CN105087647A - Recombinant adeno-associated virus vector carrying survivin antigen genes and construction method and application thereof - Google Patents

Abstract

The invention discloses a recombinant adeno-associated virus vector carrying survivin antigen genes and a construction method and application thereof. The rAAV (recombinant adeno-associated virus) vector is prepared by replacing structural genes of adeno-associated viruses in adeno-associated virus vectors with survivin antigen genes. The vector is capable of delivering the survivin antigen genes carried thereby into a monocyte-dendritic cell line so as to stimulate effector cells in an immune system. Experiments show that CTL (cytotoxic T lymphocyte) induced by DCs (dendritic cells) infected by the vector is effective in inhibiting growth of survivin antigen-positive malignant cells or killing the cells in vitro or in vivo. The vector or its related products are applicable to the preparation of drugs for treating various survivin antigen-positive tumors.

Description

A kind of carry Survivin antigen gene recombined glandulae correlation viral vectors and construction process and application

[technical field]

The present invention relates to the carrier in biological field and application thereof, particularly relate to one and carry the recombined glandulae correlation viral vectors of survivin (Survivin, also known as Survivin) antigen gene and construction process and its thereof application in the tumour medicine of the anti-Survivin positive of preparation.

[background technology]

The gene structure of adeno-associated virus (adeno-associatedvirus, AAV) is identified.Nineteen eighty-three, the people such as Samulski describe the terminal repetition fragment (upstream 5 ' end fragment of AAV, downstream 3 ' end fragment) (SamulskiRJ, SrivastavaA, BernsKI, MuzyczkaN.Rescueofadeno-associatedvirusfromrecombinantpl asmids:genecorrectionwithintheterminalrepeatsofAAV.Cell. 33:135-143.).1984, the people such as Hermonat describe low infectious particles (lip) gene and coating (cap) gene (HermonatPL of AAV, LabowMA, WrightR, BernsKI, MuzyczkaN.Geneticsofadeno-associatedvirus:isolationandpr eliminarycharacterizationofadeno-associatedvirustype2mut ants.JVirol.51:329-339.Hermonat, P.L., andMuzyczka, N.Useofadeno-associatedvirusasamammalianDNAcloningvector: transductionofneomycinresistanceintomammaliantissuecultu recells.Proc.Natl.Acad.Sci.U.S.A.81:6466-6470.).1986, the people such as Labow identify the p5 promotor (LabowMA be positioned between upstream 5 ' end fragment and rep gene, HermonatPL, BernsKI.Positiveandnegativeautoregulationoftheadeno-asso ciatedvirustype2genome.JVirol.160:251-258.).

1984, U.S. PaulL.Hermonat proved that AAV carrier can be used for the gene therapy of human diseases.At present, mainly American-European countries in the clinical trial carrying out the gene therapy human diseases based on AAV.According to U.S.'s grain and drug administration's statistics, the Gene Therapy Clinical Trials of existing ten remainders based on AAV carries out, mainly the AAV virus of carrying therapeutic gene is injected in patient body, make its expression treatment gene in vivo, thus reach the object of disease therapy.Disease mainly for treatment is the non-neoplastic diseases such as Parkinsonism, rheumatic arthritis, hemophilia, heart failure, the silent syndromes of progressive myatrophy and Olds sea.The Glybera product of European Union's approval on November 2nd, 2012 UniQure company uses in 27 member statess of European Union, this is the granted gene therapy medicaments of western countries first, and it is the genomic medicine utilizing adeno-associated virus I type (AAV-I) foreign gene-carrying to be used for the treatment of lipoprotein lipase deficiency inherited disease (LPLD).

AAV is a kind of defective virus of non-virulent, needs the gene product of other virus (as adenovirus) to assist, just can be assembled into and have infective virion.Current AAV can be divided into 12 kinds of serotypes (AAV-1 ~ AAV-12).Wherein, 2 type gland relevant viral vectors (AAV-2) because having the advantage such as no pathogenicity, host range are wide, goal gene long-term expression and become one of most potential virus vector in current gene therapy.AAV-2 full-length genome about 4700 base pair (bp), two ends are for repeating terminal fragment (TR), and centre is the structure gene of virus, comprises Rep and Cap gene.Owing to there is the unstable of AAV virus self and carrying the aspect defects such as allogenic gene (therapeutic gene) limited length, therefore be necessary that carrying out gene recombination to it forms recombinant adeno-associated virus (recombinantadeno-associatedvirus, rAAV).

The recombinant adeno-associated virus and the related products thereof that how to build the medicine that can be used for preparing disease therapy are the directions that those skilled in the art make great efforts.

[summary of the invention]

Technical problem to be solved by this invention is: make up above-mentioned the deficiencies in the prior art, propose that a kind of stability is high, recombinant adeno-associated virus (recombinantadeno-associatedvirus, the rAAV) carrier that carries survivin (Survivin) antigen gene and construction process thereof and application.

Technical problem of the present invention is solved by following technical scheme:

Carry a recombined glandulae correlation viral vectors for Survivin antigen gene, the structure gene of the adeno-associated virus in gland relevant viral vector is replaced with the recombined glandulae correlation viral vectors that Survivin antigen gene obtains.

Known gland relevant viral vector has p5 promotor, for improving the transcriptional level of goal gene, also further Survivin antigen gene can be inserted the promotor that success has built is in a kind of AAV carrier in cytomegalovirus (CMV) promotor, beta-actin promotor, SV40 early promoter.

