CN108085341A - A kind of MSGV recombinant vectors and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of MSGV recombinant vectors and its preparation method and application, the recombinant vector includes the genetic fragment being made of TRGC genes, TRDC genes and CD3 ζ genes.The present invention replaces the C areas of the α β TCR on MSGV carriers using the extracellular C areas of gamma delta T CR and the transmembrane region and intracellular region of CD3 ζ, significantly reduces the mismatch rate of external source TCR molecules and endogenous TCR.

Description

A kind of MSGV recombinant vectors and its preparation method and application

Technical field

The invention belongs to gene engineering technology fields, are related to a kind of MSGV recombinant vectors and its preparation method and application.

Background technology

In recent years, adoptive immunotherapy is carried out using the T cell (such as CAR-T, TCR-T) of genetic engineering modification to have obtainedBreakthrough, such as leukemia treating is carried out using CD19CAR-T, 90% Patients With Acute Lymphoblastic Leukemia has obtainedDirect release (S.L.Maude et al.Chimeric antigen receptor T cells for sustainedremissions in leukemia.Engl.J.Med.,371,1507-1517(2014)).It is however, immune based on T cellTreatment is faced with the safety problems such as undershooting-effect and cytokine release syndrome.On the one hand the reason for TCR-T is caused to miss the target beSince selected TCR molecules can identify the Antigenic Peptide of non-target protein, be on the other hand due to external source TCR molecules with it is endogenousTCR molecules occur mispairing and generate heterozygosis TCR, cause the generation of graft versus host disease(GVH disease) (GvHD).

At present, researcher has proposed a variety of methods for avoiding tcr gene mispairing, including introduce disulfide bond, codon optimization,The replacement of TCR C areas, Knob-into-hole, single-stranded TCR, α β TCR be transferred to gamma delta T cells, fusion signaling molecule CD3 ζ CD3 εCD28 etc..But effect of these methods on raising security, reduction mismatch rate is unsatisfactory.

A kind of it is, therefore, desirable to provide more efficiently method for avoiding tcr gene mispairing.

The content of the invention

In view of the deficiencies of the prior art, the present invention provides a kind of MSGV recombinant vectors and its preparation method and application, is used forReduce the probability that mispairing occurs with endogenous TCR molecules for external source TCR molecules.

For this purpose, the present invention uses following technical scheme：

In a first aspect, the present invention provides a kind of recombinant vector, the recombinant vector is included by TRGC genes, TRDC genesWith the genetic fragment of CD3 ζ genes composition.

In the present invention, the α β on MSGV carriers are replaced using the extracellular C areas of gamma delta T CR and the transmembrane region and intracellular region of CD3 ζThe C areas of TCR significantly reduce the mismatch rate of external source TCR molecules and endogenous TCR.

In the present invention, the extracellular C areas gene of TRGC gene representation γ TCR, the extracellular C areas base of TRDC gene representation δ TCRCause, the transmembrane region and intracellular region gene of CD3 ζ gene representation CD3 ζ.

Preferably, the genetic fragment further includes connection peptide.

Preferably, the connection peptide is P2A connection peptides.

Preferably, the recombinant vector is MSGV carriers.

In the present invention, the MSGV carriers are retroviral vector MSGV, have the C areas nucleotide sequence TRAC of α β TCRAnd TRBC.

Preferably, the genetic fragment includes TRGC genes connected in sequence, CD3 ζ genes, P2A connections peptide gene, TRDCGene and CD3 ζ genes.

Preferably, the nucleotide sequence of the TRGC genes is as shown in SEQ ID NO.1；

Nucleotide sequence shown in SEQ ID NO.1 is：

ATGCTGTCACTGCTGCTCCACGCATCAACGCTGGCAGTCCTTGGGGCTCTGTGTGTATATGGTGCAGGTCACCTAGAGCAACCTCAAATTTCCAGTACTAAAACGCTGTCAAAAACAGCCCGCCTGGAATGTGTGGTGTCTGGAATAACAATTTCTGCAACATCTGTATATTGGTATCGAGAGAGACCTGGTGAAGTCATACAGTTCCTGGTGTCCATTTCATATGACGGCACTGTCAGAAAGGAATCCGGCATTCCGTCAGGCAAATTTGAGGTGGATAGGATACCTGAAACGTCTACATCCACTCTCACCATTCACAATGTAGAGAAACAGGACATAGCTACCTACTACTGTGCCTTGAAGGAGTTGGGCAAAAAAATCAAGGTATTTGGTCCCGGAACAAAGCTTATCATTACAGATAAACAACTTGATGCAGATGTTTCCCCCAAGCCCACTATTTTTCTTCCTTCAATTGCTGAAACAAAGCTCCAGAAGGCTGGAACATACCTTTGTCTTCTTGAGAAATTTTTCCCTGATGTTATTAAGATACATTGGCAAGAAAAGAAGAGCAACACGATTCTGGGATCCCAGGAGGGGAACACCATGAAGACTAACGACACATACATGAAATTTAGCTGGTTAACGGTGCCAGAAAAGTCACTGGACAAAGAACACAGATGTATCGTCAGACATGAGAATAATAAAAACGGAGTTGATCAAGAAATTATCTTTCCTCCAATAAAGACAGATGTCATCACAATGGATCCCAAAGACAATTGTTCAAAAGATGCAAATGATACACTACTGCTGCAGCTCACAAACACCTCTGCATATTACATGTACCTCCTCCTACTCCTCAAGAGTGTGGTCTATTTTGCCATCATCACCTGCTGTCTGCTTAGAAGAACGGCTTTCTGCTGCAATGGAGAGAAATCATAA.

Preferably, the nucleotide sequence of the TRDC genes is as shown in SEQ ID NO.2；

