CN105087648A - Recombinant adeno-associated virus vector carrying MAGE-A3 gene as well as construction method and application - Google Patents
Recombinant adeno-associated virus vector carrying MAGE-A3 gene as well as construction method and application Download PDFInfo
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Abstract
The invention discloses a recombinant adeno-associated virus vector carrying an MAGE-A3 gene as well as a construction method and application. The rAAV (recombinant adeno-associated virus) is obtained by inserting the MAGE-A3 gene in the place, where an adeno-associated virus structural gene is deleted, in the adeno-associated virus vector. The rAAV can deliver the MAGE-A3 gene carried by the rAAV to a monocyte-dendritic cell line to be used for stimulating effector cells of the immune system. Experiment results show that the CTL induced by DCs infected by the virus vector of the rAAV can effectively restrain MAGE-A3 antigen-positive malignant cell growth or kill tumor cells in or out of bodies of patients. The recombinant adeno-associated virus vector or related products thereof can be used for preparing drugs for treating MAGE-A3 positive tumors.
Description
Technical field
The present invention relates to the carrier in biological field and application thereof, particularly relate to and carry melanic related antigen-A3 (Melanoma-associatedAntigen-A3; MAGE-A3) recombined glandulae correlation viral vectors of antigen gene and construction process thereof and its application in the tumour medicine preparing the anti-MAGE-A3 positive.
Background technology
Adeno-associated virus (Adeno-associatedvirus; AAV) gene structure 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 replication protein (rep) gene and envelope protein (cap) gene (1.HermonatPL of the low infectious particles of AAV, LabowMA, WrightR, BernsKI, MuzyczkaN.Geneticsofadeno-associatedvirus:isolationandpr eliminarycharacterizationofadeno-associatedvirustype2mut ants.JVirol.51:329-339.2.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 proves that AAV carrier can be used for the gene therapy (Hermonat of human diseases, P.L., andMuzyczka, N.Useofadeno-associatedvirusasamammalianDNAcloningvector: transductionofneomycinresistanceintomammaliantissuecultu recells.Proc.Natl.Acad.Sci.U.S.A.81:6466-6470.).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 has the non-neoplastic disease 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-1) foreign gene-carrying to be used for the treatment of lipoprotein lipase deficiency inherited disease (LPLD).
(AAV-2) carrier 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 terminal repetition fragment (TR), and centre is the structure gene of virus, comprises replication protein (Rep) and envelope protein (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: propose the recombinant adeno-associated virus (RecombinantAdeno-associatedvirus that a kind of stability is high, carry MAGE-A3 antigen gene; RAAV) carrier and construction process and application.
Technical problem of the present invention is solved by following technical scheme:
Carrying a recombined glandulae correlation viral vectors for MAGE-A3 antigen gene, is the structure gene of the adeno-associated virus in gland relevant viral vector is replaced with the recombined glandulae correlation viral vectors that MAGE-A3 antigen gene obtains.
Preferably, the p5 promotor in described recombined glandulae correlation viral vectors can also be replaced with the one in following three kinds of promotors: cytomegalovirus promotor, beta-actin promotor and SV40 early promoter.
A kind of construction process carrying the recombined glandulae correlation viral vectors of MAGE-A3 antigen gene, the structure gene of the adeno-associated virus in gland relevant viral vector is rejected, then on the position of rejecting, insert MAGE-A3 antigen gene, obtain recombined glandulae correlation viral vectors.
Preferably, further comprising the steps of: the p5 promotor in described recombined glandulae correlation viral vectors to be replaced with the one in following three kinds of promotors: cytomegalovirus promotor, beta-actin promotor and SV40 early promoter.
