CN1272440C - Use and constructing method for anticancer recombined gland virus with tumour cell PLK1 as target of medicine - Google Patents
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Abstract
The present invention discloses a construction scheme for artificially reforming human 5 type adenovirus (ADV5), and the specific application of a recombinant adenovirus construction body obtained by the scheme in tumor treatment. The techniques of PCR amplification point-fixed deletion, homologous recombination, transfection, adenovirus monoclone purification, etc., are used for obtaining the recombinant adenovirus construction body. The present invention is characterized in that twenty-four bases of the coding sequences of an ADV5 genome E1A conserved sequence 2 (CR2) are deleted; 28532 to 29360 nt of ADV5E3 areas are deleted, and a PLK1 cDNA segment is inserted into the area inversely. The recombinant adenovirus construction body can be selectively reproduced and proliferated in a tumor cell; the inserted reverse PLK1 cDNA segment is specifically expressed in the tumor cell; the recombinant adenovirus construction body has a strong bystander effect on the tumor cell, and therefore, the recombinant adenovirus construction body can kill specifically tumor cells without hurting normal cells. The construction body has unique practical value in the preparation of medicines and diagnostic reagents for treating tumors, and meanwhile, the recombinant adenovirus construction body also provides an ideal gene target specificity treatment application approach for tumor gene treatment.
Description
One, technical field:
The present invention relates to a kind of human 5 type adenovirus (Human adenovirus type 5, ADV5) constructing plan of recombinant chou, its technical characterictic is: the genomic 923-946nt sequence of directed disappearance ADV5 (CTTACC TGC CAC GAG GCT GGC TTT, this sequence encoding E1A protein 12 2 to 129 amino acids), on this basis, further disappearance ADV5 E3 district 28532-29360nt introduces a ClaI restriction enzyme site in this disappearance zone; In the ClaI restriction enzyme site, oppositely insert the exogenous PLK1cDNA fragment (960-161nt that is equivalent to PLK1mRNA) of 820bp, thereby obtain the recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 that tumour is had therapeutic action.Summary of the invention belongs to the cma gene field of engineering technology.
Two, background technology
Malignant tumour is becoming the primary disease of harm humans health.Show according to latest domestic epidemiology survey data: China has cancer patient more than 300 ten thousand people now, the patient who dies from malignant tumour every year is about 1,300,000 people, the malignant tumour case that 1,600,000 to 2,000,000 New Developments are arranged every year, and with 3% speed increase, cause grave danger for China people's life and health, restricted the Sustainable development of China's economy to a certain extent.At present, the treatment of most kinds of tumor still is faced with stern challenge, and conventional oncotherapy means can't fundamentally be removed malignant tumour, therefore, press on tumor therapeuticing method and innovate.
In tumor invasion under the new historical conditions that molecular mechanism is understood gradually, tumour molecular medicine theory and methodological breakthrough have caused the trial of a series of development novel tumors treatment patterns, the new drug Gleevec that is used for the treatment of chronic myelocytic leukemia formally becomes the clinical prescription medicine, a large amount of molecular target specificity anticancer compounds enter clinic trial, the weight break point of a key that indicated breaking through to of oncotherapy, and phasic results have been obtained, tentatively highlight the elementary contour that following tumour problem solves, its the most one of the solution of core to from the complicated gene regulatory network of tumour, decomposite the most key molecular target exactly, implement molecular targeted treatment.By end of the year calendar year 2001, obtain the FDA approval and enter the molecular therapy kind in clinical experiment stage above 3,000 kinds, comprise cell conduction block agent, angiogenesis inhibitor, tumor vaccine and monoclonal antibody, for the final solution of tumour problem has brought new hope, we can say, following human tumor effectively to be controlled at the development that fundamentally relies on molecular therapy perfect.
The main means of molecular therapy are divided three classes: monoclonal antibody, micromolecular compound and gene therapy.
Gene therapy has been widely used in the clinical trial treatment of kinds of tumors as having most one of part of vitality in the medical field development in recent years, demonstrates good prospects for application.From 1989 to the end of the year 2000, U.S.'s center for biologic evaluation and research (Center for Biologics Evaluations andResearch, CBER) Pi Zhun clinical gene therapy test subject is above 350, it is therapy of tumor that 70% project is wherein arranged approximately, part has entered the III clinical trial phase, obtain better curative effect, under the promotion of huge medical requirement, the gene therapy preparation might become the clinical treatment medicine by official listing in the several years, become an indispensable integral part of the existing treatment system of tumour.
One of carrier that reorganization ADV5 carrier is most widely used as therapy of tumor, its Clinical feasibility and security have obtained generally acknowledged, adenoviral gene treatment carrier has following outstanding biology advantage and clinical application advantage: (1) pattern of infection is wide, to multiple tissues-derived tumour cell, no matter be in division stage or the cell of stationary phase, all can effectively attack, therefore, anticancer spectrum is wide; (2) after ADV-TK enters cell, exist with extrachromosomal form, do not have sudden change, carcinogenic danger, clinical application safety only produces similar cold like symptoms after the use, no hemopoietic function and immunologic function inhibition; (3) clinical application is convenient, can pass through approach such as direct injection (one or more) and interventional therapy application in lacuna (abdominal cavity, thoracic cavity and cranial cavity etc.) administration, part or the knurl body; (4) in ADV-TK continuous expression 2-3 week only in cell paste, especially be fit to oncotherapy; (5) only produce slight inflammatory reaction after vein or topical application, side effect is slight; (6) adenovirus of clinical grade quantity, quality is easy to production, 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 adenovirus, 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 condition replicability adenovirus carrier is meant that carrier can optionally duplicate propagation in tumour cell, with the place of tumour cell as the production adenovirus, finally reach cracking and kill the therapeutic purpose of tumour cell, after the tumour cell cracking discharges the adenovirus 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, some authoritative medical journal is generically likened it to the SmartBombs (smart bomb) of killing tumour.In actual applications, obtained desired result preferably.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 the human 5 type adenovirus (wild-type) of wild-type, the encoding sequence of excalation (delete) E1B 55kDa, and add the translation termination signal, but keep the encoding sequence of 19kDa E1B.Under normal circumstances, wild-type adenovirus depends on the deactivation of E1B 55kDa albumen to the p53 protein function in the intracellular propagation of duplicating.At the normal cell that carries normal p53 gene, ONYX-015 can not breed in time multiplexed cell system owing to lack E1B 55kDa.On the contrary, because the inactivation rate of malignant tumor patient p53 gene is up to 50-70%, ONYX-015 thereby can in tumour cell, breed and duplicate final lysing cell, thereby ONYX-015 has the high selectivity of killing tumour cell, and normal cell is not had lethal effect.
