CN105586320A - Recombinant adeno-associated virus as well as construction method and application thereof - Google Patents
Recombinant adeno-associated virus as well as construction method and application thereof Download PDFInfo
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Abstract
The invention relates to a recombinant adeno-associated virus. The recombinant adeno-associated virus is formed by inserting a tumor antigen gene into a shuttle expression vector pAAV-MCS. The invention also provides a construction method and an application of the recombinant adeno-associated virus. According to the invention, the recombinant adeno-associated virus carrying a tumor-associated antigen gene is utilized for infecting DC cells and expresses tumor-associated antigen protein in the DC cells, and a PD-1 gene is also combined for silencing CTL cells in the application method of the recombinant adeno-associated virus.
Description
Technical field
The invention belongs to genetic engineering field, be specifically related to a kind of recombinant adeno-associated virus and construction method thereof and application.
Background technology
Immunocyte oncotherapy technology is that a kind of to excite autoimmunity system to resist with modern biotechnology means swollenThe methods for the treatment of novel, that have significant curative effect of knurl is the fourth-largest after the traditional treatment methods such as operation, radiation and chemotherapyOncotherapy technology.
Quantity research shows greatly at present, for advanced malignant tumor patient, and adoptive cellular immunotherapy toolHaving the incomparable superiority of other treatment mode, is one of effective means for the treatment of advanced malignant tumor, has very wideWealthy potential applicability in clinical practice. There is correlative study that a lot of medical institutions have carried out immunotherapy of tumors and clinical although domesticPractice, but still there are a lot of problems, be mainly manifested in: treat as main to lack specific CIK (1), and engineering level comparison fallsAfter; (2) do not grasp international advanced core technology, hospital's cell preparation quality management is undesirable comprehensively, ununifiedStandard and flow process; (3) lack large-scale clinical data support, cannot confirm the validity standard etc. These problems have restricted stateThe development of interior immunotherapy of tumors technology, brings hidden danger also to patient's treatment.
Adeno-associated virus (being called for short AVV) belongs to the member of piconavirus family, is nonencapsulated strand wire DNA virus. AAVThe about 4700bp of genome, comprise two open reading frames of upstream and downstream (ORF), be positioned at formed by 145 nucleotides respectively 2Between individual inverted terminal repeat (ITR). That adeno-associated virus has is safe, immunogenicity is low, can mediated gene movingThe advantages such as the interior expression steady in a long-term of object, thereby be considered to the most potential carrier that becomes gene therapy instrument. But at presentNot targeting strong, the recombinant adeno-associated virus that is suitable for oncotherapy.
Summary of the invention
First technical problem to be solved by this invention be immunotherapy of tumors specificity of the prior art poor, controlThe problem that therapeutic effect is undesirable, and then a kind of application of the recombinant adeno-associated virus of realizing the immunization therapy that targeting is strong is providedMethod.
Second technical problem that the present invention will solve is to provide after a kind of DC of infection cell that immunogenicity is lower, antigen causesThe recombinant adeno-associated virus that carries tumor antigen gene that the quick time is longer, DC antigen efficiency is higher.
Recombinant adeno-associated virus of the present invention, described recombinant adeno-associated virus is by tumor antigen gene is inserted into and is wornIn shuttle expression vector pAAV-MCS, the recombinant adeno-associated virus of formation.
The method that builds recombinant adeno-associated virus of the present invention, comprises the following steps:
(1) tumor antigen gene is inserted in shuttle expression carrier pAAV-MCS, described tumor antigen gene is enteredThe carrier that comprises ITR, forms recombinant plasmid;
(2) a large amount of extraction steps 1) described in recombinant plasmid, adopt calcium phosphate heavy with pHelper and pAAV-RC plasmidThe common transfection AAV-293 of shallow lake method cell, cultivates 2--3 days, collects culture medium and cell, adopts multigelation method to collect thick virusLiquid.
