restriction enzyme site place, form new carrier pCDNA3.1-Δ 6.7k/gp19k/HSV-TK; Extract Δ 920-946Ad5 TP-DNA, cut with the EcoRI enzyme, with carrier pCDNA3.1-Δ 6.7k/gp19k/HSV-TK fragment cotransfection 293 cells that cut out with the EcoRI enzyme, filter out the tumor-selective replication type adenovirus construct Ad5/ Δ E1A/ Δ 6.7k/gp19k/HSV-TK that expresses the E1A mutant.

3. the described tumor-selective replication type adenovirus of claim 1 construct is used for the application of the medicine of treatment of solid tumor in preparation.

4. the described tumor-selective replication type adenovirus of claim 1 construct is in the purposes of preparation in the gene therapy vector.

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