As the ONYX-015 of condition replicability adenovirus carrier representative products since entering clinical trial in 1996, obtained very challenging clinical efficacy, internationally famous medical journals " TheLancet " is being in the commentary of topic with " the new hope of gene therapy ", the potential applicability in clinical practice of high evaluation ONYX-015, international foremost medical journals as " Science ", ((Nature Medicine " delivered clinical test results and commentary article.At present, Pfizer (pfizer) 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, obtained very satisfied curative effect, to other tumours such as transitivity colorectal carcinoma, lung cancer, carcinoma of the pancreas, oral leukoplakia (oral leukoplakia), the clinical I-II phase of glioblastoma is tested well afoot, simultaneously, ONYX obtained with ONYX-015 be carrier some relevant gene therapy recombinant adenoviral vectors (as inserting therapeutic gene TK, patent CD), just from the preclinical phase test to clinical phase test transition.Can predict, ONYX-015 and relevant gene therapy product thereof will formally enter clinical application in the U.S. in the near future, demonstrate very good potential applicability in clinical practice in the clinical development product of gene therapy for cancer.Domestic existing 4 patent applications with the ONYX-015 similar principles: patent application " a kind of defective virus and construction process thereof " (application number 99124030.8), be positioned at the 2809-3329bp encoding sequence by disappearance E1B 55kDa, and obtain the construct of E1B 55kDa excalation in the method for disappearance position insertion stop code; Patent application " structure of disappearance apoptosis suppressor adenovirus and the application in therapy of tumor " (application number 98113494.7), excalation E1B 55kDa encoding sequence, and at this disappearance position insertion marker gene LacZ, obtain E1B 55kDa excalation and carry the adenovirus construct of marker gene simultaneously, another characteristics of this construct are disappearances that the E3 district is arranged; Patent application " a kind of acquisition and purposes that lacks the recombination adenovirus construction of E1A encoding sequence " (number of patent application 01144628.5), disappearance E1A (382-1630nt) sequence filters out the recombination adenovirus construction body that can not express the E1A functional protein; Patent application " a kind of structure and purposes of uniting disappearance 19kDa, 55kDa E1B encoding sequence recombination adenovirus construction body " (number of patent application 01144629.3), disappearance E1B 19kDa, 55kDa encoding sequence filter out the recombination adenovirus construction body that can not express the E1B functional protein.
Domestic and international existing condition replicability adenovirus carrier invention is compared with first-generation ADV5 gene therapy vector, no matter treating on the principle, still on the actual therapeutic effect, all have tangible improvement, and make a breakthrough, be called as s-generation ADV5 gene therapy vector.Yet, s-generation ADV5 gene therapy vector all has some common shortcomings: (1) is 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 ADV5 far away, as single only be 0-14% with the anticancer efficient of ONYX-015.(2) application of s-generation ADV5 gene therapy vector is still based on locally injected into tumor, in application, be subjected to great restriction, under most situations, tumour is a kind of systemic disease, needs the approach through intravenous administration, carries out systemic treatment, to control former and metastatic lesion, therefore, the selectivity of s-generation ADV5 gene therapy vector and must further strengthen the killing-efficiency of tumour cell is to adapt to the needs that improve route of administration.(3) s-generation ADV5 gene therapy vector does not all carry effective therapeutic goal gene, and the performance of treatment effect is restricted.(4) because the existing molecular target overwhelming majority lacks tumour-specific, though have relative tumor-selective with s-generation ADV5 gene therapy vector in conjunction with the strategy of exogenous promotor, therapeutic gene, but still can not avoid to normal histiocytic toxic action.
In addition, many tumour molecule medicine targets have been determined in recent years, at these molecule medicine target design monoclonal antibodies, micromolecular compound, become the oncotherapy main development tendency, wherein, part has become prescription drugs, a large amount of molecular therapy means is still arranged just in clinic trial evaluation stage, has obtained PRELIMINARY RESULTS preferably.However, because these new molecular targets and molecule thereof check means, the overwhelming majority lacks tumour-specific, consequent dose-limiting toxicity, remain the key issue that these therapies generally face and need to be resolved hurrily at present, greatly limited the solution of tumour problem.
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 third generation ADV5 gene therapy vector, the construct that obtains by this scheme has remarkable advantages such as tumour-specific duplicates, the exogenous insertion inverted defined gene of tumor specific expression, 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 deeply is familiar with and research the ADV5 adenovirus is biological based on existing.After ADV5 enters cell in 1 hour, ADV5 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 ADV5 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 the self-replacation of adenoviral gene group provides precondition; Secondly, ADV5 relies on the early gene (E1B that E1A albumen activates ADV5, E2, E3, E4) and the expression of late gene (L1-6), for the self-replacation of adenoviral gene group, the packing of adenovirus particles, the immune clearance of escaping host cell, lysing cell, release adenovirus particles, complete life cycle such as infection provides the permission environment again.This shows that ADV5 relies on the life cycle of its sequential property of E1A protein promoter, final cracking host cell.
The encoding sequence of functional protein E1A is positioned at ADV5 genome 560-1112nt, and 1229-1545nt comprises three important sequences: be respectively 677-799nt, this sequence is E1A conserved sequence 1 (CR1), is used in conjunction with transcribing cofactor P300; 917-979nt 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 E1A 917-979nt, the effect that keeps the E1A transcripting activating characteristic when reaching the proteic RB binding characteristic of deactivation E1A as far as possible, so, 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 be checked the life cycle that the adenovirus recombinant chou relies on its sequential property of E1A protein promoter, finally only forms abortive infection.This is theoretical infer by recent two independently the work of study group confirmed, (Oncogene such as Fueyo, 2000,19:2-12) made up the adenovirus recombinant chou that lacks E1A 923-946nt, observing this adenovirus recombinant chou optionally duplicates in tumour cell, its duplication characteristic is better than wild-type adenovirus, and normal cell is not had any toxic action, and list can significantly suppress tumor growth with this adenovirus recombinant chou; Heise etc. (Nature Medicine, 2000,10:1134-1139) confirm that more in depth this construct has high selectivity to tumour cell, can significantly suppress tumor growth and suppress metastases with this adenovirus recombinant chou from the vein list.
Although people's such as Fueyo and Heise previous work provides crucial theoretical clue for development third generation ADV5 gene therapy vector, their work still has tangible weak point, they develop the ADV5 gene therapy vector that and do not comprise the exogenous therapeutic gene of any insertion, have limited the performance of anti-tumour effect.For this reason, the present invention makes up a kind of novel ADV5 recombinant chou on this basis, its technical characterictic is: the genomic 923-946nt sequence of directed disappearance ADV5, and further lack ADV5 E3 district 28532-29360nt, introduce a ClaI restriction enzyme site in this disappearance zone; In the ClaI restriction enzyme site, oppositely insert the exogenous CHK1cDNA fragment (960-161nt that is equivalent to CHK1mRNA) of 820bp, thereby obtain the recombination adenovirus construction body Δ 923-946ADV5/ASCHK1 that tumour is had therapeutic action.