The present invention also provides the construction process of the recombined glandulae correlation viral vectors carrying Survivin antigen gene: in gland relevant viral vector, the structure gene of adeno-associated virus is rejected, Survivin antigen gene is inserted in the position of rejecting, obtains recombined glandulae correlation viral vectors.

Construction process provided by the present invention, it is the method using gene recombination, restriction enzyme is used first to be cut off by AAV carrier framework DNA, use DNA interconnection technique again, specific antigen gene Survivin is connected with cut-off AAV carrier DNA, obtains the recombined glandulae correlation viral vectors carrying Survivin antigen gene.The promotor of this rAAV carrier is the one in p5 promotor or following three kinds of promotors: cytomegalovirus (CMV) promotor, beta-actin promotor, SV40 early promoter.

The product that a kind of recombined glandulae correlation viral vectors to carrying Survivin antigen gene is relevant, comprise the plasmid vector of Survivin recombinant adeno-associated virus, Survivin recombinant adeno-associated virus virus vector, by the described viral vector infection of Survivin recombinant adeno-associated virus or the clone of transfection, the recombined glandulae correlation viral vectors carrying Survivin antigen gene that the plasmid vector of described Survivin recombinant adeno-associated virus is obtained by recombined glandulae correlation viral vectors or the above-mentioned construction process of the above-mentioned Survivin of carrying antigen gene obtains; The virus vector of described Survivin recombinant adeno-associated virus carries out cell cultures by the plasmid vector of described Survivin recombinant adeno-associated virus and obtains.

The preparation method of the product that a kind of recombined glandulae correlation viral vectors to carrying Survivin antigen gene is relevant, the preparation of the plasmid vector of Survivin recombinant adeno-associated virus: the recombined glandulae correlation viral vectors quiding gene engineering colon bacillus DH5 α competent cell carrying Survivin antigen gene that the recombined glandulae correlation viral vectors of the such as above-mentioned Survivin of carrying antigen gene or above-mentioned construction process are obtained, resistance screening is carried out with the LB flat board containing 100 μ g/mL penbritins, the single bacterium colony of picking white, extract plasmid and purifying, obtain the plasmid vector of Survivin recombinant adeno-associated virus, the preparation of the virus vector of Survivin recombinant adeno-associated virus: the virus vector obtaining Survivin recombinant adeno-associated virus with the plasmid vector of described Survivin recombinant adeno-associated virus and pHelper plasmid co-transfection AAVp cell, Survivin recombinant adeno-associated virus infects or the preparation of clone of transfection: obtain with the viral vector infection of described Survivin recombinant adeno-associated virus or transfection monocyte, dendritic cell or T lymphocyte, described clone comprises monocyte-dendritic cell system, T lymphocyte series.

One as above institutethe recombined glandulae correlation viral vectors carrying Survivin antigen gene stated or as above institutethe related products stated is preparing the application in antitumor drug.

Particularly, tumour is the malignant tumour of the Survivin positive.The malignant tumour of the described Survivin positive includes but not limited to lung cancer, intestinal cancer, liver cancer, mammary cancer etc.Medicine can adopt the formulation such as solvent or pulvis.The selection of described solvent is diversified, as cell culture fluid (base), physiological saline or phosphate buffered saline buffer etc.When needing, one or more pharmaceutically acceptable carriers can also be added in said medicine.Described carrier comprises thinner, the absorption enhancer and tensio-active agent etc. of pharmaceutical field routine.

Application method can be the monocyte first isolated in tumour patient body, then by the monocyte of the viral vector infection of AAV/Survivin or transfection patient.Maybe by transform have the dendritic cell of the maturation of Survivin institute stimulate generation cytotoxic T lymphocyte feedback tumour patient.

The consumption of said medicine is generally 100 μ l/5 × 10 ⁶individual monocyte/each, monthly 2 times, the course for the treatment of is generally 6 months.Dosage and the course for the treatment of all can adjust according to practical situation.

For improving curative effect, medicine of the present invention can also carry out combined therapy with microbiotic, immunostimulant and tumor-targeting drug etc.

The beneficial effect that the present invention is compared with the prior art is:

The invention provides recombinant adeno-associated virus (rAAV) carrier that a kind of stability is high, carry allogenic gene (Survivin).The Survivin antigen gene that can be carried at recombinant adeno-associated virus of the present invention (AAV/Survivin) carrier is conveyed in monocyte-dendritic cell system, and the cell that there is Survivin antigen gene is used to the effector cell of stimulating immune system (being not limited to T lymphocyte and bone-marrow-derived lymphocyte).Experiment proves, the dendritic cell infected by rAAV of the present invention and the cytotoxic T lymphocyte of inducing effectively can suppress growth or the killing off tumor cells of associated malignancies cell in vitro with in patient body, thus, the recombined glandulae correlation viral vectors of the Survivin of carrying antigen gene of the present invention or the product relevant to recombined glandulae correlation viral vectors of the present invention can be used to the cellular immunotherapy of the malignant tumour preparing anti-Survivin antigen positive.The present invention has important theoretical and practical significance in the clinical treatment and application of malignant tumour, has a extensive future.