Or, build the method for recombinant adeno-associated virus of the present invention, comprise the following steps:
(1) tumor antigen gene is inserted in shuttle expression carrier pAAV-MCS, described tumor antigen gene is enteredThe carrier that comprises ITR, forms recombinant plasmid;
(2) purifying AAV2 virion from produce viral cell, cell is through AAVproExtractionAfter Solution preliminary purification, utilize nuclease to process, then allow AAV2 particle be combined with prepacked column, through washing and washingDe-, can obtain highly purified AAV particle.
It should be noted that, tumor associated antigen (TAA) is not only tied as the antibody of tumor markers and tumour-specificShare in immune detection and diagnosis, and be the target spot that antineoplastic immune is attacked. The cloning and identification of tumor antigen gene, forThe research of tumor vaccine is laid a good foundation. The general gene of tumour antigen genes of interest refers to the cDNA sequence of certain antigen of coding,Ask this genetic fragment in mammalian cell, to express. Utilize antigen presenting cell (APC, as BMDC) submission antigenAnd the ability of activated T cell. From peripheral blood separation, amplification BMDC, then import tumor associated antigen gene, gene tableReach rear submission antigenic information with activated T cell, produce antineoplastic specificity immune response. Genes of interest is generally antigen geneFull length cDNA sequence, in order to express the holoprotein of this antigen. But there is toxic and side effect in the holoprotein of some antigen gene expression, canCan bring out autoimmune response, or holoprotein antigen submission effect is not as the egg of antigenic determinant or the expression of a certain fragment geneBai Youxiao. Need to improve by gene engineering method for this gene or select a fragment gene wherein to express. RootAccording to the characteristic of different genes of interest, determine pointedly the gene order that needs expression.
The application process of recombinant adeno-associated virus of the present invention, comprises the following steps (1): make described restructuring gland relevantVirus infections DC cell, and at DC cells tumor-associated antigen protein.
Preferably, the application process of described recombinant adeno-associated virus further comprising the steps of (2): adopt siRNA to disturbThe reticent PD-1 gene of mode, blocking-up PD-1/PD-L1 signal. The implication of described PD-1/PD-L1 is PD-1 or PD-L1, its EnglishLiterary fame is called ProgrammedDeath1.
Preferably, described step (2) is specially:
A) by have the sequential structure shown in SEQIDNo.1 siRNA-1, there is the sequence shown in SEQIDNo.2The siRNA-2 of structure, there is the siRNA-3 of the sequential structure shown in SEQIDNo.3, above-mentioned three pairs for PD-1mRNA'sSiRNA action target spot, the template DNA of synthetic hairpin like two ends pairing siRNA, the middle LOOP phase with 6 deoxynucleotidesConnect, add termination signal polyT, two ends to add respectively the enzyme of restriction enzyme A geI and EcoRI to cut position at 3 ' end respectivelyPoint cohesive end, simultaneously synthetic complementary dna chain;
B) by have the sequential structure shown in SEQIDNo.4 siRNA-1-1, there is the order shown in SEQIDNo.5The siRNA-1-2 of array structure, have the sequential structure shown in SEQIDNo.6 siRNA-2-1, there is SEQIDNo.7 instituteThe siRNA-2-2 of the sequential structure showing, have the sequential structure shown in SEQIDNo.8 siRNA-3-1, there is SEQIDThe siRNA-3-2 insertion vector of the sequential structure shown in No.9;
C) the template DNA two strands of synthetic ShRNA, builds shRNA recombinant plasmid.