PLK1 gene full name polo-like kinase 1, the key that is the cell mitogen check point is regulated kinases, its function is to promote cell to enter and finish mitotic division, in most of tumour cell unconventionality expression is arranged, and it is relevant with the genomic instability of tumour cell, existing ample evidence show PLK1 be cell necessity lifetime because of, and therefore deactivation PLK1, is chosen as molecule medicine target in the present invention with direct cell death inducing.
Compare with existing recombination adenovirus construction body, the present invention has reached following effect:
1, the proteic 923-946nt sequence of this recombination adenovirus construction body disappearance E1A, range of loss is less than the construct of people such as Fueyo and Heise report, theoretical and in fact reached the proteic RB binding characteristic of deactivation E1A in keep the effect of E1A transcripting activating characteristic as far as possible.What is more important, we oppositely insert the exogenous PLK1cDNA fragment (960-161nt that is equivalent to PLK1mRNA) of 850bp in the E36.7K/gp19K district of Δ 923-946ADV5 carrier, make the endogenous promotor of expression dependence ADV5 self the E3 district transcription unit of antisense PLK1cDNA, this promotor has the high reactivity of similar CMV promotor, the opening of the promotor adenovirus that places one's entire reliance upon is duplicated intracellular, adenovirus is not as duplicating, and exogenous antisense PLK1cDNA will not express.Because this brand-new design makes this new construct can reach the therapeutic purpose of selectively killing tumour cell by triple mechanism, that is: construct duplicating in tumour cell can make exogenous antisense PLK1cDNA copy number greatly increase; Construct carries antisense PLK1cDNA and only expresses in tumour cell, and the PLK1 genetic expression of specifically inactivating tumour cell reaches the purpose of killing tumour cell, has avoided Normocellular attack; Construct for duplicating the host, can produce high density adenovirus at tumor by local with tumour cell, forms potent bystander effect.In addition, the present invention also will provide a kind of Design Mode of use for follow-up other molecular therapies.
2, the antitumour drug such as the vincristine(VCR) of existing multiple effect m period, nvelbine, taxols etc. all are action target spot with the spindle microtubule, lack selectivity, be difficult to avoid toxicity, side effect strong, the little shortcoming of treatment window has limited the performance of clinical efficacy between host and the tumour, the present invention adopts more specific m period molecular target PLK1, is a kind of antitumour drug of brand-new effect m period.
3, tumor cell line and normal control cells in vitro experimental result are shown, this recombination adenovirus construction body does not have obvious influence to normal cell PLK1 expression of gene, and the obviously influence of the active nothing of pair cell is opposite, but its specifically inactivating PLK1 expression of gene is killed tumour cell significantly.
4, 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 recombination adenovirus construction body to being tried, and can effectively suppress metastases, and it is xicity related that animal is not had obvious treatment.
Four, description of drawings
Accompanying drawing 1: the structural representation of plasmid pXC1, this plasmid comprise human 5 type adenovirus (ADV5) 21-5790nt sequences.
Accompanying drawing 2: the structural representation of plasmid pBHGE3, this plasmid comprise the full gene group sequence except that ADV5 packaging signal (194-358nt), and insert one section artificial sequence between 194-358nt, total length 37436bp.
Accompanying drawing 3:ADV5E3 plot structure figure, 7 kinds of albumen of this regional code, the zone that lack is 28530-29355bp, is E36.7k/gp 19k zone, oppositely inserts the exogenous PLK1cDNA fragment (960-161nt that is equivalent to PLK1mRNA) of 850bp in this zone.
The plasmid structure iron of accompanying drawing 4:pCDNA3.1, this carrier are used to make up intermediate carrier pCDNA3.1-Δ E3.
Accompanying drawing 5: recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 structural representation.
Accompanying drawing 6: the sequence of recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 and structure explanation.
Five, embodiment
Except that indicating especially, used enzyme and PCR primer are all available from U.S. Gibco company in the present technique flow process.
The structure of embodiment 1:pXC1 series mutation body (Δ 923-946pXC1)
1, pXC 1 purchases the (Toronato in Microbix Biosystem Inc., Ontario, Canada, catalog number (Cat.No.): PD-01-03), this plasmid comprises human 5 type adenovirus (ADV5) 21-5790nt sequences, its plasmid structure iron is seen accompanying drawing 1, the 194-358nt of this plasmid is an ADV5 adenovirus packaging signal, and sequence 560-1112nt, 1229-1545nt are the encoding sequence of functional protein E1A, 9883-9888nt comprises the BamHI restriction enzyme site, and 1338-1343nt comprises the XbaI enzyme cutting site.
2, adopt PCR method disappearance 923-946nt 3 times.
(1) obtaining of fragment 1: primer 1=5 '-CG
GGA TCCGGG CCC CCA TTT CC-3
(be equivalent to 9883-9902nt, underscore partly is the BamHI restriction enzyme site); Primer 2=5 '-
GTC ACT GGG TGG ATC GAT CAC CTCCGG TAC-3 ' (be equivalent to 922-905nt, underscore partly is and primer 3 complementary portions);
With pXC1 is template, the performing PCR reaction, and the reaction system cumulative volume is 100 μ l, comprising: contain MgCl
210 * PCR damping fluid, 10 μ l; 2mM dNTP 10 μ l; 10 μ M primers, 11 μ l; 10 μ M primer 2s, 1 μ l; PXC1 10ng/1 μ l; 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 PCR product is 940bp (fragment 1), and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.
(2) obtaining of fragment 2: primer 3=5 '-
GAG GTG ATC GAT CCA CCC AGT GACGAC GAG-3 ' (be equivalent to 911-947nt, underscore partly is and the primer 2 complementary portion); Primer 4=5 '-TGC
TCT AGACAC AGG TGA TGT CG-3 ' (be equivalent to 1344-1325nt, underscore partly is the XbaI enzyme cutting site);
With pXC1 is template, the performing PCR reaction, and reaction conditions is the same, and product is 400bp (fragment 2), and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.
(3) obtaining of fragment 3: 50ng/2 μ l fragment 1 is mixed with 25ng/1 μ l fragment 2, 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 1400bp (fragment 3), with QIAquick 8 PCR product purification test kit (QIAGEN, German, Cat:28142) behind the purifying, use 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.
(4) 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.
(5) get 8500bp pXC1 endonuclease bamhi and the 90ng fragment 3 of 40ng, use DNA T
4Ligase enzyme is done ligation, 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 pXC1 plasmid encoding mutant body-Δ 923-946pXC1 of 121-129AA 923-946nt, is used for the clone of recombinant adenovirus.
Embodiment 2: the structure of Δ 923-946ADV5 recombinant adenovirus
1, pBHGE3 purchases the (Toronato in Microbix Biosystem Inc., Ontario, Canada, catalog number (Cat.No.): PD-01-12), this plasmid comprises the full gene group sequence except that ADV5 packaging signal (194-358nt), and between 194-358nt, insert one section artificial sequence, and total length 37436bp, its structure is seen accompanying drawing.
