CN107488677A - One plant of vaccinia virus recombinant for carrying the latent gene of membranous antigen 2 of Epstein-Barr virus and its application - Google Patents
One plant of vaccinia virus recombinant for carrying the latent gene of membranous antigen 2 of Epstein-Barr virus and its application Download PDFInfo
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Abstract
The present invention provides one plant of vaccinia virus recombinant for carrying the latent gene of membranous antigen 2 of Epstein-Barr virus and its application, the preparation method of the recombinant virus are：Structure carries EBV LMP2, the recombinant plasmid and the cells of MVA co-infections BHK 21 of green fluorescent protein foreign gene, and the MVA recombinant viruses (MVA LMP2A) for only carrying EBV LMP2 foreign genes are obtained by homologous recombination.The mouse spleen lymphocyte that IFN γ immune spot-ings detection MVA LMP2A were immunized, show that MVA LMP2A can be very good to produce EBV LMP2 specific immune responses in inducing mouse body.
Description
Technical field
The present invention relates to animal vaccine field, and in particular to only a kind of MVA-LMP2A of carrying EBV LMP2 foreign genes
Recombinant virus and its application.
Background of invention
Epstein-Barr viruses (EBV) belong to herpesviral γ subfamilies, and most of primary infections are the Childhood, without bright
Aobvious clinical symptoms, and lifelong carrying.EBV wide-scale distributions in the mankind, it is related to the tumour of many mankind, including Burkitt leaching
Bar knurl, nasopharyngeal carcinoma NPC, Hodgkin's disease HD and panimmunity suppress caused by the lymphoproliferative disorder of patient and transplant patient
The diseases such as lymthoma (PTLD)[1].Therefore the virus exist for the oncotherapy based on CTL provide one it is important potential
Target position.Latent membrane protein 1, the LMP2 of EBV expression play multiple oncogenic function, can by activate multiple signal transduction and
The expression for regulating and controlling various carcinoma genes plays a role[2].Wherein LMP2 can induce stronger CD8+CTL, and some epitopes
It has been determined that the height of the LMP2 cellular immune levels in EBV carrier's bodies is related to NPC generation[3].Therefore, it is many at present
EBV related neoplasms Therapy studies are both for LMP2.
LMP2 is the 8th latent membrane protein being identified, has continuous expression, does not cause bone-marrow-derived lymphocyte to convert, without carcinogenic
It the characteristics of property, can be very good that ctl response phosphorylation can be induced.In addition, correlative study result shows, the egg of the gene expression
In vain, inducing specific cellullar immunologic response is can be very good in patient's body, and cellular immunity plays weight in antineoplaston
Act on.Therefore, Immune inducing in vivo EBV specific CTLs, prevention and treatment NPC good target antigen can be used as by the use of LMP2.
The vaccinia virus ankara strain (Modified vaccinia virus Ankara, MVA) of modified form is German religion
Mayr Research Team is awarded in the isolated poxvirus CVA strains in Ankara areas, in the biography in chicken embryo fibroblasts generations up to a hundred
It is commissioned to train after supporting, the attenuated virus strain of acquisition.Due to the loss of about 30Kb genes in succeeding generations, make the sick host range with
It is pathogenic to substantially reduce so that MVA turn into be most widely used at present, immunogenicity is good, safe and efficient expression vector
One of.Multiple infectious disease has been widely applied to using vaccine of the genetic recombination using MVA as vector construction and swollen in recent years
In the vaccine research of knurl.2014, the recombinant MVA of EBV related antigens is carried, clinicalⅰstage result of the test shows that the vaccine has
Good security and immunogenicity, there are 8 generation immune responses in 14 patients, and should induction of Specific T cell immunity
Answer, have been enter into clinical II phase experiment [4].
