CN101760475B

CN101760475B - Recombinant retroviral vector

Info

Publication number: CN101760475B
Application number: CN2008102409703A
Authority: CN
Inventors: 秦志海; 吕继洲; 徐迎新
Original assignee: Institute of Biophysics of CAS
Current assignee: Institute of Biophysics of CAS
Priority date: 2008-12-25
Filing date: 2008-12-25
Publication date: 2012-05-02
Anticipated expiration: 2028-12-25
Also published as: CN101760475A

Abstract

The invention provides a novel recombinant retroviral vector which comprises a human interleukin 15 gene and a thymidine kinase gene (HSV TK). The recombinant retroviral vector can greatly amplifies T lymphocytes in vitro and maintains the tumor antigen specificity thereof. The vector can introduce the human interleukin 15 gene into a T lymphocyte so that the T lymphocyte can secrete human interleukin 15 per se to stimulate the multiplication of the T lymphocyte, and meanwhile, the tumor antigen specificity of the T lymphocyte is also maintained. The invention also relates to the application of the recombinant retroviral vector.

Description

Recombinant retroviral vector

Technical field

The present invention relates to a kind of novel recombinant retroviral vector, it can be applicable to the clinical tumor immunotherapy, is under the jurisdiction of medical biotechnology field.

Background technology

Malignant tumour is a kind of disease of serious threat human health.Because traditional chemotherapy, radiation therapy and operative therapy is difficult to treat the tumour patient of suffering from metastatic tumor, the tumor immunology therapy receives the extensive concern and the research of immunologist and medical worker.In the clinical treatment field, T cell adoptive transfer therapy successfully has been applied to the tumor disease (1,2) in blood system source as important passive immunization therapy.But for the treatment of solid tumor, the T cell therapy of adopting is made slow progress in recent years, indivedual reports is only arranged with these therapy for treating melanoma patients (3).At present, the limitation of T cell adoptive transfer therapy is: the T lymphocyte number quantity not sufficient that is separated in the tumour patient body; Its immunocompetence, tumour-specific not high (4).At present, general clinically way is in vitro culture tumour-specific T lymphocyte process, to add recombinant interleukin 2 (5), but the DeGrain of its clinical application.Major cause is following: interleukin-22 can cause that the T lymphocyte makes specific t cell proliferation (6) for the receptor-mediated apoptosis pathway sensitivity of Fas; Thereby interleukin-22 can also promote the generation of regulatory T cells to suppress tumour-specific T cell function, and then stops immunity system to remove tumour (7,8).The adoptive transfer immunocyte gets in the oncosis human body also has certain risk, and the immunocyte that promptly changes over to may cause serious autoimmune disorder and t cell lymphoma.This risk can increase after the T cell is gone in interleukin-22 gene or interleukin 15 gene transfection to some extent.

Therefore, in order to overcome the above-mentioned shortcoming of T cell adoptive transfer therapy, the inventor has carried out the present invention.

Summary of the invention

The objective of the invention is in order to overcome the above-mentioned shortcoming of T cell adoptive transfer therapy; A kind of novel recombinant retroviral vector is provided; The T lymphocyte of tumour patient and keep its specific for tumour antigen to improve result of treatment thereby said recombinant retroviral vector not only can increase in a large number; And can remove the cell of genetic modification simultaneously, and avoid it to breed autoimmune disorder and the lymphoma that is caused in vivo, guarantee the security of biotherapy.

Therefore, the present invention provides a kind of novel recombinant retroviral vector, it is characterized in that comprising human interleukin 15 gene (SEQ.ID.No.1; Total length 490bp, NCBI GeneBank sequence registration number is: NM000585) and thymidine kinase gene (HSV-TK, SEQ.ID.No.3; Full length sequence: 1131bp; NCBI GeneBank sequence registration number is: AB178228), and eukaryotic promoter hEF1-HTLV sequence, wherein said thymidine kinase gene is a kind of suicide gene.With this recombinant retroviral vector called after IL15-TK; The retrovirus TK-L15 Retrovirus that the inventor will carry this recombinant retroviral vector IL15-TK on December 22nd, 2008 is preserved in the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms; Da Tun road, Chaoyang District, BeiJing, China institute of microbiology of the Chinese Academy of Sciences; 100101), preservation registration number is: CGMCCNo.2810.

Recombinant retroviral vector IL15-TK of the present invention can and keep its specific for tumour antigen at external a large amount of amplification T lymphocytes: this carrier can import the human interleukin 15 gene in the T lymphocyte; Make its oneself's secretion interleukin 15 stimulate self propagation, also can keep its specific for tumour antigen simultaneously.But; The T lymphocyte of oneself's secretion interleukin 15 might cause autoimmune disorder and T lymphocytoma; Can avoid this situation to take place and suicide gene (thymidine kinase) is together imported the T lymphocyte, thereby guarantee the security of biomedical treatment.

This novel recombinant retroviral vector IL15-TK contains human interleukin 15 and thymidine kinase gene, can infect the various primary cells (primary cells) and the cell series (cell lines) that comprise people source, mouse source.Under the effect of strong promoter on the carrier, but by cells infected great expression interleukin 15 and thymidine kinase.

In one embodiment of the invention; Utilization contains recombinant retroviral vector tgls (+) HyTkEF1a-mIL4 (plasmid map is referring to Fig. 1 E) of HSV-TK, and (this plasmid is presented by German MDC laboratory Thomas doctor Blankenstein; Construction process is referring to reference 16); Enzyme cut remove to insert behind the fragment mIL4 carrier from successively win tgls (+) HyTkEF1a (also abbreviating the TK carrier hereinafter as); Recombinant retroviral vector IL2-TK of the present invention and IL15-TK have been made up on this basis; The retrovirus TK-L15 Retrovirus that wherein will carry IL15-TK is preserved in CGMCC on December 22nd, 2008, and preserving number is CGMCC No.2810.Inventor's primary study IL15-TK, IL2-TK is as the contrast of the functional study of IL15-TK.

In another embodiment of the invention; The recombinant retroviral vector IL15-TK that uses the present invention to make up distinguishes transfectional cell with IL2-TK, can effectively be promoted the propagation (Fig. 4) of mouse source former generation T cell by cells transfected excretory interleukin 15 or interleukin-22.

In a preferred embodiment of the invention, proved that the autocrine interleukin 15 can keep the lymphocytic survival of Ova antigen-specific CD4 T.

In another preferred embodiment of the present invention, proved that the tumour-specific T lymphocyte of autocrine interleukin 15 can mediate tumor rejection better, and the tumour-specific T lymphocyte of autocrine interleukin-22 can not reach above-mentioned effect.

In embodiments of the invention; The application of recombinant retroviral vector of the present invention has been described; It can be used to prepare medicine or the test kit that the clinical tumor immune adjuvant therapy is provided, and can cooperate traditional chemotherapy, radiation therapy to suppress the recurrence and the transfer of tumour.

In a preferred embodiment of the invention, the present invention provides a kind of test kit, and it comprises the recombinant retroviral vector of the present invention of treating significant quantity and pharmaceutical carrier etc.This test kit can be used to provide the clinical tumor immune adjuvant therapy, can cooperate traditional chemotherapy, radiation therapy to suppress the recurrence and the transfer of tumour.

The autocrine interleukin 15 can stimulate T lymphocyte self duplication, helps it to break away from the dependence of cytokine to external world, strengthens identification and kill capability to the tumour target cell, improves the curative effect of adoptive transfer therapy.Human interleukin 15 is the same with the human interleukin 2 to be belonged to together receptor family; But the propagation (9) of effective stimulus T lymphocyte (T lymphocytes) and natural killer cell immunocytes such as (Nature killer cells); The immunocompetence of enhancing immunity cell; And can keep the antigen-specific of immunocyte; Do not cause that the T lymphocyte loses tumour-specific and differentiation apoptosis etc. (10,11).The document that Greenberg is published on the Nature proves: in animal body in the experiment; Human interleukin 15 can be broken the lymphocytic immune tolerance state of tumour-specific T; It is run into show higher cytokine secretion, stronger target cell killing activity and better cylinder therapeutic effect (12,13) behind the tumour antigen.