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 because the plasmid fragment is very long, need be used CsCl
2-Eb ultracentrifugation plasmid purification is undertaken by the method for " molecular cloning " book.
2, homologous recombination method obtains Δ 923-946ADV5 recombination adenovirus construction body, and method is as follows:
(1), in the 15cm culture dish, plants 7.5 * 10
5(catalog number (Cat.No.): CRL-1573), nutrient solution is 10% FBS DMEM to 293 cells, and by second day, cell should be 1-1.5 * 10 for ATCC, U.S.A
6, about 70% cytogamy; Preceding 3-4 hour of transfection changes fresh medium into.
(2), preparation cotransfection DNA-calcium phosphate solution: with 2 * HBS (280mM NaCl, 43mM HEPES, 10mM KCl, the 10mM Na of 1600 μ l sterilization
2HPO
4.7H
2O, 2% dextrose, pH 7.05-7.15); Each 42 μ g of pBHGE3 and Δ 923-946pXC1; Distilled water to the 2840 μ l mixing that adds sterilization slowly adds 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.
(3), add the above-mentioned mixed solution of 500 μ l to 293 cells that contain 5ml 60mm culture dish, at 37 ℃, hatched among 5% CO2 4-6 hour, inhale and to remove aforesaid liquid, wash once with PBS.
(4), handle 1-2 minute to promote transfection efficiency, wash once, be changed to complete culture solution with PBS with containing 15% glycerine/DMEM.
(5), preparation 10% LMP agarose PBS, autoclaving is distributed into 10ml, melts in boiling water with preceding, insulation is at 45 ℃, with preceding adding 30ml 10% FBS DMEM, final concentration is 1.2%, at once use.
(6), inhale and remove nutrient solution, adding 5ml aforesaid liquid.Every 4-5 days, add the 3ml aforesaid liquid.
(7), 14-21 days, plaque occurs, selected 6-12 plaque with mark stroke circle, with the suction nozzle of 200 μ l, transferred to plaque 24 orifice plates of the 0.5ml that contains 10% FBS DMEM, filtration is 24 hours in 37 ℃.
(8), 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 filtered solution and (is approximately 10
3Adenovirus) adds, rock liquid gently 3 times, at 37 ℃, 5%CO
2In hatched 90 minutes.
(9), add perfect medium to 1ml, cell is positioned over 37 ℃, hatched among 5% CO2 5-10 days, occur until CPE completely, so-called CPE, i.e. cellulotoxic effect, cell shows as and becomes circle, floating, cell is based on kernel.If after 10 days, CPE does not occur completely, then point out tiring of adenovirus too low, need carry out second amplification of taking turns.
(10), culture plate is carried out the circulation of freezing/thawing of three-wheel, discharge adenovirus, 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 adenovirus, is approximately 5 * 10
7/ ml adenovirus.
(11), above adenovirus is increased once more, at 75cm
2Plant 5 * 10 in the culture dish
6293 cells, nutrient solution are 10ml 10% 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;
(12), get 1ml s-generation adenovirus storage liquid and add perfect medium to 1ml, be used for transfection; This MOI is about 5; Remove 75cm
2The liquid of culture dish adds above liquid, shakes gently three times; In 37 ℃, 5% CO2, hatched 90 minutes; Add 9ml 10% FBS DMEM, in 37 ℃, 5%CO2, hatched 4-7 days, be used to extract the screening that adenovirus DNA is used for positive adenovirus.
(13), because 293 cellular genome comprise complete e1a gene, pollute 293 cell DNAs when extracting positive adenovirus DNA easily, cause and identify failure, for this reason with Δ 923-946ADV5 at tumour cell Hela (ATCC, U.S.A, catalog number (Cat.No.): increase once again CCL-2), 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
3Adenovirus) adds, 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 5% CO2 5-10 days, until CPE appearance completely, cell is scraped, be collected in 1.5ml EP pipe, the centrifugal supernatant of abandoning adds 300 μ l PBS solution, carries out the circulation of freezing or thawing of three-wheel, discharge adenovirus, with lysate maximum velocity centrifugation 10 minutes, collect supernatant liquor, freeze in-80 ℃, with the test kit mini DNAisolation kit of Qiagen company, DNA is extracted in the explanation of reference reagent box.With the adenovirus DNA is template, the performing PCR reaction, upstream primer=5 '-CG GGA TCC GGG CCC CCA TIT CC-3 ', downstream primer=5 '-TGC TCT AGA CAC AGG TGA TGT CG-3 ', the reaction system cumulative volume is 100 μ l, comprising: 10 * PCR damping fluid, the 10 μ l that contain MgCl2; 2mM dNTP 10 μ l; 10 μ M upstream primers, 1 μ l; 10 μ M downstream primers, 1 μ l; Adenovirus DNA 10ng; 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 PCR product is 1400bp, and behind the conventional electrophoretic separation purifying, detectable level is used for dna sequencing, and sequencing primer is: 5 '-AGCCGGAGCA GAGAGCCTTG-3 ', pick out correct clone and be used to make up Δ 923-946ADV5/ASPLK1 recombinant adenovirus.
The structure of the subcloning vector pCDNA3.1-Δ E3 in embodiment 3:ADV5 E3 district
1, ADV5 E3 district full name is an ADV5 early region 3, this zone under the driving of endogenous promotor, the 7 kinds of albumen of encoding, sequence, structure, function following (referring to accompanying drawing 3): 12.5k, 27858-28179nt, function is not bright; 6.7k, 27547-28736nt, function is not bright; Gp19k, 28735-29215nt in conjunction with MHC I class antigen, suppresses it and presents to cell surface, consequently escapes the removing of CTL; ADP, 29419-29770nt, lysing cell discharges adenovirus; RID α, 29784-30057nt forms mixture with RID β, prevents the cracking of TNF, removes FAS antigen; RID β, 30062-30458nt; 14.7k 30453-30837 suppresses the cracking of TNF.
The purpose of this experiment is to lack 28530-29355nt zone, E3 district, and inserts exogenous therapeutic gene in the E36.7k/gp of recombinant adenovirus 19k zone.
2, adopt PCR method disappearance ADV5 E3 district 28532-29360nt 3 times.