Wherein, the sequential structure shown in described SEQIDNo.1 is as follows: CGGAGAGCTTCGTGCTAAACTGGTA; DescribedSequential structure shown in SEQIDNo.2 is as follows: TGAGCAGACGGAGTATGCCACCATT; Shown in described SEQIDNo.3Sequential structure is as follows: CAGACGGAGTATGCCACCATTGTCT;
Sequential structure shown in described SEQIDNo.4, i.e. siRNA-1-1, as follows:CcggCGGAGAGCTTCGTGCTAAACTGGTACTCGAGTACCAGTTTAGCACGAAGCTCTCCGTTTTTg；
Sequential structure shown in described SEQIDNo.5, i.e. siRNA-1-2, as follows:aattcaaaaaCGGAGAGCTTCGTGCTAAACTGGTACTCGAGTACCAGTTTAGCACGAAGCTCTCCG；
Sequential structure shown in described SEQIDNo.6, i.e. siRNA-2-1, as follows:CcggTGAGCAGACGGAGTATGCCACCATTCTCGAGAATGGTGGCATACTCCGTCTGCTCATTTTTg；
Sequential structure shown in described SEQIDNo.7, i.e. siRNA-2-2, as follows:aattcaaaaaTGAGCAGACGGAGTATGCCACCATTCTCGAGAATGGTGGCATACTCCGTCTGCTCA；
Sequential structure shown in described SEQIDNo.8, i.e. siRNA-3-1, as follows:CcggCAGACGGAGTATGCCACCATTGTCTCTCGAGAGACAATGGTGGCATACTCCGTCTGTTTTTg；
Sequential structure shown in described SEQIDNo.9, i.e. siRNA-3-2, as follows:aattcaaaaaCAGACGGAGTATGCCACCATTGTCTCTCGAGAGACAATGGTGGCATACTCCGTCTG。
Preferably, 6 deoxynucleotides described in described step (2) have the sequence knot shown in SEQIDNo.10Structure. Sequential structure shown in described SEQIDNo.10, as follows: CTCGAG.
Preferably, described step c) is specially: first-selection is dissolved in synthetic primer dry powder in annealing buffer, is placed in 90DEG C water-bath in 15min, then naturally cool to room temperature for subsequent use; VshRNA vector plasmid lenti-gag-shRNA is through restrictedRestriction endonuclease AgeI and EcoRI enzyme cut after so that its linearisation, insert siRNA-1-1, siRNA-1-2 described in segment, siRNA-2-1, siRNA-2-2, siRNA-3-1, siRNA-3-2, transformed competence colibacillus Escherichia coli, positive colony is through PCR qualification and order-checking mirrorFixed, build plasmid.
Technique scheme of the present invention, has the following advantages compared to existing technology:
(1) utilization of the present invention carries the recombinant adeno-associated virus infection DC cell of tumor associated antigen gene, thin at DCIn born of the same parents, express tumor-associated antigen protein, compare the protein or the tumor tissues holoprotein sensitization DC cell that adopt heterogenous expression,The method immunogenicity is lower, and the antigen sensibilization time is longer, and it is higher that DC antigen is offered efficiency, the antigen submission of DC to CTL cellAbility is higher;
(2) the shuttle expression carrier pAAV-MCS that the present invention uses is rAAV2 type virus, and rAAV2 type virus can be highAbundance is expressed genes of interest, obtains correct folding albumen. Expression efficiency is fast, and unconformity, to chromosome, has high security. WithRAAV2 type virus is carried tumor associated antigen gene can realize more than 90% high efficiency of infection, is suitable for carrying out DC cellThe load of infection and antigen;
(3) the present invention utilizes adeno-associated virus to carry out gene delivery and expression, can realize higher bio-safety level, AAV-The 2nd, natural defect type virus, production infection depends on several extra trans factors, and does not also find at present it and appointWhat human diseases is relevant. In AAVHelper-FreeSystem, AAV-2 inverted repeat (ITR) sequence and rep/capGene lays respectively at and lacks altogether on homotactic independent plasmid, produces restructuring wild-type virus to stop, and therefore having canReach higher biological safety level. In addition, adeno-associated virus host cell scope is wide, and adeno-associated virus can infect widelyMammalian cell and be successfully applied to the expression of the mankind and inhuman source protein. Adeno-associated virus also has high titre, heavyThe infectious titer of can produce >=107 virion/milliliters of group AAV-2, virus stock solution used titre after concentrated reaches as high as>=1012 virion/milliliters. The active division of host cell is not the necessary condition infecting, and recombinant adeno-associated virus can infectDivision stage and non-division cells. The human protein that adeno-associated virus gives expression to can be correct folding and modifying, has lengthThe gene expression potential of phase.