[accompanying drawing explanation]

Fig. 1 is the structural representation carrying the recombined glandulae correlation viral vectors of survivin (Survivin) antigen gene of the specific embodiment of the invention;

Fig. 2 is the schema that the structure of the specific embodiment of the invention carries that the recombined glandulae correlation viral vectors of survivin (Survivin) antigen gene and preparation have infective recombinant adeno-associated virus AAV/Survivin;

Fig. 3 is the PCR qualification result figure of recombinant adeno-associated virus (AAV/Survivin) carrier of the specific embodiment of the invention;

Fig. 4 is the comparison diagram of the gene order that the Survivin gene order obtained of the specific embodiment of the invention and U.S. NCIGeneBank announce;

Fig. 5 is the virus titer detected result figure of the virus vector of the recombinant adeno-associated virus (AAV/Survivin) of the specific embodiment of the invention;

Fig. 6 be the recombinant adeno-associated virus (AAV/Survivin) of the specific embodiment of the invention viral vector infection tumour patient monocyte based on kill tumor experiment schema;

Fig. 7 is the Efficiency testing result figure of the viral vector infection peripheral blood mononuclear cell of the recombinant adeno-associated virus (AAV/Survivin) of the specific embodiment of the invention;

Fig. 8 a is the detected result figure of the DC expression CD80 level of the viral vector infection of the recombinant adeno-associated virus (AAV/Survivin) of the specific embodiment of the invention;

Fig. 8 b is the detected result figure of the DC expression CD83 level of the viral vector infection of the recombinant adeno-associated virus (AAV/Survivin) of the specific embodiment of the invention;

Fig. 8 c is the detected result figure of the DC expression CD86 level of the viral vector infection of the recombinant adeno-associated virus (AAV/Survivin) of the specific embodiment of the invention;

Fig. 9 is the inspection result figure of the IFN-γ level of the CTL that the DC of the viral vector infection of the recombinant adeno-associated virus (AAV/Survivin) of the specific embodiment of the invention induces;

Figure 10 be CTL that the DC of the viral vector infection of the recombinant adeno-associated virus (AAV/Survivin) of the specific embodiment of the invention induces kill and wound Survivin antigen positive tumour cell, kill and wound specificity and the restrictive detected result comparison diagram of MHCI quasi-molecule (MHCClassI).

[embodiment]

Contrast accompanying drawing below in conjunction with embodiment the present invention is described in further details.

Contriver is successfully by the entire infrastructure gene elmination comprising rep and cap gene of the AAV-2 carried in the pBR322 plasmid (pBR-AAV2) of AAV2 type complete genome DNA, through retaining terminal repetition fragment, or retain terminal repetition fragment and p5 promotor, and insert oligonucleotide segment, with the stability of the efficiency and recombinant adeno-associated virus that improve recombinant adeno-associated virus (rAAV) DNA replication dna, obtain the basic framework of AAV-2 carrier thus.In addition, the p5 promotor in cytomegalovirus (CMV) promotor, vacuolating virus of monkey 40 (SV40) early promoter, beta actin promoter replacement AAV-2 carrier is also successfully used.And on this basis successfully preparation there is infective rAAV virion (see Chinese patent ZL201110125683.X), lay a good foundation for developing new rAAV product.

Survivin (Survivin) is one of member of IAP (Inhibitorofapoptosis, IAP) family, is to find the strongest survivin at present.Survivin gene is positioned at 17q25 karyomit(e), total length 14700bp, is made up of 4 exons and 3 introns.Due to Survivin energy inhibited apoptosis, promote DNA replication dna, shorten G1/S phase process, therefore the process LAN of Survivin in tumour cell promotes the abnormality proliferation of cell by mitotic division.Have now found that, Survivin expresses in all common cancers, comprise large bowel cancer (53.2%), mammary cancer (83.3%), small cell lung cancer (85%), nonsmall-cell lung cancer (79.1%), ovarian cancer (51.1%), cancer of the stomach (87%), liver cancer (62%), cervical cancer (77.7%), kidney (69.5%), bladder cancer (63.8%), carcinoma of the pancreas (68%), non-Hodgkin lymphoma (50%) and neurocytoma (47.2%) etc., and without expressing in normal group.Therefore this antigen has tumour-specific, is only expressed in tumour and embryonic tissue, and with the differentiation and proliferation of tumour cell and infiltration metastasis closely related.More studies have found that the multidrug resistance of this antigen high expression level and tumour is proportionate.This antigen has antigenicity, is the target antigen of cell immune response, can become a novel targets of inverse cancer cell resistance.Experiment proves, the expression blocking Survivin gene can promote the apoptosis-induced effect of chemotherapeutics to tumour, increases chemotherapy drug susceptibility, even reversing drug resistance.Therefore Survivin has become the target spot of the antineoplaston received much concern.

AAV/Survivin of the present invention inserts Survivin antigen gene in the skeleton of the gland relevant viral vector successfully built contriver to obtain recombined glandulae correlation viral vectors, and the product relevant to carrier comprises plasmid, virus and clone.

In following embodiment, method therefor is ordinary method if no special instructions, concrete steps can be see: " MolecularCloning:ALaboratoryManual " (Sambrook, J., Russell, DavidW., MolecularCloning:ALaboratoryManual, 3rdedition, 2001, NY, ColdSpringHarbor).

The synthesis of the primer, DNA sequence dna and determined dna sequence complete by LifeTechnologies company of the U.S..

The structure of embodiment 1, Survivin recombined glandulae correlation viral vectors and qualification

One, material and source thereof:

1. four kinds have AAV2 type (AAV-2) pBR322 plasmid (pBR-AAV2) of different promoters: this plasmid successfully constructs (see Chinese patent ZL201110125683.X, the reconstruction of 0056 ~ 0059 section of pBR-AAV2 plasmid, pcr amplification promotor, the promotor of amplification is inserted the medium content of pBR-AAV2 plasmid of reconstruction) by contriver.Four kinds of promotors are respectively AAVp5 promotor, cytomegalovirus (CMV) promotor, SV40 early promoter and people's beta-actin (β-actin) promotor.The feature of this plasmid is complete repetition end segment (TR) sequences in two ends, and the fragment CTGCGCTGG of 9 Nucleotide compositions is all inserted at the 75th the nucleotide sequence place of two ends TR, object improves the stability of restructuring AAV virus (rAAV) and improves the duplicating efficiency of virus, and reject the entire infrastructure gene comprising replication protein gene (rep) and envelope protein gene (cap) of AAV-2.