The adoptive transfer immunocyte gets in the oncosis human body also has certain risk, and the immunocyte that promptly changes over to may cause serious autoimmune disorder and t cell lymphoma.This risk can increase after the T cell is gone in interleukin-22 gene or interleukin 15 gene transfection to some extent.Occur for fear of this situation, we change the HSV-TK gene over to immunocyte with the interleukin 15 gene.HSV-TK (herpes simplex virus thymidine kinase; Herpes simplex virus thymidine kinase) is the suicide property gene in a kind of hsv source; (ganciclovir can will express the cell kill of this gene after GCV) meeting the chemicals DHPG.GCV is a kind of chemotherapeutics, uses clinically.Interleukin 15 gene and TK recombination import in the immunocyte together, and the security that makes immunocyte adoptive transfer therapy is increased.

At present, having two kinds of suicide systems to be applied to clinical treatment, is respectively HSV-TK (GCV) system and Isocytosine deaminase CD (5-FC) system.That expresses Isocytosine deaminase (cytosinedeaminase) can be converted into 5 Fluracils with 5 flucytosines in the cellular genome, final cell death inducing.In the present invention, the inventor concentrates HSV-TK (GCV) system of having studied.

In sum, the beneficial effect of this novel recombinant retroviral vector comprises: make T lymphocyte or NK cell autocrine interleukin 15, the effective immune stimulatory cell proliferation of ability also keeps its anti-tumor activity; Simultaneously, lymphocytic autoimmune disorder that causes of T and T lymphocytoma have effectively been avoided in the introducing of thymidine kinase (suicide gene) again, have guaranteed the security of biotechnology treatment.

Description of drawings

From below in conjunction with the detailed description of accompanying drawing to recombinant retroviral vector, above-mentioned feature and advantage of the present invention will be more obvious, wherein:

Fig. 1 shows the synoptic diagram of recombinant retroviral vector of the present invention.Wherein:

A. recombinant retroviral vector IL15-TK:1.5 ' holds LTR; 2. packaging signal Ψ signal; 3.RNA selectivity shearing site SD-SA; 4. thymidine kinase gene HSV-TK; 5. eukaryotic promoter hEF1-HTLV; 6. human interleukin 15 gene (hIL15); 7.3 ' end LTR.

B. recombinant retroviral vector IL2-TK:1.5 ' holds LTR; 2. packaging signal Ψ signal; 3.RNA selectivity shearing site SD-SA; 4. thymidine kinase gene HSV-TK; 5. eukaryotic promoter hEF1a; 6. human interleukin 2's gene (hIL2); 7.3 ' end LTR.

C. the collection of illustrative plates of the commercialization plasmid pORF-hIL15 of InvivoGen company of carrier's interleukin 15 gene, the eukaryotic promoter on it is hEF1-HTLV.

D. the collection of illustrative plates of the plasmid vector pCDNA3-hIL2 of carrier's interleukin-22 gene.

E. carry the collection of illustrative plates of recombinant retroviral vector tgls (+) HyTKEF1a-mIL4 of thymidine kinase TK suicide gene, illustrate the collection of illustrative plates of tgls (+) HyTKEF1a with this, the difference of the former with the latter is that the former has inserted mouse mIL4 gene.

Fig. 2 shows and successfully made up IL2-TK, and transfection the 293T cell of IL2-TK can secrete the human interleukin 2.A, the enzyme of the IL2-TK plasmid of structure cut digestion and measure, and through the BamHI digestion process, IL2-TK can be cut into two fragments (human interleukin 2's gene of 506bp and other carrier modules of 8200bp); B, transfection 293T emiocytosis human interleukin 2's the ELISA of IL2-TK measure, the result shows that the 293T that imports IL2-TK can secrete a large amount of people's recombinant interleukins 2.

Fig. 3 shows and successfully made up IL15-TK, and transfection the 293T cell of IL15-TK can secrete human interleukin 15.A, the enzyme of the IL15-TK plasmid of structure cut digestion and measure, and through the BamHI digestion process, IL15-TK can be cut into two fragments (human interleukin 2's gene of 512bp and other carrier modules of 8200bp); B, transfection the ELISA of 293T emiocytosis human interleukin 15 of IL15-TK measure, the result shows that the 293T that imports IL15-TK can secrete a large amount of people's recombinant interleukins 15.

The interleukin 15 of the 293T emiocytosis of Fig. 4 has shown transfection IL15-TK and IL2-TK and the propagation that interleukin-22 can effectively promote mouse source former generation Do11.10 CD4 T cell.

The cell of Fig. 5 has shown transfection recombinant retroviral vector and package carrier can be produced the retrovirus that contains IL2-TK (A) and IL15-TK (B).After extracting the RNA in the packing cell culture supernatant, utilize primer to human interleukin 15 and human interleukin 2's gene specific to detect and contain interleukin 15 and interleukin-22 recombinant retroviral vector.

Fig. 6 shows that the retrovirus that contains IL2-TK and IL15-TK can successful infected person JurkatT lymphocyte series.The Jurkat cell of retroviral infection can be secreted human interleukin 2 (A) and human interleukin 15 (B).6C and D show coloration result in the born of the same parents; Equally also the retroviral Jurkat cell of proof infection Re-IL2 (IL2-TK) can produce human interleukin 2 (6C), and the retroviral Jurkat cell of infection Re-IL15 (IL15-TK) can produce human interleukin 15 (6D).

Fig. 7 demonstration is transferred to can cause the 293T necrocytosis of expressing the TK gene in the GCV cell culture medium (0uM, 0.2uM, 1uM and 5uM).

Fig. 8 shows that the autocrine interleukin 15 can keep the lymphocytic survival of Ova antigen-specific Do11.10 CD4 T.Under the effect of T cytositimulation signal aCD3 and aCD28, the antigen-specific CD4 T cell of autocrine human interleukin 15 can be kept survival, the then easier apoptosis of autocrine human interleukin 2's CD4 T cell.

Fig. 9 shows that the tumour-specific T lymphocyte of autocrine interleukin 15 can mediate tumor rejection better.A, after to mouse model injection J558-Ova tumour the 2nd day through the adopt Ova specific C D4 T cell of vitro culture of tail vein injection, at the 6th day and the 12nd day, observe the tumor growth situation that also writes down.Mouse is divided into 4 groups, every group of T cell, the T cell of autocrine interleukin-22, contrast virus infection T cell of injecting the autocrine interleukin 15 respectively, and in same group, same mouse of identical symbology is in the tumour size of different detection times.The result shows that the Do11.10 CD4 T cell of autocrine interleukin 15 can better suppress the J558-Ova tumor growth in vivo; B, the T lymphocyte of secretion interleukin-22 does not have above-mentioned effect; And in the adoptive transfer mouse model, the Do11.10 CD4 T cell of autocrine interleukin 15 can mediate the intravital J558-Ova tumor rejection of mouse.

Embodiment

Below come further to illustrate the present invention through embodiment.But should be appreciated that said embodiment is illustrational purpose, and be not intended to limit scope of the present invention and spirit.

Embodiment 1: make up recombinant retroviral vector

The synoptic diagram of recombinant retroviral vector IL15-TK and IL2-TK is with reference to Figure 1A and 1B.

Construction of recombinant defective retroviral vector is based on that retroviral vector tgls (+) HyTKEF1a that carries the TK suicide gene (being called for short the TK carrier) carries out.Tgls (+) HyTKEF1a introduces eukaryotic promoter EF1a by tgLS (+) HyTK plasmid (14) and transforms acquisition.Last TyTK fusion gene coding hph and the HSV-1 TK enzymic activity of carrying of tgls (+) HyTKEF1a.

The IL2-TK recombinant retroviral vector makes up:

A) obtain the hIL2 gene fragment:

(plasmid map is seen Fig. 1 D with the plasmid vector pcDNA3-hIL2 of carrier's interleukin-22 gene; Its sequence is SEQ.ID.No.6; Wherein human interleukin 2's gene is positioned at base 913-1374 place) (this plasmid effluent north Wang Yong of medical university auspicious laboratory present; Referring to reference 15) be template, designer's interleukin-22 gene primer, forward primer 1 (CgC ggATCC gCgATgTACAggATgC) and reverse primer 1 (CgC ggATCC gCgCTCAAgTCAgTgT); Pcr amplification, the condition of PCR is: 94 ℃ of sex change 1 minute; 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 circulations 30 seconds; 72 ℃ 10 minutes; Obtain the hIL2 gene fragment thus.