Nucleotide sequence shown in SEQ ID NO.2 is：

GAAGTCAGCCTCATACCAAACCATCCGTTTTTGTCATGAAAAATGGAACAAATGTCGCTTGTCTGGTGAAGGAATTCTACCCCAAGGATATAAGAATAAATCTCGTGTCATCCAAGAAGATAACAGAGTTTGATCCTGCTATTGTCATCTCTCCCAGTGGGAAGTACAATGCTGTCAAGCTTGGTAAATATGAAGATTCAAATTCAGTGACATGTTCAGTTCAACACGACAATAAAACTGTGCACTCCACTGACTTTGAAGTGAAGACAGATTCTACAGATCACGTAAAACCAAAGGAAACTGAAAACACAAAGCAACCTTCAAAGAGCTGCCATAAACCCAAAGCCATAGTTCATACCGAGAAGGTGAACATGATGTCCCTCACAGTGCTTGGGCTACGAATGCTGTTTGCAAAGACTGTTGCCGTCAATTTTCTCTTGACTGCCAAGTTATTTTTCTTGTAAGGCTGACTGGCATGAGGAAGCTACACTCCTGAAGAAACCAAAGGCTTACAAAAATGCATCTCCTTGGCTTCTGACTTCTTTGTGATTCAAGTTGACCTGTCATAGCCTTGTTAAAATGGCTGCTAGCCAAACCACTTTTTCTTCAAAGACAACAAACCCAGCTCATCCTCCAGCTTGATGGGAAGACAAAAGTCCTGGGGAAGGGGGGTTTATGTCCTAACTGCTTTGTATGCTGTTTTATAAAGGGATAGAAGGATATAAAAAGATATAGGACTCTTTTTTTACTCCTACAAGTGATACACTTTGAAAATGATGTTTTGTTCCTTTTGACTTTCTTTACCTTTTGAAGTAGAAAGTGGGAACCAACAGGTTCACAGCTTCATTCCTCATGAGGCAAATAGGCCTTGGGAGAAGAAGAGCGGGTGCCCTTTTATCTAAACATGGAAGGCTCTGCTCAACTGAGCACTAGATTTGCTACAAACCAGCATCATCTTCTTCCTCCTGTCCTCACGGCTTGTCCCACCCTCTATGTTCACTTCAGGAGCCACACTAGAGATTCTGCATGGCGTGGAGGAGGACAAAGTTTCAGCACTTTCTGCCTCTCCTAATACTTTACAAATGAGATTACATTTGAATTTGCTAATACTTTATGAGCAGGCAATGAGGTTTCCAAAATCTCATCTAAATACTCTCCAATCTATTAGCAAAAATCAGAGTAAAATACAGAGGAAAGGCACTGCTTTCTGTTAATTGATTTAACATGCATGAATTAGCTCCCTCTGAGTTCCAGGCACTATGCTGAGAGTACAAAGAAGACACAAGTCTGCTTTCAAGCAACTCACTGTGAAAGTGTTTTTGAAGGGAGGAACAGAAATGAGACCCCTATCTTTCCCTATAAAAACAACATTTTTACTGTCTTTTGCCTGCCAATCTGTATTTGAAACCATTGGACACTGATTCTCTGGCCTGGGACTTTGGCATTGATGGTTTTCTGCCTTTCTTCTCAGCCTCTGCCTCTATTGCATTTATTAAACTGCATTGTGTGCCTAAAGGCACAGTAGTAAGTGGCACTGTCTTCAGTCCTTACTGGAGAGATCACCAAGTGAAAGGCTTTCTGGGTCAGAATGTGTTTCACAGAAAACCGTCCTTGAGTAAAATCTGCACCTTCTGATCTGCTGTTATCCCCATAAAAGACAAACTGAAAGGAATAATCTGGCCTTATCCGGTACCAGAATAAAT.

Preferably, the nucleotide sequence of the CD3 ζ genes is as shown in SEQ ID NO.3；

Nucleotide sequence shown in SEQ ID NO.3 is：

ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA.

Preferably, the nucleotide sequence of the P2A connections peptide gene is as shown in SEQ ID NO.4；

Nucleotide sequence shown in SEQ ID NO.4 is：

CGGGCCAAGAGATCTGGCAGCGGCGCCACCAATTTCAGCCTGCTGAAACAGGCCGGCGACGTGGAAGAGAACCCT.

Preferably, the nucleotide sequence of the genetic fragment is as shown in SEQ ID NO.5；

Nucleotide sequence shown in SEQ ID NO.5 is：

TAAGCCACCAAGTCTTCTTTTTTTGAAGACTTCTTGATGCAGATGTTTCCCCCAAGCCCACTATTTTTCTTCCTTCAATTGCTGAAACAAAGCTCCAGAAGGCTGGAACATACCTTTGTCTACTTGAGAAATTTTTCCCTGATGTTATTAAGATACATTGGCAAGAAAAGAAGAGCAACACGATTCTGGGATCCCAGGAGGGGAACACCATGAAAACTAACGACACATACATGAAATTTAGCTGGTTAACGGTGCCAGAAAAGTCACTGGACAAAGAACACAGATGTATCGTCAGACATGAGAATAATAAAAACGGAGTTGATCAAGAAATTATCTTTCCTCCAATAAAGACAGATGTCATCACAATGGATCCCAAAGACAATTGTTCAAAAGATGCAAATGATACACTACTGCTGCAGCTCACAAACACCTCTGCACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCGGGCCAAGAGATCTGGCAGCGGCGCCACCAATTTCAGCCTGCTGAAACAGGCCGGCGACGTGGAAGAGAACCCTAAGTCTTCTTTTTGAAGACTTCAGCCTCATACCAAACCATCCGTTTTTGTCATGAAAAATGGAACAAATGTCGCTTGTCTGGTGAAGGAATTCTACCCCAAGGATATAAGAATAAATCTCGTGTCATCCAAGAAGATAACAGAGTTTGATCCTGCTATTGTCATCTCTCCCAGTGGGAAGTACAATGCTGTCAAGCTTGGTAAATATGAAGATTCAAATTCAGTGACATGTTCAGTTCAACACGACAATAAAACTGTGCACTCCACTGACTTTGAAGTGAAAACAGATTCTACAGATCACGTAAAACCAAAGGAAACTGAAAACACAAAGCAACCTTCAAAGAGCTGCCATAAACCCAAAGCCATAGTTCATACCGAGAAGGTGAACATGATGTCCCTCACAGTGCTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTGAATTTGTAGATCGACGGATCCC.

Preferably, the nucleotide sequence of the recombinant vector is as shown in SEQ ID NO.6.

Second aspect, the present invention provides a kind of retrovirus expression vector, the retrovirus expression vector bagInclude recombinant vector as described in relation to the first aspect.

Preferably, the retrovirus expression vector further includes TRAV genes and TRBV genes.

In the present invention, the V areas gene of TRAV gene representation α TCR, the V areas gene of TRBV gene representation β TCR.

Preferably, the retrovirus expression vector includes TRAV genes connected in sequence, TRGC genes, CD3 ζ basesCause, connection peptide gene, TRBV genes, TRDC genes and CD3 ζ genes.

Preferably, the nucleotide sequence of the TRAV genes is as shown in SEQ ID NO.7；

The nucleotide sequence that SEQ ID NO.7 show is：

GAAGACTTCACCATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAGGAGGTGACACAGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGGCTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCAGCCACCTACCTCTGTGCTGTGAGGCCCACATCAGGAGGAAGCTACATACCTACATTTGGAAGAGGAACCAGCCTTATTGTTCATCCGGATAAACAACTTGAACTTG.

Preferably, the nucleotide sequence of the TRBV genes is as shown in SEQ ID NO.8；

The nucleotide sequence that SEQ ID NO.8 show is：

GAAGACTTCCCTATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAAACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACGTCGGGAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGAGTCAGCAAGTCTTC.

Preferably, the nucleotide sequence of the retrovirus expression vector is as shown in SEQ ID NO.9.

The third aspect, the present invention provides a kind of slow virus, the slow virus includes the reverse transcription as described in second aspectVirus expression carrier.

Fourth aspect, the present invention provides a kind of method for preparing the slow virus as described in the third aspect, including following stepSuddenly：

(1) recombinant vector of structure as described in relation to the first aspect；

(2) TRAV genes and TRBV genes are connected in the recombinant vector described in step (1), obtain retrovirus tableUp to carrier；

(3) retrovirus expression vector described in step (2) is transferred to competent cell, picking monoclonal is thin after cultureBorn of the same parents are screened；

(4) by the retrovirus expression vector after screening and packaging plasmid cotransfection mammalian cell, the slow disease is obtainedPoison.