2. people's cancerous lung tissue: the cancerous tissue deriving from excision, immunohistochemical methods confirms Survivin antigen positive.

3. gene amplification nucleotide primer: according to people living element antigen (Survivin) gene order design (GenBank:HM625836) published in U.S.'s gene pool.

Two, build the recombined glandulae correlation viral vectors (as illustrated in fig. 1 and 2) that the present embodiment carries Survivin antigen gene, detailed process comprises the following steps:

1. obtain total cDNA, concrete grammar is: adopt Trizol reagent (production of LifeTechnology company of the U.S.) to obtain total mRNA of tumor tissues.First after repeatedly being milled by the cancerous lung tissue of Survivin antigen positive, add 5mlTrizol, operate according to its specification sheets.After centrifugal acquisition supernatant liquor, with 75% (V/V) washing with alcohol secondary, then add dehydrated alcohol, centrifugation.Throw out, with deionized water dissolving, obtains total mRNA solution, concentration is adjusted to 10ng/ μ l.With the total mRNA solution of 10 μ l for template, carry out reverse transcription reaction (RT), synthesize total cDNA.Reverse transcription reaction system, be 25 μ l for total reaction system, comprise: 0.5 μ goligo (dT) 15 (Promega company of the U.S.), 0.5mMdNTPs (Promega company of the U.S.) and 200UM-MLV reversed transcriptive enzyme (Promega company of the U.S.).Reaction conditions is 37 DEG C, 1 hour, obtains total cDNA.

2. obtain SurvivincDNA, concrete grammar is: with described total cDNA for template, and pcr amplification under the guiding of primer 1:ACGCGTACCgCCAgATTTgAATCg and primer 2: CCTCgAgCCTCAATCCATggCAgC, obtains SurvivincDNA.Pcr amplification condition is: first 94 DEG C 4 minutes; Again 94 DEG C 30 seconds, 60 DEG C 35 seconds, 72 DEG C 70 seconds, totally 30 circulations; Last 72 DEG C 8 minutes, after reaction terminates, carry out 1.2% (W/V) agarose gel electrophoresis to PCR primer to detect, at the specific band of 517bp place appearance one expection, being reclaimed and obtain length after purifying by this object band is 517bpSurvivincDNA (as shown in Figure 3).Carry out determined dna sequence again, its nucleotide sequence as shown in Figure 4, proves that the SurvivincDNA gene order of pcr amplification is correct further.

3. building and carry the recombined glandulae correlation viral vectors of SurvivincDNA: carrying out after enzyme cuts, carrying out DNA ligation with restriction enzyme respectively to carrying the pBR-AAV2 plasmid of 4 kinds of different promoters and SurvivincDNA respectively.Restriction enzyme used equal purchased from American Promega company.Endonuclease reaction system is: 1 μ gpBR-AAV2 plasmid or SurvivincDNA; 10U restriction enzyme MluI and xbaI (purchased from American Promega company), 2.5 μ l10 × damping fluid C and 19.5 μ l deionized waters; Reaction conditions is: water-bath 4 hours at 37 DEG C.Ligation system is: 500ng enzyme cut after plasmid, 300ng enzyme cut after SurvivincDNA, 10IUT4DNA ligase enzyme (purchased from American Promega company), 1.5 μ l10 × T4DNA connect damping fluid and 11.5 μ l deionized waters; Reaction conditions is: at 4 DEG C 8 hours.The recombined glandulae correlation viral vectors obtaining carrying the p5 promotor of AAV and SurvivincDNA respectively, the recombined glandulae correlation viral vectors carrying CMV promoter and SurvivincDNA, carry the recombined glandulae correlation viral vectors of SV40 early promoter and SurvivincDNA, carry the recombined glandulae correlation viral vectors of beta actin promoter and SurvivincDNA.

4. by quiding gene engineering colon bacillus (E.coli) DH5 α competent cell (American I nvitrogen company) respectively of the recombined glandulae correlation viral vectors after connection, resistance screening is carried out with the LB flat board containing 100 μ g/mL penbritins, the single bacterium colony of picking white, extract plasmid and purifying, obtain the plasmid vector of a large amount of recombinant adeno-associated virus (AAV/Survivin).

Three, the qualification of the plasmid vector of recombinant adeno-associated virus

Can 1.PCR identifies: carry out pcr amplification under applying the guiding of the above-mentioned primer 1 mentioned and primer 2, occur that SurvivincDNA is for standard of perfection to increase.Pcr amplification condition and reaction system are identical as the above-mentioned.After reaction terminates, 1.2% (W/V) agarose gel electrophoresis is carried out to PCR primer and detects, the plamid vector construction success of AAV/Survivin can be judged to be at 517bp place appearance specific band.Being reclaimed and obtain length after purifying by this object band is 517bpSurvivincDNA, as shown in Figure 3.In Fig. 3: 1 is DNA molecular amount standard; 2 and 3 represent 2 positive colonies.

2.DNA sequencing: AAV/Survivin is carried out determined dna sequence.The nucleotide sequence of its sequencing result as shown in Figure 4, with gene order 99% homology of the Survivin announced in U.S. gene pool, proves that the AAV/Survivin obtained is correct, proves the plasmid vector success building AAV/Survivin further.