(4) in the application process of recombinant adeno-associated virus of the present invention, also combined PD-1 gene silencing CTL cell. CTL is thinIn born of the same parents, express negative regulatory factor PD-1, PD-1 has suppressed activation and the multiplication capacity of CTL cell, and this project adopts siRNA to disturbThe reticent PD-1 gene of mode, blocking-up PD-1/PD-L1 signal, thereby the propagation of promotion specific for tumour antigen T cell, cell expandsIncrease and reach cell therapy consumption level, feed back the tumoricidal effect of performance in patient body, implant thereby effectively improveCTL cell survival rate, intensifier target tropism immunization therapy effect.
Brief description of the drawings
For content of the present invention is more likely to be clearly understood, also combination according to a particular embodiment of the invention belowAccompanying drawing, the present invention is further detailed explanation, wherein
Fig. 1 is the cellular morphology figure in the DC of embodiment 7 and the experiment of CTL the best cultivation ratio altogether;
Fig. 2 is the testing result that in embodiment 7, flow cytometer detects CTL mature cell ratio CD4/CD25;
Fig. 3 is the testing result that in embodiment 7, flow cytometer detects CTL mature cell ratio CD8/CD69;
Fig. 4 is the detection figure of secretory volume IL-4, the IFN-r of Control group cell factor in embodiment 7;
Fig. 5 is the detection figure of secretory volume IL-4, the IFN-r of ACTL group cell factor in embodiment 7;
Fig. 6 is killing activity figure in embodiment 7.
Detailed description of the invention
The structure of embodiment 1 recombinant adeno-associated virus
The recombinant adeno-associated virus that the present embodiment builds is by tumor antigen gene is inserted into shuttle expression carrierIn pAAV-MCS, the recombinant adeno-associated virus of formation.
The construction step of described recombinant adeno-associated virus is specific as follows:
(1) tumor antigen gene is inserted in shuttle expression carrier pAAV-MCS, described tumor antigen gene is enteredThe carrier that comprises ITR, forms recombinant plasmid;
(2) a large amount of extraction steps 1) described in recombinant plasmid, adopt calcium phosphate heavy with pHelper and pAAV-RC plasmidThe common transfection AAV-293 of shallow lake method cell, cultivates 2--3 days, collects culture medium and cell, adopts multigelation method to collect thick virusLiquid.
As the interchangeable implementation of the present embodiment, the construction step of described recombinant adeno-associated virus is also replaceable is:
(1) tumor antigen gene is inserted in shuttle expression carrier pAAV-MCS, described tumor antigen gene is enteredThe carrier that comprises ITR, forms recombinant plasmid;
(2) purifying AAV2 virion from produce viral cell, cell is through AAVproExtractionAfter Solution preliminary purification, utilize nuclease to process, then allow AAV2 particle be combined with prepacked column, through washing and washingDe-, can obtain highly purified AAV particle.
After preparing according to the method described above AAV virus, can carry out according to demand quality testing, in the present embodiment, measure sickPoison titre, carry out viral quality testing:
Virus titer is measured by the method such as dot blot, ELISA conventionally, and Real-TimePCR method is more accurately with quick.Adopt AAVproTitrationKit (forRealTimePCR) kit, virus genome sequence is carried out to quantitative PCRAnalyze. Quantitatively the amplification of the ITR based on AAV2, Accurate Determining virus titer.
The application process of the recombinant adeno-associated virus described in embodiment 2:
The present embodiment adopts the described recombinant adeno-associated virus preparing in embodiment 1, makes described restructuring gland related diseasesPoison infects DC cell, and at DC cells tumor-associated antigen protein.
As the preferred embodiment of the present invention, the application process of described recombinant adeno-associated virus is further comprising the steps of(2): adopt the reticent PD-1 gene of siRNA conflicting mode, blocking-up PD-1/PD-L1 signal.