B) human interleukin 2's gene fragment is handled:

The hIL2 gene fragment that obtains through PCR in the step 1 is connected on the T carrier of promega company, then handles the hIL2 gene fragment that obtains to have the BamHI cohesive end with BamHI.

C) carry the processing of TK retroviral vector tgls (+) HyTKEF1a:

Cut tgls (+) HyTKEF1a plasmid with the BamHI enzyme, obtain 7.6Kb size BamHI cohesive end TK retroviral vector.

D) connect structure hIL2-TK retroviral vector:

The carrier segments that step b) gene fragment that has the BamHI cohesive end that obtains and the enzyme that step c) obtains are cut is connected, and obtains the hIL2-TK recombinant retroviral vector, and the system and the condition that wherein connect are following:

T4 dna ligase: 2 units

10 * ligase enzyme damping fluid: 2ul

Enzyme is cut the hIL2 fragment of processing: 80ng

Enzyme is cut the carrier segments of processing: 1ug

Distilled water: TV is mended 20ul

16 ℃ of connections are spent the night.

The IL15-TK recombinant retroviral vector makes up:

We find that the expression output of human interleukin 15 is compared with interleukin-22 much lower (result does not show) under identical promoters EF1a in the process that makes up recombinant interleukin 2 and interleukin 15 recombinant retroviral vector.In order to address this problem, we have adopted the promotor replacement policy, promptly with hEF1-HTLV promoter, fusion replacement EF1a promotor.It should be appreciated by those skilled in the art that the strategy of promotor replacement has a lot of methods to realize.For example; Can be earlier with the hIL15 gene fragment clone in plasmid with hEF1-HTLV promotor; Utilize the fusion fragment of specific PCR primer then, be cloned in the TK carrier through the standard molecule clone technology at last through pcr amplification acquisition hEF1-HTLV-hIL15.In the present embodiment, we attempt to utilize available from the hEF1-HTLV promoter, fusion on the original interleukin 15 plasmid (pORF-hIL15) of InvivoGen company and carry out promotor replacement, improve human interleukin 15 and express output.

IL15-TK recombinant retroviral vector building process is following:

A) acquisition comprises the PCR fragment of hEF1-HTLV promoter, fusion and people's recombinant interleukin 15 genes:

(plasmid map is seen Fig. 1 C with the plasmid vector pORF-hIL15 that carries hEF1-HTLV promoter, fusion and human interleukin 15 gene; Plasmid sequence is SEQ.ID.No.5) (InvivoGen company; Catalog number (Cat.No.): porf-hil15; Wherein human interleukin 15 gene hIL15 is positioned at base 690-1178 place, and the hEF1-HTLV promotor is positioned at base 1-544 place (SEQ.ID.No.4)) be template to design forward primer 2 (CCCggg gCTCCggTgCCCgTC) and reverse primer 2 (ggATCC TCAATT gCA ATC); Pcr amplification, the condition of PCR is: 94 ℃ of sex change 1 minute; 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 circulations 30 seconds; 72 ℃ 10 minutes; Obtain to comprise the PCR fragment hEF1-HTLV-hIL15 of hEF1-HTLV promoter, fusion and people's recombinant interleukin 15 genes thus.

B) the human interleukin 15 gene fragment is handled:

The hEF1-HTLV-hIL15 fragment that step 1 is obtained is connected on the T carrier of promega company, uses SmaI and BamHI double digestion to handle plasmid, and it is sticking terminal to obtain flat end of SmaI and BamHI.

C) carry the processing of TK retroviral vector tgls (+) HyTKEF1a:

Use HindIII handles tgls (+) HyTKEF1a plasmid, then mends with the T4 archaeal dna polymerase to become to put down end, and BamHI processing then obtains to glue terminal.

D) connect structure hIL15-TK retroviral vector:

The gene fragment that step b) is obtained is connected with the carrier segments that step c) obtains, and obtains the hIL15-TK recombinant retroviral vector.This hIL15-TK recombinant retroviral vector carries the reinforced promotor of hEF1-HTLV.

The system and the condition that wherein connect are following:

T4 dna ligase: 2 units

10 * ligase enzyme damping fluid: 2ul

Enzyme is cut the hEF1-HTLV-hIL15 fragment of processing: 80ng

Enzyme is cut the carrier segments of processing: 1ug

Distilled water: TV is mended 20ul

16 ℃ of connections are spent the night

The inventor has carried out preservation to the retrovirus TK-L15 Retrovirus that carries IL15-TK, and preserving number is CGMCC No.2810, and preservation date is on December 22nd, 2008.

The used gene order explanation of the present invention sees the following form 1.

Table 1: the gene order explanation that the present invention is used

SEQ.ID.No.	Title
		1	Human IL-15 (GeneBank sequence registration number: NM000585)
2	Human IL-2's (GeneBank sequence registration number: NM000586)
		3	The HSV-TK full length sequence
4	Promotor hEF1-HTLV
		5	The pORF-hIL15 plasmid
6	The pcDNA3-hIL2 plasmid

Yet, it should be appreciated by those skilled in the art that the gene fragment IL2 of insertion and IL15 can also obtain through molecular cloning method well known in the art; For example; From sample tissue, extract total RNA, design specific primers is carried out RT-PCR and can be obtained purpose insertion fragment.

Therefore, IL2-TK adopts two kinds of different promotors, i.e. EF1a and hEF1-HTLV respectively with IL15-TK.The EF1a eukaryotic promoter comes from the promotor (Eukaryotic translation elongation factor 1 alpha 1) of people's eukaryotic translation EF-1 a, and the EF1a promotor can start the expression downstream gene in a large number at positions such as human brain, placenta, lung, liver, kidney and prostate glands.After being applied to biological technical field, this promotor inorganization specificity can start expression by high level in various eukaryotic cells, be one of the most frequently used promotor of present eukaryotic expression.The reinforced promotor of hEF1-HTLV is that this pattern of fusion promotor can strengthen the stability of downstream DNA and RNA sequence with the recombinate back of EF1a promotor of the R assembly in human T-cell leukemia virus's (HTLV) the I type LTR sequence and part U5 sequence.Therefore, promoter, fusion also can increase the amount of the expression product of downstream gene through the stability of strengthening rna transcription product.

For IL2-TK, the expression of IL2 receives the control of promotor EF1a, and for IL15-TK, the hEF1-HTLV promoter, fusion has been replaced the EF1a promotor, can improve human interleukin 15 and express output.Yet experimental result proves: promoter, fusion can improve the output of interleukin 15, but does not still reach the expression level of interleukin-22.

The article that is published on the Human Gene Therapy in May, 2008 Mr. Rosenberg shows: autocrine human interleukin 2's tumour-specific CD8 T cell can not be than better mediating tumor rejection without transformed T cell.This and wes' experimental result consistent (Fig. 9).Therefore, primary study IL15-TK of the present invention.

Transcription product RNA under native state is unstable for human interleukin 15, and the amount of albumen translation product is not high under equal transcriptional level.Can use the expression level that other eukaryotic promoters can further improve human interleukin 15 instead, but the height of expression amount is disproportionate with actual medical treatment effect.This needs further check.What we can confirm is under present expression level, and the autocrine interleukin 15 can effectively be strengthened the tumor suppression effect of specific T-cells.

Embodiment 2: make up retrovirus package carrier cell

1. with 5*10 ⁵Human embryonic kidney cell line 293T spread six orifice plates, every hole adds 1640 substratum of 1ml serum-free, antibiotic-free, under pH value 7.2-7.4,37 ℃ of temperature, relative humidity 95%, 5%CO2 culture condition, hatches 12 hours.

2. in 1640 substratum of 100ul serum-free, antibiotic-free, add 3ul lipofectamine2000 transfection reagent; In 1640 substratum of 100ul serum-free, antibiotic-free, add 0.2ug recombinant retroviral vector (IL2-TK simultaneously; IL15-TK or TK) and 1ug virus package carrier 10A1, room temperature was placed 5 minutes.

3. mix and be dissolved in transfection plasmid and the transfection reagent in the substratum, room temperature held 15 minutes.