The preparation of the virus vector of embodiment 2, recombinant adeno-associated virus (rAAV) and titer determination (as shown in Figure 2 and Figure 5)

Material and source thereof:

A. the recombined glandulae correlation viral vectors carrying Survivin antigen gene of embodiment 1-1 structure.

B. containing the Rep gene of AAV and the helper plasmid pHelper of Lip/Cap gene: build (Liu by University of Arkansas of U.S. hospital attached to a medical college Gene Therapy Center professor Liu Yong, Y., Chiriva-Internati, M., Grizzi, F.Salati, E., Roman, J.J., LimS., andHermonat, P.L.RapidinductionofcytotoxicTcellresponseagainstcervica lcancercellsbyhumanpapillomavirustype16E6antigengenedeli veryintohumandendriticcellsbyanadeno-associatedvirusvect or.CancerGeneTherapy8:948-957.).

C. containing being integrated in cell chromosome and the adenoviral gene (E1 expressed, E2A, E4, VAI and VAII gene) AAVp cell strain: set up (Liu by University of Arkansas of U.S. hospital attached to a medical college Gene Therapy Center, Y., Chiriva-Internati, M., Grizzi, F.Salati, E., Roman, J.J., LimS., andHermonat, P.L.RapidinductionofcytotoxicTcellresponseagainstcervica lcancercellsbyhumanpapillomavirustype16E6antigengenedeli veryintohumandendriticcellsbyanadeno-associatedvirusvect or.CancerGeneTherapy8:948-957.).

D. lipofectamine Lipofectin: purchased from American Invotrogen company.

E.DMEM substratum and foetal calf serum (or calf serum): purchased from American Cellgro company.

F.PCRDIG labelling kit and DIG hybridization check test kit: purchased from Roche company of Switzerland.

G.DNA copy number standard: be respectively 1012 copy numbers (copies)/μ l to 109 (copies)/μ l, purchased from American ProMAGE company.

One, the preparation of the virus vector of recombinant adeno-associated virus (rAAV)

With reference to Fig. 2, recombinant adeno-associated virus (rAAV) is prepared by following method, to prepare the virus of a dish 10.0cm Tissue Culture Dish, when AAV-HEK293 cell grow in carbon dioxide cell incubator account for culture dish area 70% time, proceed as follows:

A. operate according to the operation instruction of Lipofectin: by the plasmid vector of 1.0 μ gAAV/Survivin, 1.0 μ gpHelper plasmids, 4.0 μ lLipofectin and 50.0 μ l mix containing the DMEM substratum of 5% (V/V) foetal calf serum (or calf serum), and room temperature leaves standstill 20 minutes.

B. mixed solution is added in Tissue Culture Dish, continue to be placed in carbon dioxide cell incubator and cultivate.

Hour C.72 after, all cells in results culture dish and nutrient solution.

D. thermal agitation is after 1 minute, centrifugal, retains supernatant, the i.e. virus liquid of rAAV.

E. by the virus liquid filtration sterilization of the rAAV of collection.By obtain the AAV viral nomenclature carrying tumor antigen gene-survivin gene total length SurvivincDNA be the virus vector of AAV/Survivin.

Two, the virus titer of the virus vector of recombinant adeno-associated virus (rAAV) measures

Adopt conventional spot hybridization, carry out virus titer mensuration to the virus vector of the AAV/Survivin that step one obtains, concrete grammar comprises the following steps: DNA probe only used is the specific probe for Survivin gene.

A. adopt conventional DNA phenol/chloroform extraction method, extract rAAV virion DNA.

B. nylon membrane is placed in Dot blot instrument, adds the rAAV virion DNA through alkaline denaturation, and add DNA copy number standard, vacuumize.

C., after taking out nylon membrane drying, ultraviolet is fixed.

D. with PCRDIG labelling kit and reference reagent box specification sheets prepare DIG mark specific probe, probe is " SurvivincDNA obtained in embodiment 1 ".After pcr amplification terminates, carry out 1.2% (V/V) agarose gel electrophoresis to pcr amplification product, detecting pcr amplification product under ultraviolet light, there is positive band in result, shows probe mark success.

E. use DIG hybridization check test kit and reference reagent box specification sheets, in hybrid heater, DNA hybridization is carried out to various rAAV virion DNA.The detected result of the virus vector of AAV/Survivin as shown in Figure 5.In Fig. 5,1 is standard substance, and 2-5 is 4 crowdes of AAV/Survivin, and the titre often criticizing the virus vector of AAV/Survivin can reach 10 ¹²copy/ml.

What the virus vector of embodiment 3, AAV/Survivin imported monocyte-dendritic cell system kills tumor experiment

Material and source thereof:

A. the virus vector of recombinant adeno-associated virus (rAAV): the virus vector of AAV/Survivin.

B.AIM-V cell culture medium: purchased from American LifeTechnologies company.

C. cytokine: granulocyte colony stimulating factor (GM-CSF), interleukin II, 4,7 (IL-2,4,7) and tumour necrosis factor (TNF-α) purchased from American R & D company.

The primary tumor cell of the D.Survivin positive: be respectively the liver cancer cell, lung adenocarcinoma cell, colon-cancer cell and the breast cancer cell that are separated from the tumor tissues of patient.

E.Survivin positive cell strain: 7721 hepatoma cell strains, obtains from American. tissue cell storage center (ATCC).

The negative primary cell of F.Survivin: the skin epithelial cell of antigen negative, pneumonocyte, mammary gland cell, obtain from ATCC.