Preferably, the method for step (1) described structure is that the genetic fragment shown in SEQ ID NO.5 is inserted into MSGV carriersBetween middle PacI and XhoI sites.

Preferably, the mammalian cell described in step (4) is GP2-293 cells.

As optimal technical scheme, the present invention provides a kind of method for preparing the slow virus as described in the third aspect, bagsInclude following steps：

(1) by between PacI and XhoI sites in the genetic fragment insertion MSGV carriers shown in SEQ ID NO.5, institute is obtainedState recombinant vector；

(2) using Jinmen molecule clone technology, TRAV genes and TRBV genes is connected to the restructuring described in step (1) and carriedIn body, retrovirus expression vector is obtained；

(3) retrovirus expression vector described in step (2) is transferred in competent cell, picking monoclonal after cultureCell is screened；

(4) by the retrovirus expression vector after screening and packaging plasmid cotransfection GP2-293 cells, obtain described slowVirus.

In the present invention, Jinmen molecule clone technology (Golden gate cloing) is a kind of atypical dependent on IIsThe digestion interconnection technique of type restriction enzyme, the technology are cut using BbsI restriction enzymes on the outside of identification sequenceIt cuts, generates different cohesive terminus,cohesive terminis, so as to disposably assemble two segments of TRAV genes and TRBV genes, overcome traditional multi-discIt is needed in section assembling simultaneously using the deficiency of multiple and different restriction enzymes.

5th aspect, the present invention provides a kind of pharmaceutical composition, described pharmaceutical composition is included as described in relation to the first aspectRecombinant vector, such as any one in the retrovirus expression vector of second aspect or the slow virus as described in the third aspectOr at least two combination.

Preferably, described pharmaceutical composition further includes arbitrary in pharmaceutically acceptable carrier, excipient or diluentIt is a kind of or at least two combination.

6th aspect is used to prepare T cell the present invention provides a kind of pharmaceutical composition as described in terms of the 5th and is immunizedThe drug of therapy for tumor.

Compared with prior art, the present invention has the advantages that：

(1) present invention replaces the α β on MSGV carriers using the extracellular C areas of gamma delta T CR and the transmembrane region and intracellular region of CD3 ζThe C areas of TCR, structure obtain including the pMSGV-GDZ recombinant vectors of TRGC-CD3 ζ-P2A-TRDC-CD3 ζ；

(2) using Jinmen molecule clone technology, TRAV genes and TRBV genes are connected to pMSGV-GDZ recombinant vectorsIn, the pMSGV-GDZ-NY retrovirus expression vectors that build；

(3) slow virus obtained after pMSGV-GDZ-NY retrovirus expression vectors are packed has rapid build TCRFunction, significantly reduce the mismatch rate of external source TCR molecules and endogenous TCR.

Description of the drawings

Fig. 1 is pMSGV-GDZ recombinant vector schematic diagrames, wherein, LTR- long terminal repeats；

Fig. 2 is pMSGV-GDZ recombinant vector structure charts；

Fig. 3 is pMSGV-GDZ recombinant vector cleavage maps, wherein, M-DNA standard molecular weights, 1-pMSGV-GDZ recombinant vectorsDigestion products；

Fig. 4 is pMSGV-GDZ-NY retrovirus expression vector schematic diagrames, wherein, LTR- long terminal repeats；

Fig. 5 is pMSGV-GDZ-NY retrovirus expression vector structure charts；

Fig. 6 is pMSGV-GDZ-NY retrovirus expression vector cleavage maps, wherein, M-DNA standard molecular weights, 1-PMSGV-GDZ-NY retrovirus expression vector digestion products.

Specific embodiment

The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hairIt is bright to be further described.It is understood that the specific embodiments described herein be used only for explain the present invention rather thanLimitation of the invention.

In the examples where no specific technique or condition is specified, according to the described technology of document in the art or condition,Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available fromThe conventional products of acquisition.

The structure of 1 pMSGV-GDZ recombinant vectors of embodiment

(1) design and TRGC-CD3 ζ-P2A-TRDC-CD3 ζ genetic fragment of the chemical synthesis as shown in SEQ ID NO.5；

(2) double digestion is carried out to MSGV carriers using PacI and XhoI, digestion system is shown in Table 1, to MSGV carriers after digestionCarry out gel recycling；

1 digestion system of table

Reagent	Dosage
		PacI(10U/μL)	2μL
XhoI(10U/μL)	2μL
		10 × H buffer solutions	2μL
PMSGV-GDZ carriers	1μg
		H2O	It is supplemented to 20 μ L

(3) using T4DNA ligases, the MSGV carriers after the SEQ ID NO.5 sequences of synthesis and gel recycling are carried outDNA coupled reactions, linked system are shown in Table 2, and coupled reaction keeps 30min at 22 DEG C, then inactivates 5min at 70 DEG C；

2 linked system of table

Reagent	Dosage
		MSGV endonuclease bamhis	1.6μg
SEQ ID NO.5 sequences	1μg
		T4DNA ligases (350U/ μ L)	1μL
10 × T4DNA ligase buffer solutions	2μL
		H2O	It is supplemented to 20 μ L

(4) 20 μ L connection products are added in 200 μ L competent cell DH5 α, are placed in cooled on ice 30min, 42 DEG C of water90s is bathed, then is placed in 2min on ice, is added in the LB fluid nutrient mediums of 780 μ L；By culture medium, 150rpm shaking tables are answered at 37 DEG CSoviet Union culture 1h, is then coated on the LB tablets containing amicillin resistance, and 37 DEG C are inverted culture 18h, picking positive bacterium colonyIn the LB fluid nutrient mediums containing amicillin resistance, 200rpm cultivates 12-16h at 37 DEG C, obtains expression pMSGV-GDZThe positive bacteria of recombinant vector.PMSGV-GDZ recombinant vectors are as shown in Figs. 1-2.

Using small amount plasmid extraction kit (TaKaRaMiniBEST Plasmid Purification KitVer.4.0, article No. 9760) carry out pMSGV-GDZ recombinant vectors extraction, then using PacI and XhoI restriction enzymes pairPMSGV-GDZ recombinant vectors carry out double digestion.

The electrophoretogram of digestion products is as shown in Figure 3, it can be seen that and two length are respectively the segment of 1739bp and 5602bp,Wherein the segment of 1739bp is TRGC-CD3 ζ-P2A-TRDC-CD3 ζ genetic fragments, the segment of 5602bp shown in SEQ ID NO.5For MSGV carriers, illustrate the success of pMSGV-GDZ construction of recombinant vector.