Embodiment 3PD-1 gene siRNA sequences Design and virus preparation:
Adopt the reticent PD-1 gene of siRNA conflicting mode, blocking-up PD-1/PD-L1 signal concrete grammar is as follows:
A) with reference to the design principle of siRNA, according to the PD-1mRNA nucleotide sequence of reporting in GeneBank, apply WMGWith three couples of siRNA for PD-1mRNA of two kinds of online design software designs of Qigen, siRNA action target spot sequence is as follows:
This sequence is by gene Blast examination, and confirming does not have homology with human gene extron. To there is SEQIDThe siRNA-1 of the sequential structure shown in No.1, have the sequential structure shown in SEQIDNo.2 siRNA-2, there is SEQIDThe siRNA-3 of the sequential structure shown in No.3, above-mentioned three pairs of siRNA action target spots for PD-1mRNA, synthetic hairpin likeThe template DNA of two ends pairing siRNA, the middle LOOP with 6 deoxynucleotides is connected, and adds termination signal respectively at 3 ' endPolyT, two ends add respectively the restriction enzyme site cohesive end of restriction enzyme A geI and EcoRI, simultaneously synthetic complementary DNAChain; It should be noted that, 6 described deoxynucleotides have the sequential structure shown in SEQIDNo.10, i.e. CTCGAG.
B) by have the sequential structure shown in SEQIDNo.4 siRNA-1-1, there is the order shown in SEQIDNo.5The siRNA-1-2 of array structure, have the sequential structure shown in SEQIDNo.6 siRNA-2-1, there is SEQIDNo.7 instituteThe siRNA-2-2 of the sequential structure showing, have the sequential structure shown in SEQIDNo.8 siRNA-3-1, there is SEQIDThe siRNA-3-2 insertion vector of the sequential structure shown in No.9;
Insert segment sequence as follows:
NO.5’STEMPLoopSTEMP3’
siRNA-1-1CcggCGGAGAGCTTCGTGCTAAACTGGTACTCGAGTACCAGTTTAGCACGAAGCTCTCCGTTTTTg
siRNA-1-2aattcaaaaaCGGAGAGCTTCGTGCTAAACTGGTACTCGAGTACCAGTTTAGCACGAAGCTCTCCG
siRNA-2-1CcggTGAGCAGACGGAGTATGCCACCATTCTCGAGAATGGTGGCATACTCCGTCTGCTCATTTTTg
siRNA-2-2aattcaaaaaTGAGCAGACGGAGTATGCCACCATTCTCGAGAATGGTGGCATACTCCGTCTGCTCA
siRNA-3-1CcggCAGACGGAGTATGCCACCATTGTCTCTCGAGAGACAATGGTGGCATACTCCGTCTGTTTTTg
siRNA-3-2aattcaaaaaCAGACGGAGTATGCCACCATTGTCTCTCGAGAGACAATGGTGGCATACTCCGTCTG
C) structure of the synthetic and shRNA recombinant plasmid of the template DNA two strands of ShRNA, process is: synthetic primer is dryPowder is dissolved in annealing buffer, and 90 DEG C of water-bath 15min, then naturally cool to room temperature for subsequent use. VshRNA vector plasmidLenti-gag-shRNA is so that its linearisation after restriction enzyme A geI and EcoRI enzyme are cut, and Insert Fragment link, transformsCompetence Escherichia coli, positive colony, through PCR qualification and order-checking qualification, builds plasmid for subsequent use.
Embodiment 4PD-1 gene RNA disturbs Effective target site screening test:
For verifying effect of the present invention, (over-express vector plasmid and RNAi target spot virus are carried to carry out 293T cell cotransfectionPhysique grain) experiment, experimental procedure is as follows:
(1) transfection is inoculated in well-grown 293T cell in 24 orifice plates by 1 × 104/ml for first 1 day;
(2), when 293T growth reaches 80-90% fusion, change the opti-MEM1 of 400 μ l into;
(3) 293T transfection: the amount of every hole expression vector is 0.5 μ g, lipofectamine2000 volume is 2 μ l;
(4) plasmid and lipofectamine2000 are dissolved in respectively in opti-MEM1, mix, room temperature leaves standstill 5min;
(5) plasmid good above-mentioned dilution is mixed with the lipofectamine2000 having diluted, room temperature leaves standstill 20min;
(6) above-mentioned mixed liquor is added in 293T and cultivates 6-8h, change the fresh complete culture solution containing 10% serum into;
(7) after transfection, 24h fluorescence microscopy Microscopic observation is estimated transfection efficiency;
(8) 36-48h collecting cell after transfection, extracting total protein carries out Westernblotting detection.