4. change original 293T cell culture medium with 800ul serum-free, antibiotic-free 1640 substratum.

5. the mixed solution with transfection plasmid, transfection reagent adds in the 293T cell culture medium.Hatched 4 hours for 37 ℃.

6. with containing the substratum that 10% calf serum and 10% antibiotic 1640 substratum replacing contain transfection reagent.

7. under pH value 7.2-7.4,37 ℃ of temperature, relative humidity 95%, 5%CO2 culture condition, hatched 72 hours.

8. add the 75ug/ml HYG and carry out the positive colony screening.

9.7 after it, preserve positive colony.Collect cells and supernatant and can obtain virus.

Embodiment 3: recombinant retroviral vector infected person Jurkat clone

Infection experiment carries out according to ordinary method well known by persons skilled in the art.Summary infection step is following:

1. at diameter 8cm Tissue Culture Dish middle berth 5*10 ⁶The viral packing cell that obtains among the embodiment 2.Add 5ml 1640 substratum.Hatched 48 hours under pH value 7.2-7.4,37 ℃ of temperature, relative humidity 95%, 5%CO2 culture condition.

2. collect and contain retroviral packing cell culture supernatant, use 0.45 μ m membrane filtration to eliminate cell debris.

3. the virus titer of step 2 being collected is adjusted to 2*10 ⁶/ ml adds polybrene (polybrene) to final concentration 6ng/ml.

4. with 2*10 ⁵The Jurkat cell is resuspended in the viral supernatant of 1ml step 3 (virus: target cell (number than)=10: 1).

5. at 1500g, 30 ℃ of following centrifugal 90min then place 150min in incubator.

6. change fresh 1640 substratum for the Jurkat cell that infected.

7. after infecting 72 hours, human interleukin 15 capable of using and the dyeing of human interleukin 2's intracellular antibody detect the infection rate of cell.

Through Totomycin (200ug/ml) screening in two weeks, can detect the expression of interleukin 15 in the supernatant of the Jurkat cell of infection IL15-TK virus.In like manner, also can detect the expression of interleukin-22 in the supernatant of the Jurkat cell of infection IL2-TK virus.Show that retrovirus has successfully infected the Jurkat cell.

Embodiment 4: the effect detection of recombinant retroviral vector

1. human interleukin 15 and interleukin-22 gene function detect on the recombinant retroviral vector.

Petridish middle berth 5*10 at diameter 8cm ⁶Individual 293T cell (comprise four kinds of 293T cells: transfection IL2-TK, transfection IL15-TK, transfection mock-TK and the control group that does not have transfection carrier of control group carrier), add 7ml 1640 substratum.60 hours, collecting cell culture supernatant ELISA detected interleukin-22.The result shows that the 293T cell that imports recombinant retroviral vector IL2-TK can secrete a large amount of people's recombinant interleukins 2; Secretory volume reaches 32.3ng/ml (Fig. 2 B); The 293T cell that imports recombinant retroviral vector IL15-TK can be secreted a large amount of people's recombinant interleukins 15, and secretory volume reaches 1315pg/ml (Fig. 3 B).

Collect respectively above-mentioned four kinds of 293T cells culture supernatant (transfection IL2-TK, transfection IL15-TK, transfection mock-TK and the control group that does not have transfection carrier of control group carrier).Use recombination human interleukin 2 or human interleukin 15 (concentration is respectively 0u/ml, 20u/ml and 50u/ml) to compare, relatively contain 50% culture supernatant with the method for MTT and hatch former generation CD4 T (Do11.10) cell proliferation situation.Under 450nm, read the OD value, viable count is many more, and the OD value is high more.The result of Fig. 4 shows, the interleukin-22 of the 293T emiocytosis of importing recombinant retroviral vector or the propagation that interleukin 15 can promote former generation Do11.10 CD4 T cell.

With reference to figure 6, with 2*10 ⁶The Jurkat cell suspension of/ml is spread 24 orifice plates, collecting cell culture supernatant after 60 hours, and ELISA detects the secretion of cytokine human interleukin 2 and human interleukin 15.Said Jurkat cell comprises four kinds: infection IL15-TK's, infection IL2-TK's, infection TK virus, and the control group Jurkat cell of not handling.Can find out that from Fig. 6 the retrovirus that contains IL2-TK and IL15-TK can successful infected person Jurkat T lymphocyte series.The Jurkat cell of retroviral infection can be secreted human interleukin 2 (Fig. 6 A) and human interleukin 15 (Fig. 6 B).And coloration result has equally also proved in the born of the same parents, infect the retroviral Jurkat cell of Re-IL2 (IL2-TK) and can produce human interleukin 2 (Fig. 6 C), and the retroviral Jurkat cell of infection Re-IL15 (IL15-TK) can produce human interleukin 15 (Fig. 6 D).

2. the Function detection of suicide gene TK gene on the recombinant retroviral vector.

The middle berth 3*10 in the every hole of 96 orifice plates ⁴Individual 293T cell (be transfection respectively IL15-TK carrier, the contrast 293T cell of IL2-TK carrier and any carrier of untransfected) is hatched above-mentioned 293T cell with the cell culture medium of the GCV (0uM, 0.2uM, 1uM and 5uM) that comprises four concentration gradients.At pH value 7.2-7.4,37 ℃ of temperature, relative humidity 95%, 5%CO ₂Culture condition cultivated 60 hours down, detect the viable cell number with mtt assay.

Can find out from the result of Fig. 7, compare, the 293T cell transfer of expressing the TK gene can be caused the 293T necrocytosis of expressing the TK gene in the substratum that contains GCV with the control cells of any carrier of untransfected.This explains that also IL2-TK and IL15-TK recombinant retroviral vector constructed in the embodiment of the invention 1 have all successfully changed suicide gene TK gene over to.

3. experiment in vitro proof autocrine interleukin 15 can promote the survival of activated cd4 t cell.

With reference to figure 8, with the CD4 T cell that derives from Do11.10 with every hole 5*10 ⁶/ ml is layered in the hole of 24 orifice plates.Utilize plate bonded aCD3 (500ng/ml) and free aCD28 (300ng/ml) activation antigen specific T-cells, detect t cell proliferation situation with the two dyeing of Annexin V FITC (FL1) through the flow cytometry method with PI (FL2) after 72 hours.Experiment shows: the T cell of autocrine interleukin 15 46% survival and the T cell of autocrine interleukin-22 has only 9.8% survival.

The experimental result of Fig. 8 shows that under the effect of T cytositimulation signal aCD3 and aCD28, the Ova antigen-specific CD4 T cell of autocrine human interleukin 15 can be kept survival, the then easier apoptosis of autocrine human interleukin 2's CD4 T cell.

It should be appreciated by those skilled in the art; Because interleukin 15 is a kind of multi-functional ESC; It can promote the survival of CD4 T cell, CD8 T cell and NK cell under natural condition; Specific C D4 in the booster immunization reaction, the memory effect of CD8 T cell, so, in conjunction with the experimental result of Fig. 8; Recombinant retroviral vector IL15-TK of the present invention can also promote the survival of CD4 T cell, CD8 T cell and NK cell, the specific C D4 in the booster immunization reaction, the memory effect of CD8 T cell.

4. the effect of the anti-J558-Ova of the Ova antigen-specific CD4 T cell of experimental verification autocrine interleukin 15 in the body.

Experiment is carried out through mouse T cell adoptive transfer experimental model in the body.Said mouse T cell adoptive transfer experimental model makes up as follows:

A) the 0th day the time at Balb/C mouse veutro (wild-type mice, 6 to 8 weeks of age) inoculation 2*10 ⁶The J558-Ova cell;

B) second day the time through the Ova specific C D4 T cell of tail vein injection adoptive transfer vitro culture, said cell is divided into four groups: the T cell of autocrine interleukin 15, the T cell of autocrine interleukin-22, contrast virus infection T cell and phosphate buffered saline buffer control group;

C) behind the J558-Ova tumor injection the 6th day and the 12nd day, observe also record tumor growth situation.

Experimental result shows that the T lymphocyte of autocrine interleukin 15 can better suppress J558-Ova growth of tumor (Fig. 9 A) in the mouse body, and the T lymphocyte of autocrine interleukin-22 does not have above-mentioned effect; And in the adoptive transfer mouse model, the tumor rejection of T cell mediated can be protected tumor-bearing mice (Fig. 9 B) effectively.