A large amount of extractions of 2 pMSGV-GDZ recombinant vectors of embodiment

The positive bacteria of expression pMSGV-GDZ recombinant vectors is taken out from -80 DEG C, is incubated overnight after plate streaking, next day picking2-4 monoclonal carries out small size (5mL) and shakes bacterium 6-8h；It is 1 by volume to take out bacterium solution:100 turns are shaken greatly, then using matterThe a large amount of extracts kits (Omega Endo-free Plasmid Maxi Kit, article No. D6926-03) of grain carry out plasmid and carry greatly.Plasmid, which carries greatly, to be comprised the following steps：

(1) suitable bacterium solution is taken to be placed in the centrifuge tube of new 50mL, 10min is centrifuged with 4900g at ambient temperature, is abandonedAppropriate bacterium solution is continuously added after supernatant, and to bacterium solution, all centrifugation is precipitated；

(2) 10mL buffer solution As are added in the bacterial sediment after centrifugation, in being resuspended on oscillator；

(3) 10mL buffer solution Bs are added in, 2min is stored at room temperature after turning upside down 10-15 times；

(4) 5mL buffer solution C are added in, 2min is stored at room temperature after turning upside down；

(5) obtained solution is moved into the syringe with filter membrane, be pressed in another new 50mL centrifuge tube；

(6) the ETR solution of 0.1 times of volume is added in into solution, turns upside down 7-10 times and is placed on 10-20min on ice, itAfter be transferred in 42 DEG C of water-baths and place 5min, upper solution is moved in the centrifuge tube of new 50mL, avoided after 4900g centrifugations 5minSuck lower floor's blue liquid；

(7) prepare splitter (HiBindMaxiColumn), add in 5mLGPS buffer solutions in the chimney filter of upper strata, be stored at room temperature3-10min, 4900g centrifuge 5min, abandon filtrate, chimney filter is spare；

(8) absolute ethyl alcohol of 0.5 times of volume is added in into supernatant solution, 2min is stored at room temperature after mixing；

(9) liquid is centrifuged into 4min with 4900g with the spare chimney filter of step (7), abandons filtrate until all passing through filter membrane；

(10) 10mLHB buffer solutions are added in, 4900g centrifugation 4min abandon filtrate；

(11) 15mLDNA cleaning solutions are added in, 4900g centrifugation 4min abandon filtrate；

(12) 10mLDNA cleaning solutions are added in, 4900g centrifugation 4min abandon filtrate；

(13) chimney filter is centrifuged into 10-15min with 5900g, be transferred to chimney filter in the centrifuge tube of new 50mL afterwards；

(14) 1-3mL eluents (Endotoxin-FreeElutionBuffer) are added in into filter membrane, are stored at room temperature 2min5min is centrifuged with 5900g afterwards, collects DNA solution.

The structure of 3 pMSGV-GDZ-NY retrovirus expression vectors of embodiment

(1) using Jinmen (Golden Gate) molecule clone technology, by the TCR double-strands of the NY-ESO-1 specificity of synthesisV areas DNA sequence dna (SEQ ID NO.7, SEQ ID NO.8) be connected in pMSGV-GDZ recombinant vectors, linked system is shown in Table 3；

3 linked system of table

Reagent	Dosage
		PMSGV-GDZ recombinant vectors	100ng
SEQ ID NO.7	50ng
		SEQ ID NO.8	50ng
BbsI(5U/μL)	2μL
		T4DNA ligases (350U/ μ L)	1μL
10 × T4DNA ligase buffer solutions	2μL
		H2O	It is supplemented to 20 μ L

(2) coupled reaction condition is：37 DEG C/10min, 16 DEG C/15min, totally 5 cycle；80 DEG C/5min, common 80min；After adding in the ATP buffer solutions of 1 μ L and 1 μ L exonucleases (Plasmid Safe Exonuclease), 37 DEG C/60min, 70DEG C/30min, linear DNA is digested, obtains pMSGV-GDZ-NY retrovirus expression vectors as illustrated in figures 4-5；

(3) positive-selecting

Golden Gate reaction products are transformed into Top10 Escherichia coli (Life-Technologies), take 10%Converted product is cultivated in the LB agar plates containing 100 μ g/mL ampicillins, is sieved using BsrGI restriction enzymesChoosing.

As shown in fig. 6, pMSGV-GDZ-NY retrovirus expression vectors after BsrGI digestions, obtain length isThe segment of 1573bp, 1263bp and 5274bp illustrate that pMSGV-GDZ-NY retrovirus expression vectors are built successfully.

4 retrovirus of embodiment is packed and cell transfecting

Retrovirus is packed：By the pMSGV-GDZ-NY retrovirus expression vectors of structure and packaging plasmid cotransfectionGP2-293 cells, packaging virus, is collected and concentrating virus liquid, progress titre detect the measure with MOI values, the titre of virus liquid>1.00E+08TU/mL MOI values are between 5-10.

Cell transfecting：

(1) before transfection for 24 hours, using the GP2-293 cell (cells of trypsase (0.125%) digestion exponential phaseDegrees of fusion is 85%-95%), after adjusting cell density with complete medium (DMEM, Sodium Pyruvate, glutamine, 10%FBS)Passage, is re-seeded into blake bottle and cultivates；

(2) for 24 hours afterwards using the viral transfected cells of packaging, cell fusion Du Miduda 45-65%；

(3) cell culture medium is changed to the complete medium without FBS by 2h before transfecting；

(4) CaCl is added in into the centrifuge tube of sterilizing₂, and be uniformly mixed with the DNA solution of preparation, it incubates at room temperature5min forms DNA-CaCl₂Solution；

(5) by DNA-CaCl₂Solution is slowly dropwisely added in BBS liquid, mixing；10-20min is incubated at room temperature, is formedDNA-CaCl₂- BBS solution；

(6) by DNA-CaCl₂- BBS solution is equably added dropwise in the culture solution of GP2-293 cells, in 37 DEG C, 5%CO₂Lower culture 5h；The culture solution of phosphoric acid calcium precipitate is discarded, is changed to fresh complete culture solution, continues to cultivate.

Transfection is observed after transfection 24-36h, finds to transfect successful cell up to collection disease after more than 90%, 48-54hPoison.

In conclusion the present invention replaces MSGV carriers using the extracellular C areas of gamma delta T CR and the transmembrane region and intracellular region of CD3 ζOn α β TCR C areas, structure obtains including the pMSGV-GDZ recombinant vectors of TRGC-CD3 ζ-P2A-TRDC-CD3 ζ；Utilize goldDoor molecule clone technology, TRAV genes and TRBV genes is connected in pMSGV-GDZ recombinant vectors, the pMSGV- builtGDZ-NY retrovirus expression vectors；The slow virus obtained after pMSGV-GDZ-NY retrovirus expression vectors are packed hasThere is the function of rapid build TCR, significantly reduce the mismatch rate of external source TCR molecules and endogenous TCR.

Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodimentIt is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Technical fieldTechnical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present inventionAddition, selection of concrete mode etc., all fall within protection scope of the present invention and the open scope.