(1), obtaining of fragment 1: primer 1-5 '-GGG TCA ACG GAA TCC GCG CC-3 ' (quite dried 27301-27320nt); Primer 2-5 '-
CCC ACA TAG AGT ATC GATTGCGCC TTT GGC CTA ATA-3 ' (is equivalent to 28531-28514nt; Underscore partly is and primer 3 complementary portions that ATC GAT is a Cla I restriction enzyme site);
ADV5 virus genom DNA template derives from wild-type ADV5 (Chengdu this yuan gene therapy company), and the obtaining step of adenovirus DNA is as follows:
Wild-type ADV5 Adenovirus Transfection 293 cells are at 75cm
2Inoculate 5 * 10 in the culturing bottle
6293 cells, next day, about 70% cytogamy, cell should be 1 * 10
7Individual;
Preceding 3-4 hour of transfection changes fresh medium into, draws 1 μ l wild-type ADV5 adenovirus to be checked, with 1ml DMEM mixing, makes MOI be about 20;
Remove 75cm
2The liquid of culturing bottle adds aforesaid liquid, shakes gently three times, at 37 ℃, 5% CO
2In hatched 90 minutes;
Add 9ml 10% FBS DMEM, at 37 ℃, 5% CO
2In hatched 72 hours, discard nutrient solution, give a baby a bath on the third day after its birth time with PBS, discard liquid, add the freshly prepared cell pyrolysis liquid of 800 μ l, hatched 1 hour for 37 ℃, the cell mixture after the cracking is added in the eppendorf pipe of 1.5ml, 4 ℃ 12, centrifugal 45 minutes of 000g;
Get supernatant, with isopyknic phenol/chloroform extracting once, get supernatant liquid, add the 3M NaAc of 1/10 volume, the dehydrated alcohol of 2 volumes, hatched one hour at-20 ℃, 12, centrifugal 30 minutes of 000g, 70% ethanol is washed once, with the resuspended adenovirus DNA of 25 μ l TE, on spectrophotometer, measure the concentration of DNA, DNA is frozen stand-by in-70 ℃.
With ADV5 DNA is template, the performing PCR reaction, and the reaction system cumulative volume is 100 μ l, comprising: contain MgCl
210 * PCR damping fluid, 10 μ l; 2mM dNTP 10 μ l; 10 μ M primers, 11 μ l; 10 μ M primer 2s, 1 μ l; ADV5DNA 200ng/1 μ l; Pfu high-fidelity Taq enzyme 2.5u; Add water to 100 μ l;
Reaction conditions is: 94 ℃ 30 seconds; 94 ℃ 30 seconds, 62 ℃ 1 minute, 72 ℃ totally 28 circulations in 1 minute; 72 ℃ were extended 10 minutes.
The PCR product is 1200bp (fragment 1), and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.
(2), obtaining of fragment 2: primer 3=5 '-
GCC AAA GGC GCA ATC GATACT CTATGT GGG ATA TGC TCC AG-3 ' (is equivalent to 29361-29383n; Underscore partly is and the primer 2 complementary portion that ATC GAT is a Cla I restriction enzyme site), primer 4=5 '-GGG GAA CAA AACGCA GAT AGG-3 ' (being equivalent to 30090-30120nt); The PCR reaction conditions is the same, and product is 780bp (fragment 2), and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.
(3), obtaining of fragment 3: 50ng/2 μ l fragment 1 is mixed with 25ng/1 μ l fragment 2, 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 2200bp (fragment 3), with QIAquick 8 PCR 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.
3, (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, and the structure of pCDNA3.1 is seen accompanying drawing 4 with pCDNA3.1.
4, PCR reaction product fragment 3 and enzyme are cut, pCDNA3.1 connects behind the dephosphorylation, get 1.5 μ 1 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 E3 of 28532-29360nt, is used to insert the subclone of exogenous therapeutic gene.
Embodiment 4: the exogenous PLK1cDNA fragment of oppositely inserting 820bp in carrier pCDNA3.1-Δ E3
1, the PLK1cDNA fragment is HL-60 (ATCC from the leukemia cell, U.S.A, catalog number (Cat.No.): amplification among RNA CCL-240), RNA extracts and adopts RNA to extract test kit RNeasy Mini Kit (QIAGEN, GERMAN, catalog number (Cat.No.): 74104), concrete steps are carried out with reference to the test kit specification sheets that the manufacturer provides.
2, reverse transcription condition: 5 * MMLV damping fluid, 4 μ l; 10mM dNTP 1 μ l; OligdT (50ng/ml) 1 μ l; Total RNA 2 μ l; Rnasin 0.5 μ l; MMLV reversed transcriptive enzyme 1 μ l; Add no RNA enzyme water to 20 μ l;
The reverse transcription reaction product is used for the PCR reaction.Upstream primer is 5 '-CC
ATC GATThe GGC TCCACC GGC GAA AGA GA-3 ' (161-180nt that is equivalent to the PLK1cDNA encoding sequence; Underscore partly is a Cla I restriction enzyme site); Downstream primer is 5 '-CC
ATC GATThe GCA GCT CGT TAATGG TTG GG-3 ' (960-941nt that is equivalent to the PLK1cDNA encoding sequence; Underscore partly is a Cla I restriction enzyme site), amplified fragments comprises the 960-161nt of PLK1cDNA encoding sequence, total length is about 820bp.
The reaction system cumulative volume is 100 μ l, comprising: contain MgCl
210 * PCR damping fluid, 10 μ l; 2mM dNTP 10 μ l; 10 μ M upstream primers, 1 μ l; 10 μ M downstream primers, 1 μ l; Reverse transcription reaction product 2 μ l; Pfu high-fidelity Taq enzyme 2.5u; Add water to 100 μ l;
Reaction conditions is: 94 ℃ 30 seconds; 94 ℃ 30 seconds, 62 ℃ 1 minute, 72 ℃ totally 30 circulations in 1 minute; 72 ℃ were extended 7 minutes.
The PCR product is 850bp, and after Cla I enzyme was cut, conventional electrophoretic separation purifying, detectable level were used for follow-up PCR reaction.
3, after carrier pCDNA3.1-Δ E3 uses Cla I enzyme tangent line shapeization, conventional electrophoretic separation purifying, be connected with the exogenous PLK1cDNA fragment of 850bp 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 oppositely inserting at Cla I restriction enzyme site the segmental plasmid encoding mutant body of exogenous PLK1cDNA-pCDNA3.1-Δ E3/ASPLK1 of 850bp, is used for the clone of follow-up recombinant adenovirus.
Embodiment 5: recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 obtains the obtaining according to reference " Adenovirus methods and protocol " (edited by William S.M.Wold of recombination adenovirus construction body Δ 923-946ADV5/ASPLK1, construction ofmutations in the adenovirus early region 3＜E3〉transcription units, 11-24) connection method in (ligation protocol) is carried out.