In J558-Ova and Ova specific C D4 T cell model; We have proved the antitumous effect of the CD4 T cell of autocrine interleukin 15, but the effect of the CD4 T cell of the autocrine interleukin 15 in other tumor models and CD8 T cell is still waiting check.

Should be appreciated that; Although with reference to its exemplary embodiment; The present invention is shown particularly and describe; But will be understood by those skilled in the art that, can carry out the variation of various forms and details therein, and not deviate from by the defined the spirit and scope of the present invention of accompanying Claim.

Reference:

1.Kolb，H.J.，C.Schmid，A.J.Barrett，and?D.J.Schendel2004.Graft-versus-leukemia?reactions?in?allogeneic?chimeras.Blood103：767-776.

2.Kolb，H.J.，A.Schattenberg，J.M.Goldman，B.Hertenstein，N.Jacobsen，W.Arcese，P.Ljungman，A.Ferrant，L.Verdonck，D.Niederwieser，F.vanRhee，J.Mittermueller，T.de?Witte，E.Holler，and?H.Ansari.1995.Graft-versus-leukemia?effect?of?donor?lymphocyte?transfusions?in?marrowgrafted?patients.Blood?86：2041-2050.

3.Dudley，M.E.，J.R.Wunderlich，P.F.Robbins，J.C.Yang，P.Hwu，D.J.Schwartzentruber，S.L.Topalian，R.Sherry，N.P.Restifo，A.M.Hubicki，M.R.Robinson，M.Raffeld，P.Duray，C.A.Seipp，L.Rogers-Freezer，K.E.Morton，S.A.Mavroukakis，D.E.White，and?S.A.Rosenberg.2002.Cancerregression?and?autoimmunity?in?patients?after?clonal?repopulation?withantitumor?lymphocytes.Science?298：850-854.

4.Gattinoni，L.et?al.Acquisition?of?full?effector?function?in?vitroparadoxically?impairs?the?in?vivo?antitumour?efficacy?of?adoptivelytransferred?CD8+T?cells.J.Clin.Invest.115，1616-1626(2005).

5.S?A?Rosenberg，and，M?T?Lotze?Cancer?Immunotherapy?UsingInterleukin-2?and?Interleukin-2-Activated?Lymphocytes?Annual?Review?ofImmunology.Volume?4，Page?681-709，Apr?1986

6.Yosef?Refaeli，Luk?Van?Parijs，Cheryl?A?London，Jürg?Tschopp，and?AbulK?Abbas?Biochemical?Mechanisms?of?IL-2-Regulated?Fas-Mediated?TCell?Apoptosis?Immunity?1998?8：615-623

7.Shimon?Sakaguchi，Tomoyuki?Yamaguchi，Takashi?Nomura，and?MasahiroOno?Regulatory?T?Cells?and?Immune?Tolerance?Cell，Vol?133，775-787，30?May?2008

8.Antony，P.A.et?al.CD8+T?cell?immunity?against?a?tumour/self-antigen?isaugmented?by?CD4+T?helper?cells?and?hindered?by?naturally?occurring?Tregulatory?cells.J.Immunol.174，2591-2601(2005).

9.Gattinoni，L.et?al.Acquisition?of?full?effector?function?in?vitroparadoxically?impairs?the?in?vivo?antitumour?efficacy?of?adoptivelytransferred?CD8+T?cells.J.Clin.Invest.115，1616-1626(2005).

10.Opferman，J.T.et?al.Development?and?maintenance?of?B?and?Tlymphocytes?requires?antiapoptotic?MCL-1.Nature?426，671-676(2003).

11.Hsu，C.et?al.Primary?human?T?lymphocytes?engineered?with?acodon-optimized?IL-15?gene?resist?cytokine?withdrawal-induced?apoptosisand?persist?long-term?in?the?absence?of?exogenous?cytokine.J.Immunol.175，7226-7234(2005).

12.Klebanoff，C.A.et?al.Central?memory?self/tumour-reactive?CD8+T?cellsconfer?superior?antitumour?immunity?compared?with?effector?memory?Tcells.Proc.Natl?Acad.Sci.USA?102，9571-9576(2005).

13.Teague，R.M.，B.D.Sather，J.A.Sacks，M.Z.Huang，M.L.Dossett，J.Morimoto，X.Tan，S.E.Sutton，M.P.Cooke，C.Ohlen，and?P.D.Greenberg.2006.Interleukin-15?rescues?tolerant?CD8+T?cells?for?use?inadoptive?immunotherapy?of?established?tumors.Nat?Med?12：335-341.

14.ROBERT?W.OVERELL?Dominant?Positive?and?Negative?SelectionUsing?a?Hygromycin?Phosphotransferase-Thymidine?Kinase?Fusion?GeneMOLECULAR?AND?CELLULAR?BIOLOGY，June?1991

15. the gorgeous brightness in side, the gold and jade bosom, Wang Yongxiang, Yu Danjun, Jin Shixiang, Xie Lixin, human IL-2's signal peptide gene strengthen CB 3 type VP1 dna vaccination inductive neutralizing antibodies and reply, Chinese microbiology and Journal of Immunology,, the 24th volume, the 3rd phase in 2004

16.Gabriele?Noffz，Zhihai?Qin，Manfred?Kopf，and?Thomas?Blankenstein，Neutrophils?but?Not?Eosinophils?Are?Involved?in?Growth?Suppression?ofIL-4-Secreting?Tumors.The?Journal?of?Immunology，1998，160：345-350