Embodiment 5 virus production:
Adopt the reticent PD-1 gene of siRNA conflicting mode for realizing, blocking-up PD-1/PD-L1 signal, the present embodiment is produced sickThe step of poison is as follows:
One, plasmid co-transfection, produces recombinant slow virus
1. transfection the previous day, 293T cell is inoculated in to 225cm2In Tissue Culture Flask, inoculum density 3.0 × 107Individual/mlCell, culture medium is DMEM+10%Hyclone hyclone, puts 37 DEG C containing 5CO2Incubator in overnight incubation.
2. the same day was changed liquid in transfection, continued to cultivate with the fresh DMEM culture medium containing 10% hyclone. Treat Growth of CellsDuring to the 80-90% of floor space, carry out transfection with GeneCompanionII (GeneTrans) transfection reagent box. Concrete stepsFor:
(1) each transfection bottle is got pLP1, pLP2, pLP/VSVG, the each 20ug of Lenti-gag-shRNA plasmid and 1times;TransfectingBuffer1000L+40LGeneCompanionII (transfection reagent) mixes in the aseptic Ep pipe of 1.5ml,The static 10-15min of room temperature;
(2) directly GeneCompanion/DNA mixture is added drop-wise on cell culture medium;
(3), without changing liquid, 37 DEG C containing 5%CO2Incubator in continue cultivate.
3. after transfection 24h, change complete medium.
4. after transfection 48h, collect supernatant.
Two, the purifying of virus is with concentrated
1. the virus liquid of collecting carries out centrifugally operated immediately, and 4 DEG C, 70,000g surpasses from 2h.
2. carefully outwell supernatant, tip upside down on the paper handkerchief of sterilizing, remaining nutrient solution is blotted only.
3. by PBS lytic virus precipitation.
4. 200ml virus liquid is added in to 1.5ml20% sucrose (PBS) upper, 20 DEG C, 50,000g surpasses from 2h.
5. carefully outwell supernatant, tip upside down on the paper handkerchief of sterilizing, remaining nutrient solution is blotted only.
6. by PBS (or DMEM) lytic virus precipitation.
7. packing ,-80 DEG C of preservations.
Three, the PCR of the fragment of virales qualification
Carry DNA with DNeasyBloodTissueKit (50) kit. Sample infects 293 cell 72 hours, by cellScrape the centrifugal supernatant of abandoning, add 200ulPBS2-to mix as cell liquid. Get 200ul cell liquid, last back dissolving, to 100ul, is doneFor template. Carry out PCR qualification with primer primer1 and the primer2 primer of amplified fragments, result can amplify order specificallyThe object fragment band of gene left and right size, prove that this recombinant virus carries object fragment.
Four, titer determination
Titer determination adopts ELISA to detect, and ELISA kit is QuickTiterTMLentivirusQuantitationKit (HIVp24ELISA) is CELLBIOLABS company product.
Embodiment 6PD-1 gene siRNA slow-virus infection CTL cell research:
One, siRNA virus infections optimum amount, the impact of on cell proliferation after siRNA virus infections
A, according to 1 × 106/ ml cell concentration inoculation T cell, T75 blake bottle inoculation 10ml. After 24 hours, adding virus feelsDye experiment.
B, virus infections gradient are set to 10MOI, 50MOI, and 100MOI, 200MOI, totally four concentration gradients, virus adoptsGFP contrasts virus, fluorescence microscope efficiency of infection.
C, the highest group of selection efficiency of infection, for example 100MOI, carries out concentration gradient experiment again, explores best infection and usesAmount.
D, inoculation T cell, arrange 3 groups, and 1, do not infect viral group; 2, infect empty viral group; 3, infect siRNA group. MTTAfter cell energization ability test experience detection siRNA virus infections, the impact of on cell proliferation ability, determines cell proliferation curve.