Preferably, comprise the following steps:
1) AAV-2 type pBR322 plasmid is reconstructed: use restriction enzyme Bst98I and HpaI by the structure gene excision in the AAV-2 type adeno-associated virus in AAV-2 type pBR322 plasmid, then inserted by the nucleotide sequence CGAATTCATGCGATATCGTT containing restriction enzyme EcoRI and EcoRV restriction enzyme site in described AAV-2 type pBR322 plasmid, the reservation TR sequence at two ends or the 75th nucleotide sequence place of TR sequence complete at two ends respectively all insert the fragment that 9 Nucleotide CTGCGCTGG form;
2) by promotor inserting step 1) in the AAV-2 type pBR322 plasmid of reconstruction that obtains, build the AAV-2 type pBR322 plasmid carrying promotor; Described promotor is the one in following four kinds of promotors: p5 promotor, cytomegalovirus promotor, beta-actin promotor and SV40 early promoter;
3) MAGE-A3cDNA is obtained;
4) by step 2) build described in carry AAV-2 type pBR322 plasmid and the step 3 of promotor) MAGE-A3cDNA that obtains carries out enzyme with restriction enzyme BamHI and PstI respectively and cuts, then carry out DNA ligation, obtain the recombined glandulae correlation viral vectors carrying described promotor and MAGE-A3 antigen gene.
Preferably, described step 3) in comprise the following steps: 31) from the tumor tissues of MAGE-A3 antigen positive, extract total mRNA, carry out reverse transcription reaction by total mRNA, synthesize total cDNA; 32) with described total cDNA for template, under the guiding of primer 1:GTGCTCAGACTCACTCTT and primer 2: AGTCATCATGCCTCTTGA, carry out pcr amplification, obtain described MAGE-A3cDNA.
A product relevant to the recombined glandulae correlation viral vectors carrying MAGE-A3 antigen gene, comprise the plasmid vector of MAGE-A3 recombinant adeno-associated virus, MAGE-A3 recombinant adeno-associated virus virus vector, by the described viral vector infection of MAGE-A3 recombinant adeno-associated virus or the clone of transfection; The plasmid vector of described MAGE-A3 recombinant adeno-associated virus is obtained by described or that described construction process the obtains recombined glandulae correlation viral vectors carrying MAGE-A3 antigen gene; The virus vector of described MAGE-A3 recombinant adeno-associated virus carries out cell cultures by the plasmid vector of described MAGE-A3 recombinant adeno-associated virus and obtains.
A preparation method for the product relevant to the recombined glandulae correlation viral vectors carrying MAGE-A3 antigen gene,
The preparation of the plasmid vector of MAGE-A3 recombinant adeno-associated virus: by described or that described construction process the obtains recombined glandulae correlation viral vectors quiding gene engineering colon bacillus DH5 α competent cell carrying MAGE-A3 antigen gene, 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 MAGE-A3 recombinant adeno-associated virus;
The preparation of the virus vector of MAGE-A3 recombinant adeno-associated virus: the virus vector obtaining MAGE-A3 recombinant adeno-associated virus with the plasmid vector of described MAGE-A3 recombinant adeno-associated virus and pHelper plasmid co-transfection AAVp cell;
The preparation of the viral vector infection of MAGE-A3 recombinant adeno-associated virus or the clone of transfection: by the viral vector infection of described MAGE-A3 recombinant adeno-associated virus or transfection monocyte or dendritic cell, described clone comprises monocyte-dendritic cell system.
A kind of described recombined glandulae correlation viral vectors carrying MAGE-A3 antigen gene or described related products are preparing the application in antitumor drug.
The beneficial effect that the present invention is compared with the prior art is:
The invention provides recombinant adeno-associated virus (below " carrying the recombinant adeno-associated virus of MAGE-A3 antigen gene " the being called AAV/MAGE-A3) carrier that a kind of stability is high, carry allogenic gene (MAGE-A3).The MAGE-A3 antigen gene that recombined glandulae correlation viral vectors of the present invention can be carried is conveyed in monocyte-dendritic cell system, and the cell that there is MAGE-A3 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, by the dendritic cell of the viral vector infection of AAV/MAGE-A3 of the present invention the cytotoxic T lymphocyte of inducing effectively can suppress growth or the killing off tumor cells of associated malignancies cell in patient body, thus, AAV/MAGE-A3 carrier of the present invention or the product relevant to AAV/MAGE-A3 carrier of the present invention can be used to the cellular immunotherapy of the malignant tumour preparing the anti-MAGE-A3 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 of the AAV/MAGE-A3 carrier of the specific embodiment of the invention.