SEQUENCE?LISTING

< 110>Institute of Biophysics, Academia Sinica

< 120>recombinant retroviral vector

<130>IB089435

<160>6

<170>PatentIn?version?3.1

<210>1

<211>489

<212>DNA

< 213>homo sapiens

<400>1

atgagaattt?cgaaaccaca?tttgagaagt?atttccatcc?agtgctactt?gtgtttactt 60

ctaaacagtc?attttctaac?tgaagctggc?attcatgtct?tcattttggg?ctgtttcagt 120

gcagggcttc?ctaaaacaga?agccaactgg?gtgaatgtaa?taagtgattt?gaaaaaaatt 180

gaagatctta?ttcaatctat?gcatattgat?gctactttat?atacggaaag?tgatgttcac 240

cccagttgca?aagtaacagc?aatgaagtgc?tttctcttgg?agttacaagt?tatttcactt 300

gagtccggag?atgcaagtat?tcatgataca?gtagaaaatc?tgatcatcct?agcaaacaac 360

agtttgtctt?ctaatgggaa?tgtaacagaa?tctggatgca?aagaatgtga?ggaactggag 420

gaaaaaaata?ttaaagaatt?tttgcagagt?tttgtacata?ttgtccaaat?gttcatcaac 480

acttcttga 489

<210>2

<211>462

<212>DNA

< 213>homo sapiens

<400>2

atgtacagga?tgcaactcct?gtcttgcatt?gcactaagtc?ttgcacttgt?cacaaacagt 60

gcacctactt?caagttctac?aaagaaaaca?cagctacaac?tggagcattt?actgctggat 120

ttacagatga?ttttgaatgg?aattaataat?tacaagaatc?ccaaactcac?caggatgctc 180

acatttaagt?tttacatgcc?caagaaggcc?acagaactga?aacatcttca?gtgtctagaa 240

gaagaactca?aacctctgga?ggaagtgcta?aatttagctc?aaagcaaaaa?ctttcactta 300

agacccaggg?acttaatcag?caatatcaac?gtaatagttc?tggaactaaa?gggatctgaa 360

acaacattca?tgtgtgaata?tgctgatgag?acagcaacca?ttgtagaatt?tctgaacaga 420

tggattacct?tttgtcaaag?catcatctca?acactgactt?ga 462

<210>3

<211>1131

<212>DNA

< 213>hsv

<400>3

atggcttctc?acgccggcca?acagcacgcg?cctgcgttcg?gtcaggctgc?tcgtgcgagc 60

gggcctaccg?acggccgcgc?ggcgtcccgt?cctagccatc?gccagggggc?ctccggagcc 120

cgcggggatc?cggagctgcc?cacgctgctg?cgggtttata?tagacggacc?ccacggggtg 180

gggaagacca?ccacctccgc?gcagctgatg?gaggccctgg?ggccgcgcga?caatatcgtc 240

tacgtccccg?agccgatgac?ttactggcag?gtgctggggg?cctccgagac?cctgacgaac 300

atctacaaca?cgcagcaccg?tctggaccgc?ggcgagatat?cggccgggga?ggcggcggtg 360

gtaatgacca?gcgcccagat?aacaatgagc?acgccttatg?cggcgacgga?cgccgttttg 420

gctcctcata?tcggggggga?ggctgtgggc?ccgcaagccc?cgcccccggc?cctcaccctt 480

gttttcgacc?ggcaccctat?cgcctccctg?ctgtgctacc?cggccgcgcg?gtacctcatg 540

ggaagcatga?ccccccaggc?cgtgttggcg?ttcgtggccc?tcatgccccc?gaccgcgccc 600

ggcacgaacc?tggtcctggg?tgtccttccg?gaggccgaac?acgccgaccg?cctggccaga 660

cgccaacgcc?cgggcgagcg?gcttgacctg?gccatgctgt?ccgccattcg?ccgtgtctac 720

gatctactcg?ccaacacggt?gcggtacctg?cagcgcggcg?ggaggtggcg?ggaggactgg 780

ggccggctga?cgggggtcgc?cgcggcgacc?ccgcgccccg?accccgagga?cggcgcgggg 840

tctctgcccc?gcatcgagga?cacgctgttt?gccctgttcc?gcgttcccga?gctgctggcc 900

cccaacgggg?acttgtacca?catttttgcc?tgggtcttgg?acgtcttggc?cgaccgcctc 960

cttccgatgc?atctatttgt?cctggattac?gatcagtcgc?ccgtcgggtg?tcgagacgcc 1020

ctgttgcgcc?tcaccgccgg?gatgatccca?acccgcgtca?caaccgccgg?gtccatcgcc 1080

gagatacgcg?acctggcgcg?cacgtttgcc?cgcgaggtgg?ggggagttta?g 1131

<210>4

<211>544

<212>DNA

< 213>artificial

<400>4

ggatctgcga?tcgctccggt?gcccgtcagt?gggcagagcg?cacatcgccc?acagtccccg 60

agaagttggg?gggaggggtc?ggcaattgaa?ccggtgccta?gagaaggtgg?cgcggggtaa 120

actgggaaag?tgatgtcgtg?tactggctcc?gcctttttcc?cgagggtggg?ggagaaccgt 180

atataagtgc?agtagtcgcc?gtgaacgttc?tttttcgcaa?cgggtttgcc?gccagaacac 240

agctgaagct?tcgaggggct?cgcatctctc?cttcacgcgc?ccgccgccct?acctgaggcc 300

gccatccacg?ccggttgagt?cgcgttctgc?cgcctcccgc?ctgtggtgcc?tcctgaactg 360

cgtccgccgt?ctaggtaagt?ttaaagctca?ggtcgagacc?gggcctttgt?ccggcgctcc 420

cttggagcct?acctagactc?agccggctct?ccacgctttg?cctgaccctg?cttgctcaac 480

tctacgtctt?tgtttcgttt?tctgttctgc?gccgttacag?atccaagctg?tgaccggcgc 540

ctac 544

<210>5

<211>4101

<212>DNA

< 213>artificial

<400>5

ggatctgcga?tcgctccggt?gcccgtcagt?gggcagagcg?cacatcgccc?acagtccccg 60

agaagttggg?gggaggggtc?ggcaattgaa?ccggtgccta?gagaaggtgg?cgcggggtaa 120

actgggaaag?tgatgtcgtg?tactggctcc?gcctttttcc?cgagggtggg?ggagaaccgt 180

atataagtgc?agtagtcgcc?gtgaacgttc?tttttcgcaa?cgggtttgcc?gccagaacac 240

agctgaagct?tcgaggggct?cgcatctctc?cttcacgcgc?ccgccgccct?acctgaggcc 300

gccatccacg?ccggttgagt?cgcgttctgc?cgcctcccgc?ctgtggtgcc?tcctgaactg 360

cgtccgccgt?ctaggtaagt?ttaaagctca?ggtcgagacc?gggcctttgt?ccggcgctcc 420

cttggagcct?acctagactc?agccggctct?ccacgctttg?cctgaccctg?cttgctcaac 480

tctacgtctt?tgtttcgttt?tctgttctgc?gccgttacag?atccaagctg?tgaccggcgc 540

ctacgtaagt?gatatctact?agatttatca?aaaagagtgt?tgacttgtga?gcgctcacaa 600

ttgatactta?gattcatcga?gagggacacg?tcgactacta?accttcttct?ctttcctaca 660

gctgagatca?ccggcgaagg?agggccacca?tgcgaatttc?gaaaccacat?ttgagaagta 720

tttccatcca?gtgctacttg?tgtttacttc?taaacagtca?ttttctaact?gaagctggca 780

ttcatgtctt?cattttgggc?tgtttcagtg?cagggcttcc?taaaacagaa?gccaactggg 840

tgaatgtaat?aagtgatttg?aaaaaaattg?aagatcttat?tcaatctatg?catattgatg 900

ctactttata?tacggaaagt?gatgttcacc?ccagttgcaa?agtaacagca?atgaagtgct 960

ttctcttgga?gttacaagtt?atttcacttg?agtccggaga?tgcaagtatt?catgatacag 1020

tagaaaatct?gatcatccta?gcaaacaaca?gtttgtcttc?taatgggaat?gtaacagaat 1080

ctggatgcaa?agaatgtgag?gaactggagg?aaaaaaatat?taaagaattt?ttgcagagtt 1140

ttgtacatat?tgtccaaatg?ttcatcaaca?cttcttgatt?gcaattgagc?tagcattatc 1200

cctaatacct?gccaccccac?tcttaatcag?tggtggaaga?acggtctcag?aactgtttgt 1260

ttcaattggc?catttaagtt?tagtagtaaa?agactggtta?atgataacaa?tgcatcgtaa 1320

aaccttcaga?aggaaaggag?aatgttttgt?ggaccacttt?ggttttcttt?tttgcgtgtg 1380

gcagttttaa?gttattagtt?tttaaaatca?gtacttttta?atggaaacaa?cttgaccaaa 1440

aatttgtcac?agaattttga?gacccattaa?aaaagttaaa?tgagaaacct?gtgtgttcct 1500

ttggtcaaca?ccgagacatt?taggtgaaag?acatctaatt?ctggttttac?gaatctggaa 1560