Sequence table

<110>Hua Long Bioisystech Co., Ltd of Henan Province

<120>A kind of recombinant vector and its preparation method and application

<130> 20171227

<141> 2017-12-28

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 939

<212> DNA

<213>Artificial synthesized ()

<400> 1

atgctgtcac tgctgctcca cgcatcaacg ctggcagtcc ttggggctct gtgtgtatat 60

ggtgcaggtc acctagagca acctcaaatt tccagtacta aaacgctgtc aaaaacagcc 120

cgcctggaat gtgtggtgtc tggaataaca atttctgcaa catctgtata ttggtatcga 180

gagagacctg gtgaagtcat acagttcctg gtgtccattt catatgacgg cactgtcaga 240

aaggaatccg gcattccgtc aggcaaattt gaggtggata ggatacctga aacgtctaca 300

tccactctca ccattcacaa tgtagagaaa caggacatag ctacctacta ctgtgccttg 360

aaggagttgg gcaaaaaaat caaggtattt ggtcccggaa caaagcttat cattacagat 420

aaacaacttg atgcagatgt ttcccccaag cccactattt ttcttccttc aattgctgaa 480

acaaagctcc agaaggctgg aacatacctt tgtcttcttg agaaattttt ccctgatgtt 540

attaagatac attggcaaga aaagaagagc aacacgattc tgggatccca ggaggggaac 600

accatgaaga ctaacgacac atacatgaaa tttagctggt taacggtgcc agaaaagtca 660

ctggacaaag aacacagatg tatcgtcaga catgagaata ataaaaacgg agttgatcaa 720

gaaattatct ttcctccaat aaagacagat gtcatcacaa tggatcccaa agacaattgt 780

tcaaaagatg caaatgatac actactgctg cagctcacaa acacctctgc atattacatg 840

tacctcctcc tactcctcaa gagtgtggtc tattttgcca tcatcacctg ctgtctgctt 900

agaagaacgg ctttctgctg caatggagag aaatcataa 939

<210> 2

<211> 1703

<212> DNA

<213>Artificial synthesized ()

<400> 2

gaagtcagcc tcataccaaa ccatccgttt ttgtcatgaa aaatggaaca aatgtcgctt 60

gtctggtgaa ggaattctac cccaaggata taagaataaa tctcgtgtca tccaagaaga 120

taacagagtt tgatcctgct attgtcatct ctcccagtgg gaagtacaat gctgtcaagc 180

ttggtaaata tgaagattca aattcagtga catgttcagt tcaacacgac aataaaactg 240

tgcactccac tgactttgaa gtgaagacag attctacaga tcacgtaaaa ccaaaggaaa 300

ctgaaaacac aaagcaacct tcaaagagct gccataaacc caaagccata gttcataccg 360

agaaggtgaa catgatgtcc ctcacagtgc ttgggctacg aatgctgttt gcaaagactg 420

ttgccgtcaa ttttctcttg actgccaagt tatttttctt gtaaggctga ctggcatgag 480

gaagctacac tcctgaagaa accaaaggct tacaaaaatg catctccttg gcttctgact 540

tctttgtgat tcaagttgac ctgtcatagc cttgttaaaa tggctgctag ccaaaccact 600

ttttcttcaa agacaacaaa cccagctcat cctccagctt gatgggaaga caaaagtcct 660

ggggaagggg ggtttatgtc ctaactgctt tgtatgctgt tttataaagg gatagaagga 720

tataaaaaga tataggactc tttttttact cctacaagtg atacactttg aaaatgatgt 780

tttgttcctt ttgactttct ttaccttttg aagtagaaag tgggaaccaa caggttcaca 840

gcttcattcc tcatgaggca aataggcctt gggagaagaa gagcgggtgc ccttttatct 900

aaacatggaa ggctctgctc aactgagcac tagatttgct acaaaccagc atcatcttct 960

tcctcctgtc ctcacggctt gtcccaccct ctatgttcac ttcaggagcc acactagaga 1020

ttctgcatgg cgtggaggag gacaaagttt cagcactttc tgcctctcct aatactttac 1080

aaatgagatt acatttgaat ttgctaatac tttatgagca ggcaatgagg tttccaaaat 1140

ctcatctaaa tactctccaa tctattagca aaaatcagag taaaatacag aggaaaggca 1200

ctgctttctg ttaattgatt taacatgcat gaattagctc cctctgagtt ccaggcacta 1260

tgctgagagt acaaagaaga cacaagtctg ctttcaagca actcactgtg aaagtgtttt 1320

tgaagggagg aacagaaatg agacccctat ctttccctat aaaaacaaca tttttactgt 1380

cttttgcctg ccaatctgta tttgaaacca ttggacactg attctctggc ctgggacttt 1440

ggcattgatg gttttctgcc tttcttctca gcctctgcct ctattgcatt tattaaactg 1500

cattgtgtgc ctaaaggcac agtagtaagt ggcactgtct tcagtcctta ctggagagat 1560

caccaagtga aaggctttct gggtcagaat gtgtttcaca gaaaaccgtc cttgagtaaa 1620

atctgcacct tctgatctgc tgttatcccc ataaaagaca aactgaaagg aataatctgg 1680

ccttatccgg taccagaata aat 1703

<210> 3

<211> 492

<212> DNA

<213>Artificial synthesized ()

<400> 3

atgaagtgga aggcgctttt caccgcggcc atcctgcagg cacagttgcc gattacagag 60

gcacagagct ttggcctgct ggatcccaaa ctctgctacc tgctggatgg aatcctcttc 120

atctatggtg tcattctcac tgccttgttc ctgagagtga agttcagcag gagcgcagac 180

gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 240

gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 300

agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 360

gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 420

taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 480

ccccctcgct aa 492

<210> 4

<211> 75

<212> DNA

<213>Artificial synthesized ()

<400> 4

cgggccaaga gatctggcag cggcgccacc aatttcagcc tgctgaaaca ggccggcgac 60

gtggaagaga accct 75

<210> 5

<211> 1739

<212> DNA

<213>Artificial synthesized ()

<400> 5

taagccacca agtcttcttt ttttgaagac ttcttgatgc agatgtttcc cccaagccca 60

ctatttttct tccttcaatt gctgaaacaa agctccagaa ggctggaaca tacctttgtc 120

tacttgagaa atttttccct gatgttatta agatacattg gcaagaaaag aagagcaaca 180

cgattctggg atcccaggag gggaacacca tgaaaactaa cgacacatac atgaaattta 240

gctggttaac ggtgccagaa aagtcactgg acaaagaaca cagatgtatc gtcagacatg 300

agaataataa aaacggagtt gatcaagaaa ttatctttcc tccaataaag acagatgtca 360

tcacaatgga tcccaaagac aattgttcaa aagatgcaaa tgatacacta ctgctgcagc 420

tcacaaacac ctctgcactc tgctacctgc tggatggaat cctcttcatc tatggtgtca 480

ttctcactgc cttgttcctg agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc 540

agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg 600

ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc 660

ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga 720

ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca 780

gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc cctcgccggg 840

ccaagagatc tggcagcggc gccaccaatt tcagcctgct gaaacaggcc ggcgacgtgg 900

aagagaaccc taagtcttct ttttgaagac ttcagcctca taccaaacca tccgtttttg 960

tcatgaaaaa tggaacaaat gtcgcttgtc tggtgaagga attctacccc aaggatataa 1020

gaataaatct cgtgtcatcc aagaagataa cagagtttga tcctgctatt gtcatctctc 1080

ccagtgggaa gtacaatgct gtcaagcttg gtaaatatga agattcaaat tcagtgacat 1140

gttcagttca acacgacaat aaaactgtgc actccactga ctttgaagtg aaaacagatt 1200

ctacagatca cgtaaaacca aaggaaactg aaaacacaaa gcaaccttca aagagctgcc 1260

ataaacccaa agccatagtt cataccgaga aggtgaacat gatgtccctc acagtgctct 1320

gctacctgct ggatggaatc ctcttcatct atggtgtcat tctcactgcc ttgttcctga 1380

gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag aaccagctct 1440

ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag agacgtggcc 1500

gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg 1560

aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc 1620

ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc aaggacacct 1680

acgacgccct tcacatgcag gccctgcccc ctcgctgaat ttgtagatcg acggatccc 1739

<210> 6

<211> 7341

<212> DNA

<213>Artificial synthesized ()