Fig. 2 is the schema that the structure AAV/MAGE-A3 carrier of the specific embodiment of the invention and preparation have the virus vector of infective AAV/MAGE-A3.
Fig. 3 is the PCR qualification result figure of the AAV/MAGE-A3 carrier of the specific embodiment of the invention.
The comparison diagram of the gene order that Fig. 4 announces for the MAGE-A3 antigen gene sequences that obtains in the specific embodiment of the invention and U.S. NCBIGeneBank.
Fig. 5 is the virus titer detected result figure of the virus vector of the AAV/MAGE-A3 of the specific embodiment of the invention.
Fig. 6 is the killing off tumor cells experiment flow figure based on the viral vector infection tumour patient monocyte of the AAV/MAGE-A3 of the specific embodiment of the invention.
Fig. 7 a-7d is respectively the Efficiency testing result figure containing the viral vector infection dendritic cell (DC) of the AAV/MAGE-A3 of different promotors respectively of the specific embodiment of the invention.
Fig. 8 a-8c is respectively the detected result figure of DC expression CD80, CD83 and CD86 level of the viral vector infection of the AAV/MAGE-A3 of the specific embodiment of the invention.
The CTL that the DC of the viral vector infection of the AAV/MAGE-A3 that Fig. 9 is the specific embodiment of the invention induces kills and wounds the tumour cell of MAGE-A3 antigen positive and kills and wounds specific detection result figure.
The changing conditions schematic diagram of cyfra21-1 antigen levels in serum after the CTL treatment that Figure 10 is the specific embodiment of the invention 4 routine non-small cell patients with lung adenocarcinomas are induced through the DC of the viral vector infection of AAV/MAGE-A3.
Embodiment
Below contrast accompanying drawing and combine preferred embodiment the invention will be further described.
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 AAV-2 complete genome DNA, retain terminal repetition fragment and p5 promotor, or only retain terminal repetition fragment, and insert oligonucleotide segment, to improve the stability of efficiency that rAAVDNA copies and rAAV, obtain the basic framework of AAV-2 carrier thus.The AAV-2 carrier applied has p5 promotor, is inserted wherein by MAGE-A3 antigen gene.For improving the transcriptional level of MAGE-A3 antigen gene, also this MAGE-A3 antigen gene can be inserted that the promotor that success has built be cytomegalovirus (CMV) promotor, vacuolating virus of monkey 40 (SV40) early promoter, beta-actin promotor instead of in the AAV-2 carrier of p5 promotor, also namely the promotor of the recombined glandulae correlation viral vectors of the MAGE-A3 of carrying antigen gene of the present invention is p5 promotor or CMV promoter or beta-actin promotor or SV40 early promoter.In addition, successfully having prepared on this basis and there is infective rAAV virion (see Chinese patent ZL201110125683.X), having laid a good foundation for developing new rAAV product.
MAGE-A3 antigen is distributed widely in Several Kinds of Malignancy, and (testis and placenta tissue except) does not express in the normal tissue.This antigen gene is positioned at people's X chromosome q28 district, and containing 3 exons, the molecular weight of the protein of its coding is 48000.Be made up of 314 amino acid.5 MHC-I quasi-molecule restricted epitopes and 6 MHC-II quasi-molecule restricted epitopes are had at least in its protein molecule.By corresponding MHC molecule by its information submission to T lymphocyte, thus to produce for the specific cell immunoreaction of MAGE-A3.It is the target antigen that of cellular immunotherapy is desirable that this antigen has been recognized.
Namely AAV/MAGE-A3 carrier of the present invention is insert MAGE-A3 antigen gene in the skeleton of the AAV-2 carrier successfully built contriver to obtain recombined glandulae correlation viral vectors, and the product relevant to this carrier comprises plasmid vector, virus vector and clone.Wherein, MAGE-A3 is tumor associated antigen genes.
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, AAV/MAGE-A3 carrier and qualification
One, material and source thereof:
1. four kinds have AAV2 type (AAV-2) pBR322 plasmid (pBR-AAV2) of different promoters: successfully construct (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, CMV promoter, SV40 early promoter and people's beta-actin (β-actin) promotor.The feature of this plasmid is complete terminal repetition segment (TR) sequences in two ends, or the fragment of 9 Nucleotide CTGCGCTGG compositions is all inserted respectively 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 eliminated the structure gene comprising replication protein gene (rep) and envelope protein gene (cap) of AAV-2.