acttcttgaa?aatgtaattc?ttgagttaac?acttctgggt?ggagaatagg?gttgttttcc 1620

ccccacataa?ttggaagggg?aaggaatatc?atttaaagct?atgggagggt?ttctttgatt 1680

acaacactgg?agagaaatgc?agcatgttgc?tgattgcctg?tcactaaaac?aggccaaaaa 1740

ctgagtcctt?gggttgcata?gaaagcttca?tgttgctaaa?ccaatgttaa?gtgaatcttt 1800

ggaaacaaaa?tgtttccaaa?ttactgggat?gtgcatgttg?aaacgtgggt?taattaagaa 1860

catgtgagca?aaaggccagc?aaaaggccag?gaaccgtaaa?aaggccgcgt?tgctggcgtt 1920

tttccatagg?ctccgccccc?ctgacgagca?tcacaaaaat?cgacgctcaa?gtcagaggtg 1980

gcgaaacccg?acaggactat?aaagatacca?ggcgtttccc?cctggaagct?ccctcgtgcg 2040

ctctcctgtt?ccgaccctgc?cgcttaccgg?atacctgtcc?gcctttctcc?cttcgggaag 2100

cgtggcgctt?tctcaatgct?cacgctgtag?gtatctcagt?tcggtgtagg?tcgttcgctc 2160

caagctgggc?tgtgtgcacg?aaccccccgt?tcagcccgac?cgctgcgcct?tatccggtaa 2220

ctatcgtctt?gagtccaacc?cggtaagaca?cgacttatcg?ccactggcag?cagccactgg 2280

taacaggatt?agcagagcga?ggtatgtagg?cggtgctaca?gagttcttga?agtggtggcc 2340

taactacggc?tacactagaa?gaacagtatt?tggtatctgc?gctctgctga?agccagttac 2400

cttcggaaaa?agagttggta?gctcttgatc?cggcaaacaa?accaccgctg?gtagcggtgg 2460

tttttttgtt?tgcaagcagc?agattacgcg?cagaaaaaaa?ggatctcaag?aagatccttt 2520

gatcttttct?acggggtctg?acgctcagtg?gaacgaaaac?tcacgttaag?ggattttggt 2580

catgagatta?tcaaaaagga?tcttcaccta?gatcctttta?aattaaaaat?gaagttttaa 2640

atcaatctaa?agtatatatg?agtaaacttg?gtctgacagt?taccaatgct?taatcagtga 2700

ggcacctatc?tcagcgatct?gtctatttcg?ttcatccata?gttgcctgac?tccccgtcgt 2760

gtagataact?acgatacggg?agggcttacc?atctggcccc?agtgctgcaa?tgataccgcg 2820

agacccacgc?tcaccggctc?cagatttatc?agcaataaac?cagccagccg?gaagggccga 2880

gcgcagaagt?ggtcctgcaa?ctttatccgc?ctccatccag?tctattaatt?gttgccggga 2940

agctagagta?agtagttcgc?cagttaatag?tttgcgcaac?gttgttgcca?ttgctacagg 3000

catcgtggtg?tcacgctcgt?cgtttggtat?ggcttcattc?agctccggtt?cccaacgatc 3060

aaggcgagtt?acatgatccc?ccatgttgtg?caaaaaagcg?gttagctcct?tcggtcctcc 3120

gatcgttgtc?agaagtaagt?tggccgcagt?gttatcactc?atggttatgg?cagcactgca 3180

taattctctt?actgtcatgc?catccgtaag?atgcttttct?gtgactggtg?agtactcaac 3240

caagtcattc?tgagaatagt?gtatgcggcg?accgagttgc?tcttgcccgg?cgtcaatacg 3300

ggataatacc?gcgccacata?gcagaacttt?aaaagtgctc?atcattggaa?aacgttcttc 3360

ggggcgaaaa?ctctcaagga?tcttaccgct?gttgagatcc?agttcgatgt?aacccactcg 3420

tgcacccaac?tgatcttcag?catcttttac?tttcaccagc?gtttctgggt?gagcaaaaac 3480

aggaaggcaa?aatgccgcaa?aaaagggaat?aagggcgaca?cggaaatgtt?gaatactcat 3540

actcttcctt?tttcaatatt?attgaagcat?ttatcagggt?tattgtctca?tgagcggata 3600

catatttgaa?tgtatttaga?aaaataaaca?aataggggtt?ccgcgcacat?ttccccgaaa 3660

agtgccacct?gacgtctaag?aaaccattat?tatcatgaca?ttaacctata?aaaataggcg 3720

tatcacgagg?ccctttcgtc?tcgcgcgttt?cggtgatgac?ggtgaaaacc?tctgacacat 3780

gcagctcccg?gagacggtca?cagcttgtct?gtaagcggat?gccgggagca?gacaagcccg 3840

tcagggcgcg?tcagcgggtg?ttggcgggtg?tcggggctgg?cttaactatg?cggcatcaga 3900

gcagattgta?ctgagagtgc?accatatgga?tctcgagcgg?ccgcaataaa?atatctttat 3960

tttcattaca?tctgtgtgtt?ggttttttgt?gtgaatcgta?actaacatac?gctctccatc 4020

aaaacaaaac?gaaacaaaac?aaactagcaa?aataggctgt?ccccagtgca?agtgcaggtg 4080

ccagaacatt?tctctatcga?a 4101

<210>6

<211>5914

<212>DNA

< 213>artificial

<400>6

gacggatcgg?gagatctccc?gatcccctat?ggtcgactct?cagtacaatc?tgctctgatg 60

ccgcatagtt?aagccagtat?ctgctccctg?cttgtgtgtt?ggaggtcgct?gagtagtgcg 120

cgagcaaaat?ttaagctaca?acaaggcaag?gcttgaccga?caattgcatg?aagaatctgc 180

ttagggttag?gcgttttgcg?ctgcttcgcg?atgtacgggc?cagatatacg?cgttgacatt 240

gattattgac?tagttattaa?tagtaatcaa?ttacggggtc?attagttcat?agcccatata 300

tggagttccg?cgttacataa?cttacggtaa?atggcccgcc?tggctgaccg?cccaacgacc 360

cccgcccatt?gacgtcaata?atgacgtatg?ttcccatagt?aacgccaata?gggactttcc 420

attgacgtca?atgggtggac?tatttacggt?aaactgccca?cttggcagta?catcaagtgt 480

atcatatgcc?aagtacgccc?cctattgacg?tcaatgacgg?taaatggccc?gcctggcatt 540

atgcccagta?catgacctta?tgggactttc?ctacttggca?gtacatctac?gtattagtca 600

tcgctattac?catggtgatg?cggttttggc?agtacatcaa?tgggcgtgga?tagcggtttg 660

actcacgggg?atttccaagt?ctccacccca?ttgacgtcaa?tgggagtttg?ttttggcacc 720

aaaatcaacg?ggactttcca?aaatgtcgta?acaactccgc?cccattgacg?caaatgggcg 780

gtaggcgtgt?acggtgggag?gtctatataa?gcagagctct?ctggctaact?agagaaccca 840

ctgcttactg?gcttatcgaa?attaatacga?ctcactatag?ggagacccaa?gcttggtacc 900

gagctcggat?ccatgtacag?gatgcaactc?ctgtcttgca?ttgcactaag?tcttgcactt 960

gtcacaaaca?gtgcacctac?ttcaagttct?acaaagaaaa?cacagctaca?actggagcat 1020

ttactgctgg?atttacagat?gattttgaat?ggaattaata?attacaagaa?tcccaaactc 1080

accaggatgc?tcacatttaa?gttttacatg?cccaagaagg?ccacagaact?gaaacatctt 1140

cagtgtctag?aagaagaact?caaacctctg?gaggaagtgc?taaatttagc?tcaaagcaaa 1200

aactttcact?taagacccag?ggacttaatc?agcaatatca?acgtaatagt?tctggaacta 1260

aagggatctg?aaacaacatt?catgtgtgaa?tatgctgatg?agacagcaac?cattgtagaa 1320

tttctgaaca?gatggattac?cttttgtcaa?agcatcatct?caacactgac?ttgaggatcc 1380

actagtaacg?gccgccagtg?tgctggaatt?ctgcagatat?ccatcacact?ggcggccgct 1440

cgagcatgca?tctagagggc?cctattctat?agtgtcacct?aaatgctaga?gctcgctgat 1500

cagcctcgac?tgtgccttct?agttgccagc?catctgttgt?ttgcccctcc?cccgtgcctt 1560

ccttgaccct?ggaaggtgcc?actcccactg?tcctttccta?ataaaatgag?gaaattgcat 1620

cgcattgtct?gagtaggtgt?cattctattc?tggggggtgg?ggtggggcag?gacagcaagg 1680

gggaggattg?ggaagacaat?agcaggcatg?ctggggatgc?ggtgggctct?atggcttctg 1740

aggcggaaag?aaccagctgg?ggctctaggg?ggtatcccca?cgcgccctgt?agcggcgcat 1800

taagcgcggc?gggtgtggtg?gttacgcgca?gcgtgaccgc?tacacttgcc?agcgccctag 1860

cgcccgctcc?tttcgctttc?ttcccttcct?ttctcgccac?gttcgccggc?tttccccgtc 1920

aagctctaaa?tcggggcatc?cctttagggt?tccgatttag?tgctttacgg?cacctcgacc 1980