1, the extraction of TP DNA
(1), gets recombination adenovirus construction body Δ 923-946ADV5 2 * 10
8, be dissolved in 10ml DMEM, make MOI be about 10; 75cm one bottle of 80-90% fusion
2The culture dish kind is gone into 293 cells, and this moment, cell was about 2 * 10
7Cell adds the above liquid of 1ml, shakes gently three times, at 37 ℃, 5% CO
2In hatched 90 minutes, add 10% FBS DMEM to total amount of liquid be 10ml, at 37 ℃, 5% CO
2In hatched 4-5 days, obtain 2 * 10
10Adenovirus, centrifugal, supernatant discarded adds 10ml 10% FBSDMEM, carries out the circulation of freezing and thawing of three-wheel, discharges adenovirus, and concentration is 2 * 10
9/ ml, with lysate with the volume packing of 1ml frozen be 10 the pipe.Amplification for the second time: before the transfection, get the above-mentioned frozen storing liquid of 1 pipe and add 9ml DMEM, concentration is 2 * 10
9/ ml, transfection 10 * 75cm
2The cell of culture dish is at the 75cm of ten bottles of 80-90% fusions
2Culture dish 293 cells respectively are 2 * 10 approximately
7Cell, each adds the above liquid of 1ml, shakes gently three times, at 37 ℃, 5% CO
2In hatched 90 minutes, add 10%FBS DMEM to total amount of liquid be 10ml, at 37 ℃, 5% CO
2In hatch 4-5 days, centrifugal, supernatant discarded repeats above transfection ten times, obtains altogether 2 * 10
13Adenovirus, frozen in-80 ℃, preserve to be purified.
(2), the collection of 293 cells, concentrated and frozen: with 30ml PBS resuspending cell precipitation, cell suspension is merged the centrifuge tube that places 2 50ml, at liquid nitrogen or ethanol, alternate freezing and thawing is three times in 37 ℃ of water-baths, make cell rupture, dissolving, discharge Δ 923-946ADV5, centrifugal 5 minutes of 2500rpm is to remove cell debris on generic centrifuge, the centrifuging and taking supernatant freezes in-70 ℃ to be purified.
(3), the separation and purification of Δ 923-946ADV5: Δ 923-946ADV5 adopts cesium chloride density gradient centrifugation to carry out purifying, can obtain the adenovirus of extreme high purity.
Supernatant is placed CsCl
2In the liquid (Gibco, U.S.A, catalog number (Cat.No.): 456-32), and on two SW-40TL rotors centrifugal 4 ℃, 35,000rpm (150,000 * g), 1 hour.
The preparation of CsCl2 gradient centrifugation liquid:
A:1.5gm/ml CsCl
2Gradient centrifugation liquid, 30g CsCl
2, being dissolved among the PBS, its final volume is 42.5ml.
B:1.35gm/ml CsCl
2Gradient centrifugation liquid is got 15ml A liquid, adds PBS 7ml, and its final volume is 21ml.
C:1.25gm/ml CsCl
2Gradient centrifugation liquid is got 11ml A liquid, adds PBS 9ml, and its final volume is 20ml.
Filtration sterilization accurately takes by weighing the liquid weight of each pipe after degerming, and carries out the adjustment of proportion where necessary.
The preparation of gradient centrifugation pipe: the solution of above-mentioned preparation is added in the 12 volumetrical centrifuge tubes in the following manner, and the volume of its adding and order are as follows: 0.5ml 1.5gm/ml CsCl
2Gradient centrifugation liquid; 3.0ml 1.35gm/ml CsCl
2Gradient centrifugation liquid; 3.0ml 1.25gm/ml CsCl
2Gradient centrifugation liquid.
After gradient forms, add 6ml cell pyrolysis liquid supernatant, with the weight of each pipe of PBS balance at each pipe.
After centrifugal, adenovirus will be taken present 1.35gm/ml CsCl out of with one deck white area
2Gradient centrifugation liquid and 1.25gm/ml CsCl
2Between the gradient centrifugation liquid.By suction sucking-off upper strata protein, with the careful sucking-off of Pasteur's moral suction pipe middle level gland-containing virus part, in aseptic 100ml centrifuge tube of its income.
With the centrifugal adenovirus solution that obtains for the first time with 1.35gm/ml CsCl
2Gradient centrifugation liquid is diluted to 72ml, and it is dispensed in 6 aseptic centrifuge tubes, after the counterweight, carries out single gradient centrifugation with above-mentioned condition, and centrifugation time extends to 18 hours.
After above-mentioned centrifugal spending the night, Δ 923-946ADV5 adenovirus will form one deck white area band once more.By suction sucking-off upper strata protein, with the careful sucking-off of Pasteur's moral suction pipe middle level gland-containing virus part, in aseptic 100ml centrifuge tube of its income, its harvest yield is approximately 15ml.
(4), dialysis method is removed residual cesium chloride and other small-molecule substances:
Residual cesium chloride and the small molecular weight impurity in the extract removed in dialysis.Dialyzate: 10mmolTris-HCl pH 7.5; 1mmol MgCl
2Dialysis volume 1000ml * 3 times.
Dialysis process: bar magnet is added in the dialyzate, put 4 ℃ of precoolings, stir dialysis then.Can prepare 100 * Tris-HCl/MgCl
2Storage liquid is standby, faces with before being diluted to 1 * dialyzate.Centrifugal product before dialysis earlier the dialyzate with equivalent diluted rearmounted dialysis tubing, in order to avoid precipitate because of adenovirus excessive concentration dialysis back produces.The dialysis tubing that is used to dialyse is facing with preceding with deionized water immersion 30 minutes, and then with the sterilization distilled water wash, clamp the end opening of dialysis tubing, dialysis tubing is placed 1 liter of diluent, seal the dialysis bottle bottleneck with tinfoil paper, in case the direct sunshine photograph is placed on the magnetic stirring apparatus and stirs dialysed overnight at 4 ℃, similarity condition repeats above dialysis 2 times.The gained adenovirus is purification of adenoviral.
2, column chromatography purification Δ 923-946ADV5 adenovirus TP DNA:
The column chromatography instrument (Hubei Province's scientific instrument company, catalog number (Cat.No.): SSM-213) go up to prepare 250ml Sepharose 4B (Sigma, U.S.A, catalog number (Cat.No.): 4B-200) chromatography column, put 4 ℃, regulate flow velocity to 18ml/h;
In the adenopathy venom, add isopyknic proteinase inhibitor pefabloc (Boehringger Mannheim that contains 2mmol/L, catalog number (Cat.No.): 8M hydrochloric acid guanidinium isothiocyanate (Sigma 1429876), U.S.A, catalog number (Cat.No.): G9284), after putting on ice 10min, be splined on chromatography column, according to the ratio of absorbance A 260/280, collect TP-DNA (A260/280=1.8-2.0), detect be kept at after the DNA concentration 4 ℃ standby.
3, ligation
PCDNA3.1-Δ E3/ASPLK1 plasmid is cut with the EcoRI enzyme, obtains 10 μ g and comprises the insertion dna fragmentation at interior EcoRI endonuclease bamhi, and dephosphorylation reaction back endonuclease bamhi separates the back recovery at 1% agarose gel electrophoresis, is used for ligation;
After 2.5 μ g Δ 923-946ADV5 TP DNA cuts with the EcoRI enzyme, add 5 μ g and comprise the insertion dna fragmentation at interior EcoRI endonuclease bamhi and 10U T4 ligase enzyme, under 16 ℃ of conditions, conventional ligation.