2. people's melanoma cancer tissue: the cancerous tissue deriving from excision, immunohistochemical methods turns out to be MAGE-A3 antigen positive.
3. gene amplification nucleotide primer: according to human melanoma antigen-A3 (MAGE-A3) gene order design (U.S. NCBI gene pool: BC000340) published in U.S.'s gene pool.
Two, the recombined glandulae correlation viral vectors (as illustrated in fig. 1 and 2) that the present embodiment carries MAGE-A3 antigen gene is built.
The construction process that the present embodiment provides, it is the method using conventional gene recombination, restriction enzyme is used first to be cut off by AAV-2 carrier framework DNA, use DNA interconnection technique again, specific antigen gene MAGE-A3 is connected with cut-off AAV-2 carrier DNA, obtains AAV/MAGE-A3 carrier.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 melanoma cancer tissue of MAGE-A3 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.Concentration, with deionized water dissolving, is adjusted to 10ng/ μ l by throw out (i.e. total mRNA), obtains total mRNA solution.With the total mRNA solution of 10 μ l for template, carry out reverse transcription reaction, synthesize total cDNA, the reaction system of reverse transcription reaction, for 25 μ l, comprising: 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 MAGE-A3cDNA, concrete grammar is: total cDNA is at primer 1 (as SEQIDNO:1): GTGCTCAGACTCACTCTT and primer 2 (as SEQIDNO:2): carry out pcr amplification under the guiding of AGTCATCATGCCTCTTGA, obtain MAGE-A3cDNA.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 962bp place appearance one expection, this object band is reclaimed and obtains the MAGE-A3cDNA (as SEQIDNO:3) that length is 962bp after purifying, be illustrated in figure 3 the PCR detected result of constructed AAV/MAGE-A3 carrier, result shows that MAGE-A3cDNA successfully inserts AAV-2 carrier, wherein 1 represents DNA molecular amount standard, and 2-5 is 4 positive colonies.Carry out determined dna sequence again, its nucleotide sequence as shown in Figure 4, gene order 99% homology of the MAGE-A3cDNA sequence that recombinant adeno-associated virus (rAAV) carries and the MAGE-A3 that U.S. NCBIGeneBank announces, proves that the MAGE-A3 antigen gene sequences of pcr amplification is correct further.
3. build AAV/MAGE-A3 carrier: carry 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 MAGE-A3cDNA respectively.Restriction enzyme used equal purchased from American Promega company.Endonuclease reaction system is: 1 μ gpBR-AAV2 plasmid or MAGE-A3cDNA; 10U restriction enzyme BamHI and PstI (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 pBR-AAV2 plasmid, 300ng enzyme cut after MAGE-A3cDNA, 10IUT
4dNA ligase (purchased from American Promega company), 1.5 μ l10 × T
4dNA connects 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 MAGE-A3cDNA respectively, the recombined glandulae correlation viral vectors carrying CMV promoter and MAGE-A3cDNA, carry the recombined glandulae correlation viral vectors of SV40 early promoter and MAGE-A3cDNA and carry the recombined glandulae correlation viral vectors of beta-actin promotor and MAGE-A3cDNA.
4. by quiding gene engineering colon bacillus (E.coli) DH5 α competent cell (American I nvitrogen company) respectively of the AAV/MAGE-A3 carrier after connection, resistance screening is carried out with the LB flat board containing 100 μ g/mL penbritins (Amp), the single bacterium colony of picking white, extract plasmid and purifying, obtain the plasmid vector of a large amount of AAV/MAGE-A3.
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 MAGE-A3cDNA 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/MAGE-A3 can be judged to be at 962bp place appearance specific band.Being reclaimed and obtain length after purifying by this object band is 962bpMAGE-A3cDNA (as shown in Figure 3).
2.DNA sequencing: AAV/MAGE-A3 is carried out determined dna sequence.The nucleotide sequence of its sequencing result as shown in Figure 4, proves the plasmid vector success building AAV/MAGE-A3 further.