Two, the reticent genes of interest PD-1 of siRNA virus infections T cell expresses and changes (mRNA level, protein level)
According to 1 × 106/ ml cell concentration inoculation T cell, T75 blake bottle inoculation 10ml. After 24 hours, infect and use according to the bestAmount virus inoculation. 1000IU/mlIL-2GT-T551 culture medium amplifying cells, the next day sample 1ml, RT-PCR detects PD-1 baseBecause of the variation of mRNA level, Westernblot detects the variation of PD-1 gene protein level. With a blood sample, nothing is setInfection group and viral infection group in contrast.
| Gene | Forword | Reverse |
| PD-1 | 5′-CTCAGGGTGACAGAGAGAAG-3′ | 5′-GACACCAACCACCAGGGTTT-3′ |
Three, in the whole preparation of CTL, PD-1 expresses, jamming effectiveness (establishing control group)
At siRNA virus infections T cell, reticent genes of interest PD-1 expresses in the experiment changing, and fetches cell system on defeated same dayAgent 1ml, adopts the reticent genes of interest PD-1 of siRNA virus infections T cell and expresses the method that variation experiment the inside is described, and emphasis detectsBefore cell feeds back, whether also keep PD-1 gene silencing, whether stably express has been described after siRNA slow-virus infection.
Four, PD-1siRNA disturbs with PD-1 monoclonal antibody and hatches CTL cell, relatively both cytokine secretions, cell phenotype,Cell killing effect test
FDA has ratified the PD-1 antibody of Merck, and the PD-1 of BMS ratifies in Japan. Order the PD-1 antibody of Merck. According to1×106/ ml cell concentration inoculation T cell, after 24 hours, is divided into three groups and tests: a, control group; The sense of b, PD-1siRNA virusDye group; C, PD-1 antibody incubation group (search pertinent literature, determine PD-1 incubated cell consumption).
1., after three days (or five days, because slow virus expression time be 1---3 days), get cell culture medium and detect IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNF-a, IFN-r cytokine secretion amount.
2. get cell carry out flow cytometer detect show marker change, CD3, CD4, CD8, CD56, CD69.
3. get equal number cell, taking K562 cell as target cell, kill knurl experiment according to target effect than 1:10, compare threeGroup is killed knurl difference on effect.
Embodiment 7DC and the test of CTL co-culture of cells:
One, DC and CTL the best are cultivated ratio altogether
In order to bring into play the maximal efficiency of DC cellular antigens submission, select suitable T cell and DC co-culture of cells, maximumThe generation of degree ground induction ACTL cell, so design the optimal proportion that this experimental exploring DC and CTL cultivate altogether.
Test as an example of the DC cell of load C EA antigen example, AAV-CEA adeno-associated virus infects DC cell, the 6th day withThe T cell of cultivating carries out common culture experiment. Be total to according to DC and T cell 1:1,1:5,1:10,1:20,1:50,1:100 ratioCulture experiment. While being cultured to altogether d14 days, harvesting, flow cytometer detects CTL mature cell ratio CD8/CD69, CD4/cd25。
Result:
DC and T cell 1:20 best results as shown in Figure 1, are the cell morphological characteristic of every day. After DC and T mixing with cells(D7), D8 days T cells are around DC cell agglomerate, and cell mass is more and more subsequently, and T cell is all gathered in DC cell peripheral substantially(D8-D14). As shown in Figure 2,3, flow cytometer detects that CTL mature cell ratio CD8/CD69 is 57.51%, CD4/cd25Be 5.32%.
Two, cultivate ripe ACTL biological characteristic research
The biological characteristics of ripe ACTL is cultivated in research, cultivate the ACTL cell of d14 days, and control group (does not causeThe cell of quick DC and T co-culture of cells) the following index of sampling detection:
(1) the secretory volume IL-4 of cell factor, IFN-r in culture supernatant
As shown in Figure 4,5, the secretory volume of the IL-4 of ACTL significantly declines testing result, and the secretory volume of IFN-r significantly goes upAdjust.
(2) kill knurl experiment
Get equal number cell, taking K562 cell as target cell, kill knurl experiment, relatively contrast according to target effect than 1:10Group and ACTL group are killed knurl difference on effect.
Testing result as shown in Figure 6, for the killing activity of ACTL is far above control group, is more than 50%.
Obviously, above-described embodiment is only for example is clearly described, and the not restriction to embodiment. RightIn those of ordinary skill in the field, can also make on the basis of the above description other multi-form variation orVariation. Here without also giving exhaustive to all embodiments. And the apparent variation of being extended out thus orVariation is still among the protection domain in the invention.