Although above-mentioned vaccine has carried out the clinical trial of I phase, but currently without the text of any infrastest data and correlation
Support is offered, structure and immunological effect research of this experiment to MVA-LMP2A vaccine strains is inquired into.EBNA1 is EBV weights
The albumen for the cause cell transformation wanted, and LMP2 genes do not cause the conversion of bone-marrow-derived lymphocyte, non-carcinogenesis, are one and well may be used
To trigger the albumen of ctl response, it has also become immunization therapy NPC good target antigen, so this experiment is safe etc. from vaccine strain
From the aspect of, only select LMP2 antigens.The MVA-LMP2A recombinant viruses of this research and establishment, using MVA as carrier, are only included
EBV LMP2 foreign genes, do not carry any foreign gene in addition, can be used as EBV related neoplasms treatment vaccine researchs and application
Lay a good foundation.
Bibliography：
[1] Zuo Jianmin, Zhou Ling, the once Chinese experiments of the Research Review of firm .EB virus lays dormants memebrane protein 2 and clinical virology
Magazine, 2003, Vol 17:296-298.
[2]Li,J.,Liu,X.,Liu,M,et a.Methylation and expression of Epstein–Barr
virus latent membrane protein 1,2A and 2B in EBV-associated gastric
carcinomas and cell lines.Digestive and Liver Disease,Vol.48(6):673-680,2016.
[3] Epstein-Barr virus specific T-cells are to target antigen in Zhou Ling, Yao Qingyun, Lee S. Nasopharyngeal Carcinoma Patients and normal population
Identification and response virus journals, 2001, Vol.17:7-10.
[4] Njuguna, I.N., Ambler, G., Reilly, M., et al.PedVacc 002:A phase I/II
randomized clinical trial of MVA.HIVA vaccine administered to infants born to
human immunodeficiency virus type 1-positive mothers in Nairobi.Vaccine,
Vol.32(44):5801-5808,2014.
The content of the invention
The basic background of related recombinant vaccine development based on this laboratory using EBV LMP2 as target spot, structure carry
EBV LMP2, green fluorescent protein (GFP) foreign gene recombinant plasmid (PZL-GFP-LMP2) and MVA co-infections BHK-21
Cell, the MVA recombinant viruses (MVA-GFP- of foreign gene-carrying EBV LMP2 and GFP simultaneously are obtained by homologous recombination
LMP2A).The MVA-GFP-LMP2A of acquisition takes off GFP genes by autologous restructuring, is screened by several wheels, identification obtains only
Carry the MVA recombinant viruses (MVA-LMP2A) of EBV LMP2 foreign genes.Identified by PCR, the methods of indirect immunofluorescence,
MVA-LMP2A can be very good to express EBV LMP2 foreign genes.IFN-γ immune spot-ing (ELISOPT) detects MVA-
The mouse spleen lymphocyte that LMP2A was immunized, test result indicates that MVA-LMP2A can be very good to produce in inducing mouse body
EBV LMP2 specific immune responses.
It is an object of the present invention to provide one kind to carry Epstein-Barr virus latent membrane protein2 (EBVLMP2) foreign gene, and not
Carry the EBVLMP2 vaccinia virus recombinants of any foreign gene in addition to EBVLMP2.
According to a preferred embodiment of the invention, wherein MVA-LMP2A recombinant viruses are lacked from highly attenuated, duplication
Ankara strain vaccinia virus strain structure of swaged.The EBV LMP2 of carrying are gene order (the SEQ ID that length scale is 1.5kb
NO.7).According to a preferred embodiment of the invention, wherein MVA-LMP2A recombinant viruses do not carry in addition to target gene
Other foreign genes.
According to a preferred embodiment of the invention, wherein the foreign gene-carrying EBV-LMP2 used plasmid is
PZL-GFP-LMP2 recombinant plasmids.
It is a further object to provide a kind of application of vaccine as defined above in EBV related neoplasms are treated.
According to a preferred embodiment of the invention, wherein described EBV related neoplasms are that EBV LMP2 expression is positive
Related neoplasms.
According to a preferred embodiment of the invention, wherein MVA-LMP2A recombinant viruses both can be directly as independent
Vaccine candidate vaccine, the EBV LMP2 that can be deducted a percentage again using MVA as carrier load to presenting cells, stem cell, T lymphocytes
Deng inducing specific cellullar immunologic response.