The preparation of the virus vector of embodiment 2, recombinant adeno-associated virus (rAAV) and virus titer measure (as shown in Fig. 2,5)
Material and source thereof:
A. the AAV/MAGE-A3 carrier of embodiment 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 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 expressing adenoviral gene (E1, 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 6X10
9copy number (copies)/μ l to 6X10
10copy number (copies)/μ l, purchased from American Promega 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 AAVp 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/MAGE-A3,1.0 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 liquor, i.e. rAAV virus liquid.
The rAAV virus liquid filtration sterilization of E. will collect.By obtain the rAAV viral nomenclature carrying tumor antigen gene-human melanoma antigen full length gene MAGE-A3cDNA be the virus vector of AAV/MAGE-A3.
Two, the virus titer of the virus vector of AAV/MAGE-A3 measures
Adopt conventional spot hybridization, carry out virus titer mensuration to the virus vector of the AAV/MAGE-A3 that step one obtains, concrete grammar comprises the following steps: DNA probe only used is the specific probe for MAGE-A3 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 the specific probe of DIG mark prepared by reference reagent box specification sheets, probe be " in embodiment 1 acquisition MAGE-A3cDNA ".After pcr amplification terminates, carry out 1.2% (W/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 rAAV viral DNA.As shown in Figure 5, the virus titer of the virus vector of AAV/MAGE-A3 is at least 6X10 to the virus titer detected result of the virus vector of AAV/MAGE-A3
10copy number/ml.
What the virus vector of embodiment 3, AAV/MAGE-A3 imported monocyte-dendritic cell system kills tumor experiment
Material and source thereof:
The virus vector of the virus vector of A.rAAV: AAV/MAGE-A3.
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.MAGE-A3 positive: be respectively the malignant melanoma cell, lung adenocarcinoma cell and the breast cancer cell that are separated from the tumor tissues of patient.
E.MAGE-A3 positive cell strain: A375 melanoma cell strain, obtains from American. tissue cell storage center (ATCC).
The negative primary cell of F.MAGE-A3: the skin epithelial cell of antigen negative, lung, mammary gland cell, obtain from ATCC.
One, tumor experiment step is killed
As shown in Figure 6, the whole process killing tumor experiment based on the viral vector infection tumour patient monocyte of AAV/MAGE-A3 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 Flask, be placed in constant temperature CO2gas incubator and cultivate 2 hours.
B. remove suspension cell, retain attached cell (monocyte, monocyte, Mo).Suspension cell and peripheral blood lymphocyte, after itself and AIM-V substratum being mixed, continue cultivation for subsequent use.Monocyte is the precursor cells of dendritic cell (DendriticCells, DC), becomes DC through the induction of the cell in vitro factor.
C. in monocyte, add the virus vector of the AAV/MAGE-A3 that the embodiment of the present invention 2 obtains, add-on is 100 ~ 1000MOI (being 100MOI in this example), 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 5 days, the 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 7-9 days, the cytotoxic T lymphocyte (CTL) that results activate detects.
Two, the Characteristic and function of dendritic cell (DC) detects
The Efficiency testing of the viral vector infection dendritic cell of A.AAV/MAGE-A3
Adopt conventional fluorescence antibody mark staining, with the specific fluorescent antibody (purchased from American BD company) for MAGE-A3 mark above-mentioned acquisition by the dendritic cell of the viral vector infection of AAV/MAGE-A3 of the present invention (DC), then carry out Flow Cytometry and detect the quantity of positive cell.The Flow Cytometry detected result of the efficiency of the viral vector infection DC of AAV/MAGE-A3 is as shown in Fig. 7 a-7d, promotor in Fig. 7 a-7d is respectively p5 promotor, CMV promoter, SV40 early promoter and beta-actin promotor, and the efficiency infecting DC is respectively 91.5%, 92.1%, 88.35%, 87.0%.Namely point out the DC of 90 about percent can express MAGE-A3 antigen, prove that the virus vector of rAAV of the present invention has higher efficiency of infection and DC antigen uptaking efficiency.