One, tumor experiment is killed

As shown in Figure 6, the whole process killing tumor experiment based on the viral vector infection tumour patient monocyte of the rAAV of the Survivin of carrying antigen gene of the present invention is comprised the following steps:

A. tumour patient 50-150 milliliter peripheral blood is got, peripheral blood mononuclear cell (PBMC) is obtained according to a conventional method with hemocyte separometer (or lymphocyte separation medium), after mixing with AIM-V substratum, add Tissue Culture Dish, be placed in constant temperature CO2gas incubator and cultivate 2 hours.

B. isolated cell, removing suspension cell, retains attached cell (monocyte, monocyte, Mo).Suspension cell and peripheral blood lymphocyte, after itself and AIM-V substratum being mixed, continue cultivation for subsequent use.

C. in monocyte, add the virus vector of the rAAV that one (or multiple, effect is better) embodiment of the present invention 1-2 obtains, add-on is about 100-1000MOI, adds GM-CSF (800IU/mL) more simultaneously, continues cultivation 4 hours.

D. the old substratum of removing step C, supplements the AIM-V substratum containing GM-CSF, IL-4 (800IU/mL) and TNF-α (20IU/mL), continues to cultivate.

E. cultivate after 6 days, the dendritic cell (DC) that results are ripe, and mix with cultivated peripheral blood lymphocyte, in AIM-V substratum, add IL-2 (20IU/mL) and IL-7 (500IU/mL), continue to cultivate.

F., after being cultured to 6-12 days, the cytotoxic T lymphocyte (CTL) that results activate detects.

Two, the detection of dendritic cell (DC) and cytotoxic T lymphocyte (CTL)

The Efficiency testing of the viral vector infection peripheral blood mononuclear cell of A.rAAV

Adopt conventional fluorescence antibody mark staining, the monocyte infected by rAAV of the present invention obtained by specific fluorescent antibody (the purchased from American BD company) markers step one for tumor associated antigen-survivin (Survivin) or immature DC, then carry out the quantity of flow cytomery positive cell.Wherein, recombinant adeno-associated virus AAV/Survivin infects the Efficiency testing result of peripheral blood mononuclear cell as shown in Figure 7.The efficiency of infection of Fig. 7 to be promotor be virus vector of the AAV/Survivin of cytomegalovirus (CMV) promotor, the efficiency of the viral vector infection peripheral blood mononuclear cell of AAV/Survivin is 98.5%, namely the peripheral blood mononuclear cell of about 98.5% by rAAV virus infection, can prove that the virus vector of AAV/Survivin of the present invention has higher efficiency of infection.Point out the DC of about 90% to carry out angtigen presentation simultaneously, stimulate T lymphocyte.The efficiency of the viral vector infection dendritic cell of the AAV/Survivin of other 3 kinds of promotors is also all about 90%.

B. the detection of CD molecular level expressed of dendritic cell (DC)

DC expresses the level of CD80, CD83 and CD86 and the function of DC is proportionate.By the detection method identical with steps A, namely adopt the fluorescently-labeled antibody for these three kinds of CD molecules (purchased from American BD company) to detect the level that the DC that step one obtains expresses CD80, CD83 and CD86 respectively, the DC stimulated with Survivin albumen and non-stimulated DC is contrast.Wherein, the DC of the viral vector infection of AAV/Survivin expresses the detected result of CD80, CD83 and CD86 level as shown by figures 8 a-8 c, be the mean level (ML) of CD80, CD83 and CD86 of the DC expression of the viral vector infection of the AAV/Survivin of p5 promotor for promotor, the CD molecular level expressed by the DC of the viral vector infection of AAV/Survivin higher (expression efficiency is respectively 42.07%, 82.54% and 74.21%).After proving that the rAAV of the Survivin antigen gene building and prepare infects peripheral blood lymphocytes, the DC's induced is powerful.In addition, the mean level (ML) of DC expression CD80, CD83 and CD86 of the viral vector infection of three kinds of AAV/Survivin of CMV promoter, SV40 early promoter, beta-actin promotor is higher, and prompting can be reacted by effective stimulus Th1, cell immune response.

C. the detection of IFN-γ (IFN-γ) level expressed of cytotoxic T lymphocyte (CTL)

The expression level of the function of CTL and the ability of killing tumor cell and IFN-γ is proportionate.With the method similar with steps A detect the CTL that induces by the DC of the viral vector infection of rAAV of the present invention express the level (DC stimulated with Survivin albumen and non-stimulated DC the CTL that induces for contrasting) of IFN-γ, after DC and peripheral blood lymphocyte mixed culture terminate, harvested cell, traditional Intracellular cytokine staining methods is adopted to carry out cell fluorescence dye marker, antibody used is the fluorescent-labeled antibody (purchased from American BD company) for IFN-γ, finally utilizes flow cytomery result.Wherein, by the DC of the viral vector infection of AAV/Survivin the IFN-γ expression level of CTL of inducing as shown in Figure 9, the DC being the viral vector infection of the AAV/Survivin of SV40 early promoter for promotor stimulates the CTL produced to express IFN-γ mean level (ML), the CTL that induces by the DC of the viral vector infection of AAV/Survivin express the level of IFN-γ apparently higher than contrast.Prove to be built by the present invention and prepare carry the DC that tumour antigen-survivin gene rAAV infects the CTL that induces powerful.In addition, the level that the CTL that the DC of the viral vector infection of three kinds of AAV/Survivin of p5 promotor, CMV promoter, beta-actin promotor induces expresses IFN-γ is also higher, and prompting cell immune response is comparatively strong, that is the killing activity of CTL is high.