4, connect product cotransfection 293 cells
In the 60mm culture dish, plant 5 * 10
5293 cells, nutrient solution are 10% FBS DMEM, and by second day, cell should be 1 * 10
6, about 70% cytogamy; Preceding 3-4 hour of transfection changes fresh medium into.
To connect product and mix, add aqua sterilisa, add 2.5M 50 μ l CaCl to cumulative volume 450 μ l with 20 μ g salmon sperm dnas
22 * HBS (280mM NaCl, 43mM HEPES, 10mM KCl, 10mM Na in 500 μ l sterilization
2HPO
4.7H
2O, 2% dextrose, pH 7.05-7.15) in slowly add DNA/CaCl
2, precipitate 45-60 minute, form slightly turbid precipitation.
Add the above-mentioned mixed solution of 500 μ l to 293 cells of 60mm culture dish, in 37 ℃, 5% CO2, hatched 1/2 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 10% LMP agarose PBS, autoclaving is distributed into 10ml, melts in boiling water with preceding, and insulation is at 45 ℃, and with preceding adding 30ml 10% FBS DMEM, final concentration is 1.2%, at once use.
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, and selected 6-12 plaque with mark stroke circle, with the suction nozzle of 200 μ l, transferred to plaque 24 orifice plates of the 0.5ml that contains 10% FBS DMEM, filters 24 hours in 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 filtered solution and (is approximately 10
3Adenovirus) adds, rock liquid gently 3 times, at 37 ℃, 5% CO
2In hatched 90 minutes.
5, the picking of plaque and evaluation
Obtain recombination adenovirus construction body by above experimental procedure, rotaring redyeing 293 cell again, extract adenovirus DNA, Δ 923-946ADV5/ASPLK1 is identified in order-checking, positive adenovirus should lack ADV5 923-946nt sequence, and disappearance ADV5 E3 district 28532-29360nt, comprises exogenous PLK1cDNA fragment (960-161nt that is equivalent to PLK1mRNA) in this zone, other genome structures and wild-type ADV5 are identical, and its sequence and structure are seen accompanying drawing 6.
Embodiment 6: recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 characterized
1, the external tumour-specific of the recombination adenovirus construction body evaluation of duplicating
The purpose of this experiment is to identify the duplicate characteristics of Δ 923-946ADV5/ASPLK1 recombinant adenovirus in a series of tumours and normal cell, experiment will be with the positive contrast of wild-type ADV5, with the negative contrast of ADV-TK (being the replication-defective adenoviral that this study group makes up voluntarily).Selecting cell for use is that (catalog number (Cat.No.): CCL-185), Hela, human osteoblast cell's oncocyte are U-2OS (P53+, RB-to A549 for ATCC, U.S.A; ATCC, U.S.A, catalog number (Cat.No.): HTB-96), the metastatic human adenoma cell is HS700T (P53-, RB-; ATCC, U.S.A, catalog number (Cat.No.): HTB-147), CCL188 DLD-1 (P53-, RB-; ATCC, U.S.A, catalog number (Cat.No.): CCL-221), MCF-7 (P53+, RB+; ATCC, U.S.A, catalog number (Cat.No.): HTB-22).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, these normal cells separate from clinical samples and obtain, and select 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 bone marrow mononuclearcell, 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, 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 1% FBS DMEM substratum to 1ml, cell is positioned in 37 ℃, 5% CO2 hatched 3-10 days, the required time of complete CPE appears in observation of cell.
Experimental result shows, positive control adenovirus wild-type adenovirus hominis 5 non-selectivities ground cracking tumour and normal cell; (ADV-TK can not any cell of cracking for replication-defective adenoviral, recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 is the cracking tumour cell optionally, its effect significantly is better than wild-type adenovirus hominis 5, and this recombination adenovirus construction body is to the obviously influence of normal control cells system's nothing simultaneously.The result fully shows recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 selectively killing tumour cell, and concrete data see the following form:
| Wild-type ADV5 | ADV-TK | Δ923-946ADV5/ASPLK1 | |
| A549 | |||
| The 3rd day | 50％ | 1％ | 92％ |
| The 6th day | 100％ | 2％ | 100％ |
| The | 4％ | ||
| Hela | |||
| The 3rd day | 60％ | 1％ | 93％ |
| The 6th day | 100％ | 2％ | 100％ |
| The | 4％ | ||
| U-2OS | |||
| The 3rd day | 60％ | 1％ | 92％ |
| The 6th day | 100％ | 2％ | 100％ |
| The |
4％ | ||
| HS700T | |||
| The 3rd day | 60％ | 1％ | 93％ |
| The 6th day | 100％ | 2％ | 100％ |
| The |
4％ | ||
| DLD-1 | |||
| The 3rd day | 60％ | 1％ | 92％ |
| The 6th day | 100％ | 2％ | 100％ |
| The |
4％ | ||
| MCF-7 | |||
| The 3rd day | 60％ | 1％ | 92％ |
| The 6th day | 100％ | 1％ | 100％ |
| The |
1％ | ||
| Vascular endothelial cell | |||
| The 3rd day | 60％ | 1％ | 4％ |
| The 6th day | 100％ | 2％ | 4％ |
| The |
4％ | 4％ | |
| Lung tracheole epithelial cell | |||
| The 3rd day | 60％ | 1％ | 3％ |
| The 6th day | 100％ | 2％ | 3％ |
| The |
4％ | 6％ | |
| Prostate epithelial cell | |||
| The 3rd day | 60％ | 1％ | 4％ |
| The 6th day | 100％ | 2％ | 4％ |
| The |
4％ | 5％ | |
| BMNC | |||
| The 3rd day | 60％ | 1％ | 2％ |
| The 6th day | 100％ | 2％ | 2％ |
| The |
4％ | 6％ |
2, for further carrying out detection by quantitative Δ 923-946ADV5/ASPLK1 recombination adenovirus construction body 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 is 10% FBS DMEM, by second day, cell should be 2 * 10
5, about 70% cytogamy is inhaled and is removed liquid, adds Δ 923-946ADV5/ASPLK1 10MOI 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 1% FBS DMEM substratum, cell is positioned in 37 ℃, 5% CO2 hatched 6 days to 1ml.
Add 300 μ l PBS solution, carry out the circulation of freezing or thawing of three-wheel, discharge adenovirus,, collect supernatant liquor, freeze, in 293 cells, detect the titre of adenovirus with TCID50 in-80 ℃ with lysate maximum velocity centrifugation 10 minutes.
The result shows and duplicates the back by Δ 923-946ADV5/ASPLK1 in tumour cell it is tired and is better than initial 500-1000 doubly, and infectious titer does not have considerable change in normal cell, and the result shows that fully this recombination adenovirus construction body has the characteristic of duplicating at specifically inside tumor cell.