B. the detection of the CD molecular level 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 level of by the DC of the viral vector infection of of the present invention AAV/MAGE-A3 being expressed CD80, CD83 and CD86 of the fluorescently-labeled antibody for these three kinds of CD molecules (purchased from American BD company) to above-mentioned acquisition to carry out conventional Flow Cytometry respectively and detect.The Flow Cytometry detected result of the expression level of CD80, CD83 and CD86 tri-kinds of CD molecules as shown by figures 8 a-8 c, the CD molecular level expressed by the DC of the viral vector infection of AAV/MAGE-A3 higher (expression efficiency is respectively 70.1%, 50.6% and 81.0%).After the viral vector infection DC of the constructed AAV/MAGE-A3 with preparing of result prompting, the DC induced stimulates Th1 reaction, and namely cell immune response is powerful.
Three, cytotoxic T lymphocyte (CTL) killing tumor cell test
After the DC of the viral vector infection of AAV/MAGE-A3 and lymphocyte mixed culture terminate, by the cytotoxic T lymphocyte (CytotoxicTlymphocytes that induces by the DC of the viral vector infection of AAV/MAGE-A3, CTL) by after 20:1 (lymphocyte: tumour cell or lymphocyte: normal cell) and the tumour cell of MAGE-A3 antigen positive or the cytomixis of MAGE-A3 antigen negative, adopt traditional
51cr (chromium-51) fragmentation test, detects the activity of CTL killing tumor cell and kills and wounds specificity.Wherein
51the cytotoxic T lymphocyte (CTL) that the DC of viral vector infection that Cr (chromium-51) fragmentation test detects AAV/MAGE-A3 induces kills and wounds the tumour cell of MAGE-A3 antigen positive and kills and wounds specific detection result as shown in Figure 9, and killer cell result as shown in the figure repeats the mean value that this test obtains for 6 times.Result shows, the CTL that induces by the DC of the viral vector infection of AAV/MAGE-A3 of the present invention can the tumour cell of cracking effectively (killing and wounding) the MAGE-A3 positive, comprise melanoma cell, lung carcinoma cell, breast cancer cell and A375 cell strain, killed tumour cell is on average more than 70%.With the cell of the skin epithelial cell of MAGE-A3 antigen negative, pneumonocyte, mammary gland cell for contrast, then with above-mentioned identical method detect above-mentioned the CTL that induces by the DC of the viral vector infection of the AAV/MAGE-A3 specificity of killing and wounding.Detected result as shown in Figure 9, built by the present invention and prepare (DC of the viral vector infection of AAV/MAGE-A3 the CTL that induces to the cell of these MAGE-A3 antigen negatives without lethal effect, prove the DC of the viral vector infection of the AAV/MAGE-A3 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, prove that this lethal effect has the antigen-specific of height, meet the feature of the lethal effect of CTL completely.。
Comprehensive above-mentioned detected result, prove the DC of the viral vector infection being built and prepare the recombinant adeno-associated virus carrying MAGE-A3 antigen gene by the present invention the tumour cell of CTL to the MAGE-A3 positive of inducing there is obvious cracking (killing and wounding) effect, can be used for preparing antitumor drug.
The clinical trial of embodiment 4, oncotherapy
The present embodiment provides a kind of recombined glandulae correlation viral vectors of the MAGE-A3 of carrying antigen gene or its related products preparing the application in antitumor drug, especially the application in the malignant tumor medicine of the anti-MAGE-A3 positive, wherein the malignant tumour of the MAGE-A3 positive includes but not limited to the melanoma of the MAGE-A3 positive, lung cancer, mammary cancer and intestinal cancer etc.
The activeconstituents of antitumor drug is the recombined glandulae correlation viral vectors of the above-mentioned MAGE-A3 of carrying antigen gene or the product relevant to the recombined glandulae correlation viral vectors carrying MAGE-A3 antigen gene.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, can also add one or more pharmaceutically acceptable carriers in said medicine, described carrier can comprise the thinner of pharmaceutical field routine, absorption enhancer and tensio-active agent etc.
Application method can be the monocyte first isolated in tumour patient body, again by the monocyte of the viral vector infection of AAV/MAGE-A3 or transfection patient, maybe the cytotoxic T lymphocyte feedback tumour patient having the dendritic cell of the maturation of MAGE-A3 antigen gene institute to stimulate generation will be transformed.For improving curative effect, said medicine can also carry out combined therapy with microbiotic, immunostimulant and tumor-targeting drug etc.