Specifically, it is provided by the present invention be a kind of only foreign gene-carrying EBV LMP2 recombinant MVA structure and
Using comprising the steps：
1) the carrying GFP built using this laboratory PZL-GFP plasmids, the plasmid is in GFP and multiple cloning sites two
Comprising two sections the sequence of homologous recombination can occur with MVA for end, and one section and GFP are included between GFP and multiple cloning sites
The sequence of autologous restructuring can occur for the other end, so as to slough marker gene GFP, obtain and only carry foreign aid's gene EBV
LMP2 recombinant virus.From two restriction enzymes of Pst1 and Not1, EBV LMP2 are inserted into PZL-GFP plasmids, obtained
PZL-GFP-LMP2 recombinant plasmids (as shown in Figure 1)；
2) PZL-GFP-LMP2 recombinant plasmids and MVA co-infection BHK-21 cells, recombinant plasmid occur homologous with virus
After sequence restructuring, fluorescence microscope visible cell after a few wheel screenings, obtains in green, i.e. expressing green fluorescent protein
Pure MVA-GFP-LMP2A viruses；
3) autologous restructuring can occur for the homologous sequence according to the recombinant virus GFP genes both ends obtained, to having obtained
The MVA-GFP-LMP2A viruses obtained carry out continuous passage and screening, and GFP genes are sloughed in acquisition, redgreen fluorescent protein expression
MVA-LMP2A recombinant viruses.The detection methods such as PCR methods, IIF are carried out to the MVA-LMP2A recombinant viruses of acquisition
Identification；
4) mouse, IFN-γ immunodotting is immunized using the vaccinia virus recombinant MVA-LMP2A for carrying EBV LMP2 genes
The mouse spleen lymphocyte that method (Elispot) detection MVA-LMP2A was immunized, detects MVA-LMP2A recombinant virus inducing mouses
EBV LMP2 specific immune response effects caused by vivo.
Experimental result shows that the vaccinia virus recombinant MVA-LMP2A of the carrying EBV LMP2 foreign genes of acquisition can be very
Good expression EBV LMP2, after mouse is immunized, EBV LMP2 specific immune responses can be produced in inducing mouse body well.
Beneficial effect
Beneficial effects of the present invention include：
(1) structure carries the PZL-GFP-LMP2 recombinant plasmids of GFP, LMP2 foreign gene, and the plasmid can be very good table
Up to EBV LMP2 genes, homologous recombination can occur with MVA viruses.In addition, the plasmid includes one between GFP and LMP2 sequences
The longer sequence of autologous restructuring can occur with the GFP other ends for section, be easy to slough marker gene GFP；
(2) PZL-GFP-LMP2 recombinant plasmids and rMVA co-infection BHK-21 cells, the MVA-GFP-LMP2A diseases of acquisition
It can be very good to express LMP2, GFP after malicious infection cell, obvious green fluorescence can be observed under fluorescence microscope, can be quick
Detection foreign gene expression；
(3) autologous restructuring occurs for MVA-GFP-LMP2A viruses, obtains the MVA- for only carrying EBV LMP2 foreign genes
LMP2A recombinant viruses, candidate vaccine is provided for the vaccine using EBV LMP2 as target spot development；
(4) MVA-LMP2A recombinant viruses can produce preferable EBV LMP2 specific immune responses with inducing mouse, be
The treatment of EBV related neoplasms provides reference.