3, the evaluation of the external selective inactivation tumour cell PLK1 expression characterization of recombination adenovirus construction body
The purpose of this experiment is to identify that Δ 923-946ADV5/ASPLK1 recombinant adenovirus is in the influence to a series of tumours and normal cell PLK1 expression, it is A549 that cell is selected in experiment for use, Hela, human osteoblast cell's oncocyte be U-2OS (P53+, RB-), the metastatic human adenoma cell is HS700T (P53-, RB-), and CCL188 DLD-1 (P53-, RB-), MCF-7 (P53+, RB+).The clone of selecting for use has different P53, 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 bone marrow mononuclearcell, 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, 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 1% FBS DMEM substratum, cell is positioned over 37 ℃ of 5% FBS DMEM substratum to 1ml, cell is positioned in 37 ℃, 5% CO2 hatched 3-10 days, extract the variation that total protein of cell detects the PLK1 protein expression level to 1ml.
Experimental result shows, recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 selective inactivation tumour cell PLK1 protein expression is to can not detection level, and this recombination adenovirus construction body is that the PLK1 protein expression does not have obvious influence to normal control cells simultaneously.The result fully shows recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 selective inactivation tumour cell PLK1 expression of gene.
4, recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 anti-tumor in vivo CHARACTERISTICS IDENTIFICATION
The tumour cell animal model: select the MCF-7 tumor growth good, the better tumor animal of overall health of patients, the cervical vertebra dislocation is put to death.Aseptic condition takes out down the knurl piece, cuts into the MCF-7 knurl piece in 1-2mm footpath with scalpel, and trochar is inoculated in the nude mouse bilateral, the visible tumour in an about week laboratory animal oxter, back, when tumor growth to 6-7mm, by knurl volume size hierarchical grouping.Every group of 5-8 animal is with 1 * 10
8Injection in the direct tumour of pfu adenovirus, continuous 5 days, the treatment back was with vernier caliper measurement tumour length and width footpath, and (90 days or gross tumor volume are greater than 1cm until the experiment terminal point
3).
Experimental result shows that when the experiment terminal point, positive control adenovirus wild-type adenovirus hominis 5 tumor control rates are 80% ± 2%; Replication-defective adenoviral (ADV-TK) tumor control rate is-50% ± 2%; Recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 tumor control rate is 92% ± 4%, and the result shows that fully recombination adenovirus construction body 923-946ADV5/ASPLK1 has clear and definite anti-tumor in vivo effect.
The recombination adenovirus construction body intravenous administration: select tumor growth good, the better tumor animal of overall health of patients, the cervical vertebra dislocation is put to death.Aseptic condition takes out down the knurl piece, cuts into the knurl piece in 1-2mm footpath with scalpel, and trochar is inoculated in the nude mouse bilateral, the visible tumour in an about week laboratory animal oxter, back, when tumor growth to 4-5mm, by knurl volume size hierarchical grouping.Every group of 5-8 animal is with 1 * 10
8The intravenous injection of pfu adenovirus, continuous 5 days, treatment back was with vernier caliper measurement tumour length and width footpath, and (60 days or gross tumor volume are greater than 1cm until the experiment terminal point
3), experimental result shows that when the experiment terminal point, positive control adenovirus wild-type adenovirus hominis 5 tumor control rates are 60% ± 2%; Replication-defective adenoviral (ADV-ASPLK1) tumor control rate is-70% ± 2%; Recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 tumor control rate is 93% ± 4%, and the result shows that fully recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 intravenous administration has clear and definite anti-tumor in vivo effect.
The recombination adenovirus construction body intravenous administration suppresses the tumour micrometastasis: select MDA-MB-231 tumour (ATCC, U.S.A, catalog number (Cat.No.): HTB-26) well-grown, the better tumor animal of overall health of patients, cervical vertebra dislocation execution.Aseptic condition takes out down the knurl piece, cuts into the knurl piece in 1-2mm footpath with scalpel, and trochar is inoculated in nude mice breast portion, the visible tumour in an about week laboratory animal oxter, back, when tumor growth to 4-5mm, by the big or small hierarchical grouping of knurl volume.Every group of 5-8 animal is with 1 * 10
8The intravenous injection of pfu adenovirus, continuous 5 days, treatment back was with vernier caliper measurement tumour length and width footpath, and (90 days or gross tumor volume are greater than 1.2cm until the experiment terminal point
3), take out tumour, tracheae, lymphonodi pulmonales, the pathology detection micrometastasis.Experimental result shows that when the experiment terminal point, positive control adenovirus wild-type adenovirus hominis 5 tumor control rates are 60% ± 2%, and the incidence of metastases is 45% ± 5%; Replication-defective adenoviral (ADV-TK) tumor control rate is-70% ± 2%, and the incidence of metastases is 100%; Recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 tumor control rate is 94% ± 5%, and the incidence of metastases is 0%.The result shows that fully recombination adenovirus construction body 923-946ADV5/ASPLK1 intravenous administration has the effect of clear and definite anti-tumor in vivo and inhibition metastases.
Claims (3)
1. recombination adenovirus construction body, its technical characterictic is: this construct called after Δ 923-946ADV5/ASPLK1, it has lacked ADV5923-946nt sequence C TT ACC TGC CAC GAGGCT GGC TTT, this sequence encoding E1A protein 12 2 to 129 amino acids, simultaneously, it has lacked ADV5E3 district 28532-29360nt, introduce a ClaI restriction enzyme site in this disappearance zone, and in the ClaI restriction enzyme site, oppositely inserting the cDNA fragment of the 960-161nt that is equivalent to PLK1mRNA, other genome structures and wild-type ADV5 are identical.
2. make up the method for the described recombination adenovirus construction body of claim 1, it is characterized in that: with the coding region 923-946nt sequence C TT ACCTGC CAC GAG GCT GGC TTT of E1A in the pcr amplification fixed point deletion techno-absence pXC1 plasmid, form new carrier Δ 923-946pXC1, Δ 923-946pXC1 plasmid and shuttle vectors pBHGE3 cotransfection 293 cells filter out the recombination adenovirus construction body Δ 923-946ADV5 that expresses E1A mutant functional protein; Adopt pcr amplification fixed point deletion techno-absence ADV5E3 district 28532-29360nt, and at ClaI restriction enzyme site of this disappearance zone introducing, form new carrier pCDNA3.1-Δ E3, oppositely insert the cDNA fragment of the 960-161nt that is equivalent to PLK1mRNA at ClaI restriction enzyme site place, form new carrier pCDNA3.1-Δ E3/ASPLK1; Extract Δ 923-946ADV5 TP DNA, cut with the EcoRI enzyme, with carrier pCDNA3.1-Δ E3/ASPLK1 fragment cotransfection 293 cells that cut out with the EcoRI enzyme, filter out the recombination adenovirus construction body Δ 923-946ADV5/ASPLK1 that expresses E1A mutant functional protein.
3. the described recombination adenovirus construction body of claim 1 is used for the application of the medicine of oncotherapy in preparation.
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