Three, cytotoxic T lymphocyte (CTL) killing tumor cell test

After being terminated by the DC of the viral vector infection of AAV/Survivin and lymphocyte mixed culture, by the cytotoxic T lymphocyte (CytotoxicTlymphocytes that induces by the DC of the viral vector infection of AAV/Survivin, CTL) after mixing with the tumour cell of the Survivin positive by 20:1 (lymphocyte: tumour cell), adopt traditional 51Cr (chromium-51) fragmentation test, detect the activity of CTL killing tumor cell, MHCclassI is restricted and kill and wound specificity.Detected result as shown in Figure 10, the CTL that induces by the DC of the viral vector infection of AAV/Survivin of the present invention can the tumour cell of cracking effectively (killing and wounding) the Survivin positive, kill rate can reach more than 60%, and the effect that tumour cell is killed in prompting is stronger.

With the liver of Survivin antigen negative, mammary gland and pneumonocyte for contrast, then with above-mentioned identical method detect above-mentioned by the DC of the viral vector infection of AAV/Survivin the specificity of CTL killing tumor cell of inducing.As shown in Figure 10, the rAAV (AAV/Survivin) carrying Survivin antigen gene built by the present invention and prepare viral vector infection DC the CTL that induces to the cell of Survivin antigen negative without lethal effect, show that this lethal effect has Survivin antigen-specific, prove the DC of the viral vector infection of the AAV/Survivin being built by the present invention and prepare the CTL that induces there is antigen-specific, namely to the cell of antigen negative without lethal effect.

For primary tumor cell and the cell strain of the above-mentioned Survivin positive, after first using MHCclassI antibody (purchased from American R & D company) to carry out pre-treatment, adopt traditional 51Cr (chromium-51) fragmentation test again, detect the activity of CTL killing tumor cell.Detected result as shown in Figure 10, shows that the tumor cytotoxicity effect of the Survivin positive is not obvious, is not is not substantially killed and wounded, prove that this lethal effect has MHCClassI restricted, is the feature of the lethal effect of CTL.This result prove the DC of the viral vector infection of the AAV/Survivin being built by the present embodiment and prepare the CTL that induces to have MHCClassI restricted.

Comprehensive above-mentioned detected result, prove the DC of the viral vector infection being built and prepare the rAAV carrying tumour antigen-survivin Survivin specific antigen by the present embodiment the tumour cell of CTL to the Survivin positive of inducing there is good curative effect, can be used for preparing antitumor drug.

The clinical experiment of embodiment 4, oncotherapy

The technology of application the invention described above, by the dendritic cell (DC) be separated from tumour patient by the viral vector infection of AAV/Survivin in embodiment 1-3 the cytotoxic T lymphocyte (CTL) of inducing feed back the cancer patients of 22 routine III-IV phase Survivn antigen positives, comprising 6 routine non-small cell adenocarcinomas of lung, 3 routine primary hepatocarcinoma, 8 routine mammary cancer and 5 routine intestinal cancer.

Infused cells number is 1 × 10 ⁸-5 × 10 ⁸.Treat the course for the treatment of: 12 months, monthly 2 times.Result for the treatment of reduces with the imaging examination knurl lifting capacity of patient; Blood serum tumor markers decline 50% and symptom are improved as judging criterion.Through the observation of 12 months, as shown in the table, the CTL that induces by the DC of the viral vector infection of rAAV of the present invention can play certain curative effect in most of tested patients's body, can effectively reduce or eliminate knurl lifting capacity, reduce blood serum tumor markers level, even recover normal and improve symptom.Patient over the course for the treatment of, occurs without obvious toxic side effect.This test-results show the CTL prepared by this kind of technology not only there is certain curative effect and also security higher, can be used for preparing antitumor drug.

Observation of curative effect result after cancer patients's treatment of table one, 22 routine III-IV phase Survivn antigen positives

Numbering

Diagnosis

Clinical stages

Knurl lifting capacity

Blood serum tumor markers

Symptom

Situation

1

Mammary cancer

III

Reduce

Reduce

Nothing

2

Mammary cancer

III

Disappear

Recover normal

Nothing

3

Mammary cancer

III

Disappear

Recover normal

Nothing

4

Mammary cancer

III

Increase

Raise

Nothing

Dead

5

Mammary cancer

IV/IV

Stable

Reduce

Nothing

6

Mammary cancer

IV

Increase

Raise

Exist

7

Mammary cancer

IV

Increase

Raise

Exist

Dead

8

Mammary cancer

IV

Reduce

Reduce

Exist

9

Colorectal carcinoma

III

Disappear

Recover normal

Nothing

10

Colorectal carcinoma

IV

Reduce

Reduce

Nothing

11

Colorectal carcinoma

IV

Reduce

Reduce

Exist

Dead

12

Colorectal carcinoma

IV

Reduce

Stable

Nothing

13

Colorectal carcinoma

IV

Stable

Raise

Exist

14

Lung cancer

III

Disappear

Recover normal

Nothing

15

Lung cancer

III

Reduce

Reduce

Nothing

16

Lung cancer

III

Reduce

Recover normal

Nothing

17

Lung cancer

III

Increase

Stable

Exist

Dead

18

Lung cancer

IV

Reduce

Reduce

Nothing

19

Lung cancer

IV

Reduce

Stable

Exist

Dead

20

Liver cancer

IV

Disappear

Recover normal

Nothing

21

Liver cancer

IV

Stable

Reduce

Nothing

22

Liver cancer

IV

Stable

Stable

Exist

Industrial applicability

Experiment proves, by the dendritic cell of the viral vector infection of Survivin recombinant adeno-associated virus rAAV of the present invention and the cytotoxic T lymphocyte of inducing effectively can suppress growth or the killing off tumor cells of associated malignancies cell in patient body, thus, Survivin recombined glandulae correlation viral vectors of the present invention or the product relevant to Survivin recombined glandulae correlation viral vectors of the present invention can be used to prepare antitumor drug, in malignant tumour as lung cancer, epithelial cell malignant tumour, prostate cancer, mammary cancer, colorectal carcinoma, cancer of the stomach, ovarian cancer, nasopharyngeal carcinoma, cervical cancer, have great importance in the clinical treatment of liver cancer etc. and application.

Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, make some substituting or obvious modification without departing from the inventive concept of the premise, and performance or purposes identical, all should be considered as belonging to protection scope of the present invention.

Claims (9)

1. carry a recombined glandulae correlation viral vectors for Survivin antigen gene, it is characterized in that: the structure gene of the adeno-associated virus in gland relevant viral vector is replaced with the recombined glandulae correlation viral vectors that Survivin antigen gene obtains.

2. recombined glandulae correlation viral vectors according to claim 1, is characterized in that: the p5 promotor of described adeno-associated virus replaces with the one in following three kinds of promotors: cytomegalovirus promotor, beta-actin promotor, SV40 early promoter.

3. one kind is carried the construction process of the recombined glandulae correlation viral vectors of Survivin antigen gene, it is characterized in that: in gland relevant viral vector, the structure gene of adeno-associated virus is rejected, Survivin antigen gene is inserted in the position of rejecting, obtains recombined glandulae correlation viral vectors.

4. construction process according to claim 3, is characterized in that: further comprising the steps of: the p5 promotor of described adeno-associated virus is replaced with the one in following three kinds of promotors: cytomegalovirus promotor, beta-actin promotor, SV40 early promoter.

5. construction process according to claim 4, is characterized in that: comprise the following steps:

1) AAV2 type pBR322 plasmid is reconstructed: use restriction enzyme Bst98I and HpaI by the structure gene excision in the AAV2 type adeno-associated virus in AAV2 type pBR322 plasmid, then insert in plasmid by the nucleotide sequence CGAATTCATGCGATATCGTT containing restriction enzyme EcoRI and EcoRV restriction enzyme site, the 75th the nucleotide sequence place retaining the TR sequence at two ends or the TR sequence at two ends all inserts the fragment CTGCGCTGG be made up of 9 Nucleotide;

2) by promotor inserting step 1) in the AAV2 type pBR322 plasmid of reconstruction that obtains, build the AAV2 type pBR322 plasmid carrying promotor; Described promotor is the one in p5 promotor or following three kinds of promotors: cytomegalovirus promotor, beta-actin promotor or SV40 early promoter;

3) SurvivincDNA is obtained;

4) by step 2) plasmid that builds and step 3) SurvivincDNA that obtains carries out enzyme with restriction enzyme MluI and xbaI respectively and cuts, then carry out DNA ligation, obtain the recombined glandulae correlation viral vectors carrying promotor and Survivin antigen gene.

6. construction process according to claim 5, is characterized in that: described step 3) in comprise the following steps: 31) obtain total mRNA from the tumor tissues of Survivin antigen positive, carry out reverse transcription reaction by total mRNA, synthesize total cDNA; 32) with described total cDNA for template, under the guiding of primer 1:ACGCGTACCGCCAGATTTGAA and primer 2: CCTCGAGCCTCAATCCAT, carry out pcr amplification, the SurvivincDNA obtained.

7. a product relevant to the recombined glandulae correlation viral vectors carrying Survivin antigen gene, is characterized in that: comprise the plasmid vector of Survivin recombinant adeno-associated virus, Survivin recombinant adeno-associated virus virus vector, by the described viral vector infection of Survivin recombinant adeno-associated virus or the clone of transfection; The plasmid vector of described Survivin recombinant adeno-associated virus by described in claim 1 ~ 2 or any one of claim 3 ~ 6 described in the recombined glandulae correlation viral vectors carrying Survivin antigen gene that obtains of construction process obtain; The virus vector of described Survivin recombinant adeno-associated virus carries out cell cultures by the plasmid vector of described Survivin recombinant adeno-associated virus and obtains.

8. a preparation method for the product relevant to the recombined glandulae correlation viral vectors carrying Survivin antigen gene, is characterized in that:

The preparation of the plasmid vector of Survivin recombinant adeno-associated virus: by as described in claim 1 ~ 2 or any one of claim 3 ~ 6 as described in the recombined glandulae correlation viral vectors quiding gene engineering colon bacillus DH5 α competent cell carrying Survivin antigen gene that obtains of construction process, resistance screening is carried out with the LB flat board containing 100 μ g/mL penbritins, the single bacterium colony of picking white, extract plasmid and purifying, obtain the plasmid vector of Survivin recombinant adeno-associated virus;

The preparation of the virus vector of Survivin recombinant adeno-associated virus: the virus vector obtaining Survivin recombinant adeno-associated virus with the plasmid vector of described Survivin recombinant adeno-associated virus and pHelper plasmid co-transfection AAVp cell;

The preparation of the viral vector infection of Survivin recombinant adeno-associated virus or the clone of transfection: obtain with the viral vector infection of described Survivin recombinant adeno-associated virus or transfection monocyte, dendritic cell or T lymphocyte, described clone comprises monocyte-dendritic cell system, T lymphocyte series.

9. relevant product described in the recombined glandulae correlation viral vectors carrying Survivin antigen gene as claimed in claim 1 or claim 7 is preparing the application in antitumor drug.