The present embodiment also provides a kind of method of killing in vitro tumour, comprises the following steps:
1) by spontaneous monocyte-dendritic cell in tumour place system (this system produce by the mode of manual simulation or in tumour patient body) by the viral vector infection of AAV/MAGE-A3 or transfection, or by the product treatment relevant to AAV/MAGE-A3 carrier, obtain the cell after processing separately;
2) by step 1) in monocyte-dendritic cell after process add in the system of tumour place and kill tumour; Or the monocyte after not processed T lymphocyte and described process-dendritic cell mixed culture is formed Antigen-specific cytotoxic T lymphocyte, then this Antigen-specific cytotoxic T lymphocyte is added in the system of tumour place kill tumour; Maybe processed T lymphocyte and not processed monocyte-dendritic cell are added in the system of tumour place and kill tumour.
Also namely, give a tumour patient and feed back MAGE-A3 Antigen-specific cytotoxic T lymphocyte, the spontaneous T lymphocyte that this cell origin comes from patient produces with the monocyte-dendritic cell mixed culture deriving from this patient.Before mixed culture, these have been carried viral vector infection or the transfection of the recombinant adeno-associated virus of MAGE-A3 antigen gene by the present invention in monocyte-dendritic cell, or by the product treatment relevant to recombined glandulae correlation viral vectors of the present invention; Or, give a tumour patient and feed back the monocyte-dendritic cell deriving from patient.Before feedback, these have been carried viral vector infection or the transfection of the recombinant adeno-associated virus of MAGE-A3 antigen gene by the present invention in monocyte-dendritic cell, or by the product treatment relevant to recombined glandulae correlation viral vectors of the present invention.
Be specially in this example: by the dendritic cell (DC) be separated from tumour patient by the viral vector infection of AAV/MAGE-A3 in embodiment 3 the cytotoxic T lymphocyte (CTL) of inducing feed back 4 routine non-small cell patients with lung adenocarcinomas.
Infused cells number is 1 × 10
8-5 × 10
8(in this example, infusion amount is 100 μ l/5 × 10 to individual CTL cell
6individual monocyte/each).Treat the course for the treatment of: be generally 6 months, monthly 2 times, the state of an illness can be kept to monthly 1 time after improving, and can be kept to further and treat every 1-3 month 1 time (dosage and the course for the treatment of all can adjust according to practical situation).Result for the treatment of is obviously reduced to judging criterion with the serum cyfra21-1 antigen of patient.Figure 10 is the changing conditions of the serum cyfra21-1 antigen levels of 4 routine non-small cell patients with lung adenocarcinomas after the CTL treatment that the DC of the viral vector infection of rAAV/MAGE-A3 induces.Observe through the research Clinical Treatment Tests of 6 months, as shown in the figure, result shows that the serum cyfra21-1 level of patient obviously declines, and even recovers normal.The CTL that induces by the DC of the viral vector infection of rAAV of the present invention can play certain curative effect in patient body, effectively can reduce the level of serum cyfra21-1 antigen, even make it turn out cloudy, and occur without obvious toxic side effect over the course for the treatment of without patient.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.
industrial applicability
Experiment proves, by the dendritic cell of the viral vector infection of MAGE-A3 recombinant adeno-associated virus 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, MAGE-A3 recombined glandulae correlation viral vectors of the present invention or the product relevant to MAGE-A3 recombined glandulae correlation viral vectors of the present invention can be used to prepare antitumor drug, malignant tumour as but be not limited to melanoma, lung cancer, mammary cancer and intestinal cancer etc. clinical treatment and application in have great importance.
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 MAGE-A3 antigen gene, it is characterized in that: be the structure gene of the adeno-associated virus in gland relevant viral vector is replaced with the recombined glandulae correlation viral vectors that MAGE-A3 antigen gene obtains.
2. recombined glandulae correlation viral vectors according to claim 1, is characterized in that: the p5 promotor in described recombined glandulae correlation viral vectors is replaced with the one in following three kinds of promotors: cytomegalovirus promotor, beta-actin promotor and SV40 early promoter.