<400> 6

aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60

atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag 120

cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 180

gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt 240

ttccagggtg ccccaaggac ctgaaaatga ccctgtgcct tatttgaact aaccaatcag 300

ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa 360

cccctcactc ggcgcgccag tcctccgata gactgcgtcg cccgggtacc cgtattccca 420

ataaagcctc ttgctgtttg catccgaatc gtggactcgc tgatccttgg gagggtctcc 480

tcagattgat tgactgccca cctcgggggt ctttcatttg gaggttccac cgagatttgg 540

agacccctgc ctagggacca ccgacccccc cgccgggagg taagctggcc agcggtcgtt 600

tcgtgtctgt ctctgtcttt gtgcgtgttt gtgccggcat ctaatgtttg cgcctgcgtc 660

tgtactagtt agctaactag ctctgtatct ggcggacccg tggtggaact gacgagttcg 720

gaacacccgg ccgcaaccct gggagacgtc ccagggactt cgggggccgt ttttgtggcc 780

cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg gtgcaccccc cttagaggag 840

ggatatgtgg ttctggtagg agacgagaac ctaaaacagt tcccgcctcc gtctgaattt 900

ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg tctgctgcag catcgttctg 960

tgttgtctct gtctgactgt gtttctgtat ttgtctgaga atatgggccc gggctagcct 1020

gttaccactc ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac 1080

aaccagtcgg tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca 1140

acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt 1200

aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg 1260

acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag 1320

cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc 1380

ccgcctcgat cctcccttta tccagccctc actccttctc taggcgcccc catatggcca 1440

tatgagatct tatatggggc acccccgccc cttgtaaact tccctgaccc tgacatgaca 1500

agagttacta acagcccctc tctccaagct cacttacagg ctctctactt agtccagcac 1560

gaagtctgga gacctctggc ggcagcctac caagaacaac tggaccgacc ggtggtacct 1620

cacccttacc gagtcggcga cacagtgtgg gtccgccgac accagactaa gaacctagaa 1680

cctcgctgga aaggacctta cacagtcctg ctgaccaccc ccaccgccct caaagtagac 1740

ggcatcgcag cttggataca cgccgcccac gtgaaggctg ccgaccccgg gggtggacca 1800

tcctctagac cctgcaggtt aattaagcca ccaagtcttc tttttttgaa gacttcttga 1860

tgcagatgtt tcccccaagc ccactatttt tcttccttca attgctgaaa caaagctcca 1920

gaaggctgga acataccttt gtctacttga gaaatttttc cctgatgtta ttaagataca 1980

ttggcaagaa aagaagagca acacgattct gggatcccag gaggggaaca ccatgaaaac 2040

taacgacaca tacatgaaat ttagctggtt aacggtgcca gaaaagtcac tggacaaaga 2100

acacagatgt atcgtcagac atgagaataa taaaaacgga gttgatcaag aaattatctt 2160

tcctccaata aagacagatg tcatcacaat ggatcccaaa gacaattgtt caaaagatgc 2220

aaatgataca ctactgctgc agctcacaaa cacctctgca ctctgctacc tgctggatgg 2280

aatcctcttc atctatggtg tcattctcac tgccttgttc ctgagagtga agttcagcag 2340

gagcgcagac gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct 2400

aggacgaaga gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg 2460

gggaaagccg agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa 2520

gatggcggag gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca 2580

cgatggcctt taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat 2640

gcaggccctg ccccctcgcc gggccaagag atctggcagc ggcgccacca atttcagcct 2700

gctgaaacag gccggcgacg tggaagagaa ccctaagtct tctttttgaa gacttcagcc 2760

tcataccaaa ccatccgttt ttgtcatgaa aaatggaaca aatgtcgctt gtctggtgaa 2820

ggaattctac cccaaggata taagaataaa tctcgtgtca tccaagaaga taacagagtt 2880

tgatcctgct attgtcatct ctcccagtgg gaagtacaat gctgtcaagc ttggtaaata 2940

tgaagattca aattcagtga catgttcagt tcaacacgac aataaaactg tgcactccac 3000

tgactttgaa gtgaaaacag attctacaga tcacgtaaaa ccaaaggaaa ctgaaaacac 3060

aaagcaacct tcaaagagct gccataaacc caaagccata gttcataccg agaaggtgaa 3120

catgatgtcc ctcacagtgc tctgctacct gctggatgga atcctcttca tctatggtgt 3180

cattctcact gccttgttcc tgagagtgaa gttcagcagg agcgcagacg cccccgcgta 3240

ccagcagggc cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga 3300

tgttttggac aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa 3360

ccctcaggaa ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga 3420

gattgggatg aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct 3480

cagtacagcc accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgctg 3540

aatttgtaga tcgacggatc cctcgagctc aagcttcgaa ttctgcagtc gacggtaccg 3600

cgggcccggg atccgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg 3660

aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg 3720

aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag agacagcaga 3780

atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac 3840

agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc 3900

agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc 3960

ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct 4020

cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtgt atccaataaa 4080

ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag 4140

tgattgacta cccgtcagcg ggggtctttc atgggtaaca gtttcttgaa gttggagaac 4200

aacattctga gggtaggagt cgaatattaa gtaatcctga ctcaattagc cactgttttg 4260

aatccacata ctccaatact cctgaaatcc atcgatggag ttcattatgg acagcgcaga 4320

aagagctggg gagaattgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 4380

ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 4440

ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 4500

ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 4560

gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 4620

atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 4680

caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 4740

cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 4800

taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 4860

ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 4920

tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 4980

gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 5040

ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 5100

aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 5160

aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 5220

agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 5280

cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 5340

gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 5400

atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 5460

gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 5520

tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 5580

gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 5640

ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 5700

actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 5760

ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 5820

tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 5880

cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 5940

ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 6000

ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 6060

tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 6120

agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 6180

atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 6240

gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 6300

aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 6360

tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 6420

aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 6480

gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 6540

ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 6600

acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 6660

gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg 6720

caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc 6780

cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 6840

ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg 6900

ttttcccagt cacgacgttg taaaacgacg gccagtgcca cgctctccct tatgcgactc 6960

ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa 7020

tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc 7080

cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat 7140

gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg 7200

tccggcgtag aggcgattta aagacaggat atcagtggtc caggctctag ttttgactca 7260

acaatatcac cagctgaagc ctatagagta cgagccatag ataaaataaa agattttatt 7320

tagtctccag aaaaaggggg g 7341

<210> 7

<211> 430

<212> DNA

<213>Artificial synthesized ()