ccaaaaaact?tgattagggt?gatggttcac?gtagtgggcc?atcgccctga?tagacggttt 2040

ttcgcccttt?gacgttggag?tccacgttct?ttaatagtgg?actcttgttc?caaactggaa 2100

caacactcaa?ccctatctcg?gtctattctt?ttgatttata?agggattttg?gggatttcgg 2160

cctattggtt?aaaaaatgag?ctgatttaac?aaaaatttaa?cgcgaattaa?ttctgtggaa 2220

tgtgtgtcag?ttagggtgtg?gaaagtcccc?aggctcccca?ggcaggcaga?agtatgcaaa 2280

gcatgcatct?caattagtca?gcaaccaggt?gtggaaagtc?cccaggctcc?ccagcaggca 2340

gaagtatgca?aagcatgcat?ctcaattagt?cagcaaccat?agtcccgccc?ctaactccgc 2400

ccatcccgcc?cctaactccg?cccagttccg?cccattctcc?gccccatggc?tgactaattt 2460

tttttattta?tgcagaggcc?gaggccgcct?ctgcctctga?gctattccag?aagtagtgag 2520

gaggcttttt?tggaggccta?ggcttttgca?aaaagctccc?gggagcttgt?atatccattt 2580

tcggatctga?tcaagagaca?ggatgaggat?cgtttcgcat?gattgaacaa?gatggattgc 2640

acgcaggttc?tccggccgct?tgggtggaga?ggctattcgg?ctatgactgg?gcacaacaga 2700

caatcggctg?ctctgatgcc?gccgtgttcc?ggctgtcagc?gcaggggcgc?ccggttcttt 2760

ttgtcaagac?cgacctgtcc?ggtgccctga?atgaactgca?ggacgaggca?gcgcggctat 2820

cgtggctggc?cacgacgggc?gttccttgcg?cagctgtgct?cgacgttgtc?actgaagcgg 2880

gaagggactg?gctgctattg?ggcgaagtgc?cggggcagga?tctcctgtca?tctcaccttg 2940

ctcctgccga?gaaagtatcc?atcatggctg?atgcaatgcg?gcggctgcat?acgcttgatc 3000

cggctacctg?cccattcgac?caccaagcga?aacatcgcat?cgagcgagca?cgtactcgga 3060

tggaagccgg?tcttgtcgat?caggatgatc?tggacgaaga?gcatcagggg?ctcgcgccag 3120

ccgaactgtt?cgccaggctc?aaggcgcgca?tgcccgacgg?cgaggatctc?gtcgtgaccc 3180

atggcgatgc?ctgcttgccg?aatatcatgg?tggaaaatgg?ccgcttttct?ggattcatcg 3240

actgtggccg?gctgggtgtg?gcggaccgct?atcaggacat?agcgttggct?acccgtgata 3300

ttgctgaaga?gcttggcggc?gaatgggctg?accgcttcct?cgtgctttac?ggtatcgccg 3360

ctcccgattc?gcagcgcatc?gccttctatc?gccttcttga?cgagttcttc?tgagcgggac 3420

tctggggttc?gaaatgaccg?accaagcgac?gcccaacctg?ccatcacgag?atttcgattc 3480

caccgccgcc?ttctatgaaa?ggttgggctt?cggaatcgtt?ttccgggacg?ccggctggat 3540

gatcctccag?cgcggggatc?tcatgctgga?gttcttcgcc?caccccaact?tgtttattgc 3600

agcttataat?ggttacaaat?aaagcaatag?catcacaaat?ttcacaaata?aagcattttt 3660

ttcactgcat?tctagttgtg?gtttgtccaa?actcatcaat?gtatcttatc?atgtctgtat 3720

accgtcgacc?tctagctaga?gcttggcgta?atcatggtca?tagctgtttc?ctgtgtgaaa 3780

ttgttatccg?ctcacaattc?cacacaacat?acgagccgga?agcataaagt?gtaaagcctg 3840

gggtgcctaa?tgagtgagct?aactcacatt?aattgcgttg?cgctcactgc?ccgctttcca 3900

gtcgggaaac?ctgtcgtgcc?agctgcatta?atgaatcggc?caacgcgcgg?ggagaggcgg 3960

tttgcgtatt?gggcgctctt?ccgcttcctc?gctcactgac?tcgctgcgct?cggtcgttcg 4020

gctgcggcga?gcggtatcag?ctcactcaaa?ggcggtaata?cggttatcca?cagaatcagg 4080

ggataacgca?ggaaagaaca?tgtgagcaaa?aggccagcaa?aaggccagga?accgtaaaaa 4140

ggccgcgttg?ctggcgtttt?tccataggct?ccgcccccct?gacgagcatc?acaaaaatcg 4200

acgctcaagt?cagaggtggc?gaaacccgac?aggactataa?agataccagg?cgtttccccc 4260

tggaagctcc?ctcgtgcgct?ctcctgttcc?gaccctgccg?cttaccggat?acctgtccgc 4320

ctttctccct?tcgggaagcg?tggcgctttc?tcaatgctca?cgctgtaggt?atctcagttc 4380

ggtgtaggtc?gttcgctcca?agctgggctg?tgtgcacgaa?ccccccgttc?agcccgaccg 4440

ctgcgcctta?tccggtaact?atcgtcttga?gtccaacccg?gtaagacacg?acttatcgcc 4500

actggcagca?gccactggta?acaggattag?cagagcgagg?tatgtaggcg?gtgctacaga 4560

gttcttgaag?tggtggccta?actacggcta?cactagaagg?acagtatttg?gtatctgcgc 4620

tctgctgaag?ccagttacct?tcggaaaaag?agttggtagc?tcttgatccg?gcaaacaaac 4680

caccgctggt?agcggtggtt?tttttgtttg?caagcagcag?attacgcgca?gaaaaaaagg 4740

atctcaagaa?gatcctttga?tcttttctac?ggggtctgac?gctcagtgga?acgaaaactc 4800

acgttaaggg?attttggtca?tgagattatc?aaaaaggatc?ttcacctaga?tccttttaaa 4860

ttaaaaatga?agttttaaat?caatctaaag?tatatatgag?taaacttggt?ctgacagtta 4920

ccaatgctta?atcagtgagg?cacctatctc?agcgatctgt?ctatttcgtt?catccatagt 4980

tgcctgactc?cccgtcgtgt?agataactac?gatacgggag?ggcttaccat?ctggccccag 5040

tgctgcaatg?ataccgcgag?acccacgctc?accggctcca?gatttatcag?caataaacca 5100

gccagccgga?agggccgagc?gcagaagtgg?tcctgcaact?ttatccgcct?ccatccagtc 5160

tattaattgt?tgccgggaag?ctagagtaag?tagttcgcca?gttaatagtt?tgcgcaacgt 5220

tgttgccatt?gctacaggca?tcgtggtgtc?acgctcgtcg?tttggtatgg?cttcattcag 5280

ctccggttcc?caacgatcaa?ggcgagttac?atgatccccc?atgttgtgca?aaaaagcggt 5340

tagctccttc?ggtcctccga?tcgttgtcag?aagtaagttg?gccgcagtgt?tatcactcat 5400

ggttatggca?gcactgcata?attctcttac?tgtcatgcca?tccgtaagat?gcttttctgt 5460

gactggtgag?tactcaacca?agtcattctg?agaatagtgt?atgcggcgac?cgagttgctc 5520

ttgcccggcg?tcaatacggg?ataataccgc?gccacatagc?agaactttaa?aagtgctcat 5580

cattggaaaa?cgttcttcgg?ggcgaaaact?ctcaaggatc?ttaccgctgt?tgagatccag 5640

ttcgatgtaa?cccactcgtg?cacccaactg?atcttcagca?tcttttactt?tcaccagcgt 5700

ttctgggtga?gcaaaaacag?gaaggcaaaa?tgccgcaaaa?aagggaataa?gggcgacacg 5760

gaaatgttga?atactcatac?tcttcctttt?tcaatattat?tgaagcattt?atcagggtta 5820

ttgtctcatg?agcggataca?tatttgaatg?tatttagaaa?aataaacaaa?taggggttcc 5880

gcgcacattt?ccccgaaaag?tgccacctga?cgtc 5914

Claims

1. recombinant retroviral vector; Said recombinant retroviral vector is characterised in that; It comprises human interleukin 15 gene and herpes simplex virus thymidine kinase HSV-TK gene, and the reinforced eukaryotic promoter hEF1-HTLV that is used for the expressing human interleukin 15.

2. the application of the described recombinant retroviral vector of claim 1, it is used to prepare the medicine that promotes activated CD4+T cell survival.

3. test kit, it comprises described recombinant retroviral vector of the claim 1 of treating significant quantity and pharmaceutical carrier.

4. retrovirus TK-L15 Retrovirus, its preserving number is CGMCC No.2810.