Claims (8)
1. a recombinant adeno-associated virus, is characterized in that, described recombinant adeno-associated virus is by tumor antigen gene is insertedEnter in shuttle expression carrier pAAV-MCS the recombinant adeno-associated virus of formation.
2. a method that builds recombinant adeno-associated virus claimed in claim 1, is characterized in that, comprises the following steps:
(1) tumor antigen gene is inserted in shuttle expression carrier pAAV-MCS, described tumor antigen gene is entered and compriseThe carrier of ITR, forms recombinant plasmid;
(2) a large amount of extraction steps 1) described in recombinant plasmid, adopt calcium phosphate precipitation method with pHelper and pAAV-RC plasmidCommon transfection AAV-293 cell, cultivates 2--3 days, collects culture medium and cell, adopts multigelation method to collect thick virus liquid.
3. a method that builds recombinant adeno-associated virus claimed in claim 1, is characterized in that, comprises the following steps:
(1) tumor antigen gene is inserted in shuttle expression carrier pAAV-MCS, described tumor antigen gene is entered and compriseThe carrier of ITR, forms recombinant plasmid;
(2) purifying AAV2 virion from produce viral cell, cell is at the beginning of AAVproExtractionSolutionAfter step purifying, utilize nuclease to process, then allow AAV2 particle be combined with prepacked column, through washing and wash-out, get final productTo highly purified AAV particle.
4. an application process for the recombinant adeno-associated virus described in claim 1, is characterized in that, comprises the following steps(1): make described recombinant adeno-associated virus infect DC cell, and at DC cells tumor-associated antigen protein.
5. the application process of recombinant adeno-associated virus according to claim 4, is characterized in that, further comprising the steps of(2): adopt the reticent PD-1 gene of siRNA conflicting mode, blocking-up PD-1/PD-L1 signal.
6. the application process of recombinant adeno-associated virus according to claim 5, is characterized in that, described step (2) is concreteFor:
A) by have the sequential structure shown in SEQIDNo.1 siRNA-1, there is the sequential structure shown in SEQIDNo.2SiRNA-2, there is the siRNA-3 of the sequential structure shown in SEQIDNo.3, above-mentioned three couples of siRNA for PD-1mRNAAction target spot, the template DNA of synthetic hairpin like two ends pairing siRNA, the middle LOOP with 6 deoxynucleotides is connected, pointDo not add termination signal polyT, two ends to add respectively the restriction enzyme site viscosity of restriction enzyme A geI and EcoRI at 3 ' endEnd, simultaneously synthetic complementary dna chain;
B) by have the sequential structure shown in SEQIDNo.4 siRNA-1-1, there is sequence shown in SEQIDNo.5 knotThe siRNA-1-2 of structure, have the sequential structure shown in SEQIDNo.6 siRNA-2-1, have shown in SEQIDNo.7The siRNA-2-2 of sequential structure, have the sequential structure shown in SEQIDNo.8 siRNA-3-1, there is SEQIDNo.9The siRNA-3-2 insertion vector of shown sequential structure;
C) the template DNA two strands of synthetic ShRNA, builds shRNA recombinant plasmid.
7. the application process of recombinant adeno-associated virus according to claim 6, is characterized in that, institute in described step (2)6 deoxynucleotides stating have the sequential structure shown in SEQIDNo.10.
8. the application process of recombinant adeno-associated virus according to claim 7, is characterized in that, described step is c) concreteFor: first-selection is dissolved in synthetic primer dry powder in annealing buffer, is placed in the water-bath 15min of 90 DEG C, then naturally coolingFor subsequent use to room temperature; VshRNA vector plasmid lenti-gag-shRNA after restriction enzyme A geI and EcoRI enzyme are cut so thatIts linearisation, inserts siRNA-1-1, siRNA-1-2 described in segment, siRNA-2-1, siRNA-2-2, siRNA-3-1, siRNA-3-2, transformed competence colibacillus Escherichia coli, positive colony, through PCR qualification and order-checking qualification, builds plasmid.
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