<400> 7

gaagacttca ccatggagac cctcttgggc ctgcttatcc tttggctgca gctgcaatgg 60

gtgagcagca aacaggaggt gacacagatt cctgcagctc tgagtgtccc agaaggagaa 120

aacttggttc tcaactgcag tttcactgat agcgctattt acaacctcca gtggtttagg 180

caggaccctg ggaaaggtct cacatctctg ttgcttattc agtcaagtca gagagagcaa 240

acaagtggaa ggcttaatgc ctcgctggat aaatcatcag gacgtagtac tttatacatt 300

gcagcttctc agcctggtga ctcagccacc tacctctgtg ctgtgaggcc cacatcagga 360

ggaagctaca tacctacatt tggaagagga accagcctta ttgttcatcc ggataaacaa 420

cttgaacttg 430

<210> 8

<211> 423

<212> DNA

<213>Artificial synthesized ()

<400> 8

gaagacttcc ctatgagcat cggcctcctg tgctgtgcag ccttgtctct cctgtgggca 60

ggtccagtga atgctggtgt cactcagacc ccaaaattcc aggtcctgaa aacaggacag 120

agcatgacac tgcagtgtgc ccaggatatg aaccatgaat acatgtcctg gtatcgacaa 180

gacccaggca tggggctgag gctgattcat tactcagttg gtgctggtat cactgaccaa 240

ggagaagtcc ccaatggcta caatgtctcc agatcaacca cagaggattt cccgctcagg 300

ctgctgtcgg ctgctccctc ccagacatct gtgtacttct gtgccagcag ttacgtcggg 360

aacaccgggg agctgttttt tggagaaggc tctaggctga ccgtactgag tcagcaagtc 420

ttc 423

<210> 9

<211> 8110

<212> DNA

<213>Artificial synthesized ()

<400> 9

aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60

atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag 120

cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 180

gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt 240

ttccagggtg ccccaaggac ctgaaaatga ccctgtgcct tatttgaact aaccaatcag 300

ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa 360

cccctcactc ggcgcgccag tcctccgata gactgcgtcg cccgggtacc cgtattccca 420

ataaagcctc ttgctgtttg catccgaatc gtggactcgc tgatccttgg gagggtctcc 480

tcagattgat tgactgccca cctcgggggt ctttcatttg gaggttccac cgagatttgg 540

agacccctgc ctagggacca ccgacccccc cgccgggagg taagctggcc agcggtcgtt 600

tcgtgtctgt ctctgtcttt gtgcgtgttt gtgccggcat ctaatgtttg cgcctgcgtc 660

tgtactagtt agctaactag ctctgtatct ggcggacccg tggtggaact gacgagttcg 720

gaacacccgg ccgcaaccct gggagacgtc ccagggactt cgggggccgt ttttgtggcc 780

cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg gtgcaccccc cttagaggag 840