3. one kind is carried the construction process of the recombined glandulae correlation viral vectors of MAGE-A3 antigen gene, it is characterized in that: the structure gene of the adeno-associated virus in gland relevant viral vector is rejected, then on the position of rejecting, insert MAGE-A3 antigen gene, obtain recombined glandulae correlation viral vectors.
4. construction process according to claim 3, is characterized in that: further comprising the steps of: the p5 promotor in described recombined glandulae correlation viral vectors is replaced with the one in following three kinds of promotors: cytomegalovirus promotor, beta-actin promotor and SV40 early promoter.
5. construction process according to claim 4, is characterized in that: comprise the following steps:
1) AAV-2 type pBR322 plasmid is reconstructed: use restriction enzyme Bst98I and HpaI by the structure gene excision in the AAV-2 type adeno-associated virus in AAV-2 type pBR322 plasmid, then inserted by the nucleotide sequence CGAATTCATGCGATATCGTT containing restriction enzyme EcoRI and EcoRV restriction enzyme site in described AAV-2 type pBR322 plasmid, the reservation TR sequence at two ends or the 75th nucleotide sequence place of TR sequence complete at two ends respectively all insert the fragment that 9 Nucleotide CTGCGCTGG form;
2) by promotor inserting step 1) in the AAV-2 type pBR322 plasmid of reconstruction that obtains, build the AAV-2 type pBR322 plasmid carrying promotor; Described promotor is the one in following four kinds of promotors: p5 promotor, cytomegalovirus promotor, beta-actin promotor and SV40 early promoter;
3) MAGE-A3cDNA is obtained;
4) by step 2) build described in carry AAV-2 type pBR322 plasmid and the step 3 of promotor) MAGE-A3cDNA that obtains carries out enzyme with restriction enzyme BamHI and PstI respectively and cuts, then carry out DNA ligation, obtain the recombined glandulae correlation viral vectors carrying described promotor and MAGE-A3 antigen gene.
6. construction process according to claim 5, is characterized in that: described step 3) in comprise the following steps: 31) from the tumor tissues of MAGE-A3 antigen positive, extract total mRNA, carry out reverse transcription reaction by total mRNA, synthesize total cDNA; 32) with described total cDNA for template, under the guiding of primer 1:GTGCTCAGACTCACTCTT and primer 2: AGTCATCATGCCTCTTGA, carry out pcr amplification, obtain described MAGE-A3cDNA.
7. a product relevant to the recombined glandulae correlation viral vectors carrying MAGE-A3 antigen gene, is characterized in that: comprise the plasmid vector of MAGE-A3 recombinant adeno-associated virus, MAGE-A3 recombinant adeno-associated virus virus vector, by the described viral vector infection of MAGE-A3 recombinant adeno-associated virus or the clone of transfection; The plasmid vector of described MAGE-A3 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 MAGE-A3 antigen gene that obtains of construction process obtain; The virus vector of described MAGE-A3 recombinant adeno-associated virus carries out cell cultures by the plasmid vector of described MAGE-A3 recombinant adeno-associated virus and obtains.
8. a preparation method for the product relevant to the recombined glandulae correlation viral vectors carrying MAGE-A3 antigen gene, is characterized in that:
The preparation of the plasmid vector of MAGE-A3 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 MAGE-A3 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 MAGE-A3 recombinant adeno-associated virus;
The preparation of the virus vector of MAGE-A3 recombinant adeno-associated virus: the virus vector obtaining MAGE-A3 recombinant adeno-associated virus with the plasmid vector of described MAGE-A3 recombinant adeno-associated virus and pHelper plasmid co-transfection AAVp cell;
The preparation of the viral vector infection of MAGE-A3 recombinant adeno-associated virus or the clone of transfection: by the viral vector infection of described MAGE-A3 recombinant adeno-associated virus or transfection monocyte or dendritic cell, described clone comprises monocyte-dendritic cell system.
9. the recombined glandulae correlation viral vectors of the MAGE-A3 of a carrying antigen gene according to claim 1 or related products according to claim 7 are preparing the application in antitumor drug.
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