ggatatgtgg ttctggtagg agacgagaac ctaaaacagt tcccgcctcc gtctgaattt 900

ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg tctgctgcag catcgttctg 960

tgttgtctct gtctgactgt gtttctgtat ttgtctgaga atatgggccc gggctagcct 1020

gttaccactc ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac 1080

aaccagtcgg tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca 1140

acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt 1200

aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg 1260

acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag 1320

cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc 1380

ccgcctcgat cctcccttta tccagccctc actccttctc taggcgcccc catatggcca 1440

tatgagatct tatatggggc acccccgccc cttgtaaact tccctgaccc tgacatgaca 1500

agagttacta acagcccctc tctccaagct cacttacagg ctctctactt agtccagcac 1560

gaagtctgga gacctctggc ggcagcctac caagaacaac tggaccgacc ggtggtacct 1620

cacccttacc gagtcggcga cacagtgtgg gtccgccgac accagactaa gaacctagaa 1680

cctcgctgga aaggacctta cacagtcctg ctgaccaccc ccaccgccct caaagtagac 1740

ggcatcgcag cttggataca cgccgcccac gtgaaggctg ccgaccccgg gggtggacca 1800

tcctctagac cctgcaggtt aattaagcca ccatggagac cctcttgggc ctgcttatcc 1860

tttggctgca gctgcaatgg gtgagcagca aacaggaggt gacacagatt cctgcagctc 1920

tgagtgtccc agaaggagaa aacttggttc tcaactgcag tttcactgat agcgctattt 1980

acaacctcca gtggtttagg caggaccctg ggaaaggtct cacatctctg ttgcttattc 2040

agtcaagtca gagagagcaa acaagtggaa ggcttaatgc ctcgctggat aaatcatcag 2100

gacgtagtac tttatacatt gcagcttctc agcctggtga ctcagccacc tacctctgtg 2160

ctgtgaggcc cacatcagga ggaagctaca tacctacatt tggaagagga accagcctta 2220

ttgttcatcc ggataaacaa cttgatgcag atgtttcccc caagcccact atttttcttc 2280

cttcaattgc tgaaacaaag ctccagaagg ctggaacata cctttgtcta cttgagaaat 2340

ttttccctga tgttattaag atacattggc aagaaaagaa gagcaacacg attctgggat 2400

cccaggaggg gaacaccatg aaaactaacg acacatacat gaaatttagc tggttaacgg 2460

tgccagaaaa gtcactggac aaagaacaca gatgtatcgt cagacatgag aataataaaa 2520

acggagttga tcaagaaatt atctttcctc caataaagac agatgtcatc acaatggatc 2580

ccaaagacaa ttgttcaaaa gatgcaaatg atacactact gctgcagctc acaaacacct 2640

ctgcactctg ctacctgctg gatggaatcc tcttcatcta tggtgtcatt ctcactgcct 2700

tgttcctgag agtgaagttc agcaggagcg cagacgcccc cgcgtaccag cagggccaga 2760

accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt ttggacaaga 2820

gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct caggaaggcc 2880

tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt gggatgaaag 2940

gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt acagccacca 3000

aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgccgggcc aagagatctg 3060

gcagcggcgc caccaatttc agcctgctga aacaggccgg cgacgtggaa gagaaccctg 3120

gccctatgag catcggcctc ctgtgctgtg cagccttgtc tctcctgtgg gcaggtccag 3180

tgaatgctgg tgtcactcag accccaaaat tccaggtcct gaaaacagga cagagcatga 3240

cactgcagtg tgcccaggat atgaaccatg aatacatgtc ctggtatcga caagacccag 3300

gcatggggct gaggctgatt cattactcag ttggtgctgg tatcactgac caaggagaag 3360

tccccaatgg ctacaatgtc tccagatcaa ccacagagga tttcccgctc aggctgctgt 3420

cggctgctcc ctcccagaca tctgtgtact tctgtgccag cagttacgtc gggaacaccg 3480

gggagctgtt ttttggagaa ggctctaggc tgaccgtact gagtcagcct cataccaaac 3540

catccgtttt tgtcatgaaa aatggaacaa atgtcgcttg tctggtgaag gaattctacc 3600

ccaaggatat aagaataaat ctcgtgtcat ccaagaagat aacagagttt gatcctgcta 3660

ttgtcatctc tcccagtggg aagtacaatg ctgtcaagct tggtaaatat gaagattcaa 3720

attcagtgac atgttcagtt caacacgaca ataaaactgt gcactccact gactttgaag 3780

tgaaaacaga ttctacagat cacgtaaaac caaaggaaac tgaaaacaca aagcaacctt 3840

caaagagctg ccataaaccc aaagccatag ttcataccga gaaggtgaac atgatgtccc 3900

tcacagtgct ctgctacctg ctggatggaa tcctcttcat ctatggtgtc attctcactg 3960

ccttgttcct gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc 4020

agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat gttttggaca 4080

agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac cctcaggaag 4140

gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag attgggatga 4200

aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc agtacagcca 4260

ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgctga atttgtagat 4320

cgacggatcc ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga 4380

tccgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac 4440

ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga aaatacataa 4500

ctgagaatag agaagttcag atcaaggtta ggaacagaga gacagcagaa tatgggccaa 4560

acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc 4620

agatgcggtc ccgccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca 4680

aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct 4740

gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggcgcg 4800

ccagtcctcc gatagactgc gtcgcccggg tacccgtgta tccaataaac cctcttgcag 4860

ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac 4920

ccgtcagcgg gggtctttca tgggtaacag tttcttgaag ttggagaaca acattctgag 4980

ggtaggagtc gaatattaag taatcctgac tcaattagcc actgttttga atccacatac 5040

tccaatactc ctgaaatcca tcgatggagt tcattatgga cagcgcagaa agagctgggg 5100

agaattgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 5160

gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 5220

gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 5280

ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 5340

ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 5400

cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 5460

gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 5520

tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 5580

ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 5640

atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 5700

gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 5760

tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 5820

cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 5880

cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 5940

tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 6000

cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 6060

cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 6120

gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 6180

gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 6240

gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 6300

ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 6360

atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 6420

agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 6480

ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 6540

tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 6600

ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 6660

caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 6720

gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 6780

atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 6840

accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 6900

aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 6960

gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 7020

tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 7080

aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 7140

ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 7200

aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 7260

tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt 7320

cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct 7380

gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg 7440

tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg 7500

gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt 7560

caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 7620

ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc 7680

acgacgttgt aaaacgacgg ccagtgccac gctctccctt atgcgactcc tgcattagga 7740

agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca 7800

aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac 7860

aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat 7920

aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga 7980

ggcgatttaa agacaggata tcagtggtcc aggctctagt tttgactcaa caatatcacc 8040

agctgaagcc tatagagtac gagccataga taaaataaaa gattttattt agtctccaga 8100

aaaagggggg 8110

Claims (10)

1. a kind of recombinant vector, which is characterized in that the recombinant vector is included by TRGC genes, TRDC genes and CD3 ζ genomesInto genetic fragment.

2. recombinant vector according to claim 1, which is characterized in that the genetic fragment further includes connection peptide；

Preferably, the connection peptide is P2A connection peptides；

Preferably, the recombinant vector is MSGV carriers；

Preferably, the genetic fragment includes TRGC genes connected in sequence, CD3 ζ genes, P2A connections peptide gene, TRDC genesWith CD3 ζ genes.

3. recombinant vector according to claim 1 or 2, which is characterized in that the nucleotide sequence such as SEQ of the TRGC genesShown in ID NO.1；

Preferably, the nucleotide sequence of the TRDC genes is as shown in SEQ ID NO.2；

Preferably, the nucleotide sequence of the CD3 ζ genes is as shown in SEQ ID NO.3；

Preferably, the nucleotide sequence of the P2A connections peptide gene is as shown in SEQ ID NO.4；

Preferably, the nucleotide sequence of the genetic fragment is as shown in SEQ ID NO.5；

Preferably, the nucleotide sequence of the recombinant vector is as shown in SEQ ID NO.6.

4. a kind of retrovirus expression vector, which is characterized in that the retrovirus expression vector includes such as claim1-3 any one of them recombinant vectors；

Preferably, the retrovirus expression vector further includes TRAV genes and TRBV genes；

Preferably, the retrovirus expression vector includes TRAV genes connected in sequence, TRGC genes, CD3 ζ genes, companyConnect peptide gene, TRBV genes, TRDC genes and CD3 ζ genes；

Preferably, the nucleotide sequence of the TRAV genes is as shown in SEQ ID NO.7；

Preferably, the nucleotide sequence of the TRBV genes is as shown in SEQ ID NO.8；

Preferably, the nucleotide sequence of the retrovirus expression vector is as shown in SEQ ID NO.9.

5. a kind of slow virus, which is characterized in that the slow virus carries including retrovirus expression as claimed in claim 4Body.

A kind of 6. method for preparing slow virus as claimed in claim 5, which is characterized in that comprise the following steps：

(1) structure such as claim 1-3 any one of them recombinant vectors；

(2) TRAV genes and TRBV genes are connected in the recombinant vector described in step (1), obtain retrovirus expression loadBody；

(3) retrovirus expression vector described in step (2) is transferred to competent cell, after culture picking monoclonal cell intoRow screening；

(4) by the retrovirus expression vector after screening and packaging plasmid cotransfection mammalian cell, the slow virus is obtained.

7. according to the method described in claim 6, it is characterized in that, the method for step (1) described structure is by SEQ ID NO.5In shown genetic fragment insertion MSGV carriers between PacI and XhoI sites；

Preferably, the mammalian cell described in step (4) is GP2-293 cells.

8. the method according to claim 6 or 7, which is characterized in that comprise the following steps：

(1) by between PacI and XhoI sites in the genetic fragment insertion MSGV carriers shown in SEQ ID NO.5, obtain described heavyGroup carrier；

(2) using Jinmen molecule clone technology, TRAV genes and TRBV genes are connected to the recombinant vector described in step (1)In, obtain retrovirus expression vector；

(3) retrovirus expression vector described in step (2) is transferred in competent cell, picking monoclonal cell after cultureIt is screened；

(4) by the retrovirus expression vector after screening and packaging plasmid cotransfection GP2-293 cells, the slow disease is obtainedPoison.

9. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes such as claim 1-3 any one of them weightsIt is any one in group carrier, retrovirus expression vector as claimed in claim 4 or slow virus as claimed in claim 5Kind or at least two combination；

Preferably, described pharmaceutical composition further includes any one in pharmaceutically acceptable carrier, excipient or diluentOr at least two combination.

10. a kind of pharmaceutical composition as claimed in claim 9 is used to prepare the drug of T cell Immuno Suppressive Therapy tumour.