Brief description of the drawings
Fig. 1：PZL-GFP-LMP2 construction of recombinant plasmid schematic diagrames；
Fig. 2：PZL-GFP-LMP2 recombinant plasmid digestion qualification figures, wherein, 1.MarkerGsDL2502,2. negative controls,
3.T-LMP2 positive control digestion results, 4.PZL-GFP-LMP2 digestion results.As a result the LMP2 clip sizes for showing to obtain are
1490bp is identical with positive control clip size, i.e., qualification result is consistent with expected results, and illustration purpose fragment is successively inserted into matter
Grain PZL-GFP, can carry out follow-up test；
Fig. 3：The structure schematic diagram of MVA-GFP-LMP2A recombinant viruses；
Fig. 4：Fluorescence microscope result, wherein, 1：MVA infects BHK-21；2:MVA-GFP-LMP2A infects BHK-
21, into green under fluorescence microscope；
Fig. 5：3MVA-GFP-LMP2A PCR qualification result electrophoretograms, wherein, M:Marker GsDL2501；1:It is negative right
According to a group TK identified for genes；2:The wild poison group TK identified for genes of rMVA；3:MVA-GFP-LMP2A group TK identified for genes；4:Negative control
Group GFP identified for genes；5:The wild poison group GFP identified for genes of rMVA；6:MVA-GFP-LMP2A group GFP identified for genes；7:Negative control
Group LMP2 identified for genes；8:The wild poison group LMP2 identified for genes of rMVA；9:MVA-GFP-LMP2A group LMP2 identified for genes.
Fig. 6 MVA-LMP2A recombinant virus fluorescence microscopes, wherein, 1：MVA-LMP2A fluorescence microscope results, i.e.,
GFP MVA-LMP2A recombinant viruses are taken off, redgreen fluorescence occurs.2：MVA-LMP2A visible ray results show, sick cell
Occur becoming big, be rounded.
Fig. 7 MVA-LMP2A PCR qualification result electrophoretograms, wherein, M:Marker GsDL2502；1:MVA-LMP2A is recombinated
Viral LMP2 identified for genes；2:LMP2 gene negatives compare；3:LMP2 gene masculine control groups；4:MVA-LMP2A recombinant viruses
GFP identified for genes；5:GFP gene negatives compare；6:GFP gene masculine control groups.
Fig. 8 MVA-LMP2A indirect immunofluorescence results, wherein, 1：MVA-LMP2A groups；2：RAd5-LMP2 groups；
Fig. 9 MVA-LMP2A electromicroscopic photographs
Figure 10：Elispot testing result block diagrams；
Embodiment
In this application, unless otherwise specified, implementation of the invention is all using molecular biology, microbiology, recombinant DNA
With immunology routine techniques, these technologies are all that those skilled in the art are grasped.
Experiment material
Bacillus coli DH 5 alpha, plasmid pDC316, pBHG, B95-8,293 cell strains are in Disease Control and Prevention in China
Once firm academician laboratory preserves for worry viral disease prevention and control institute.Condition of culture is containing 10%FBS, 1% mycillin, 1% paddy ammonia
The DMEM nutrient solutions of acid amides.Wherein FBS is purchased from Hyclone companies purchased from GIBCO companies of the U.S., DMEM, other nutrient solutions by
China Sickness Prevention Control Center Virus Disease Prevention Control Institute liquor room provides.
The a large amount of extracts kit QIAGEN PlasmidegaKits of plasmid are purchased from German QIAGEN companies；It is conventional restricted
Restriction endonuclease EcoRI and XhoI, nucleic acid gel purification kit, PCR kit are purchased from precious bioengineering (Dalian) Co., Ltd；Even
Connect enzyme and be purchased from NEB companies of the U.S.；Eucaryon transfection reagent FuGene HD are purchased from Promega companies of the U.S.；RPMI1640 cell culture
Base, DMEM cell culture mediums and sterile PBS are purchased from HyClone companies of the U.S.；Hyclone is purchased from GIBCO companies of the U.S.；PVDF
Film MultiScreenHTS is purchased from MABTECH companies of Sweden；Tris alkali (SERVA companies), boric acid (Shanghai Yunling chemical plant,
AR), EDTA (Sigma is original-pack), tryptone (OXOID companies), yeast extract (OXOID companies), goat anti-mouse EBNA1
Monoclonal antibody is purchased from Santa Cruz Biotechnology companies of the U.S., horseradish peroxidase (HRP) mark goat anti-mouse
IgG, streaming antibody are purchased from Bioisystech Co., Ltd of Zhong Shan Golden Bridge.Other analysis pure chemistry reagents are by Chinese Disease Control and Prevention Center virus
Disease is provided.
1st, embodiment 1：Structure and identification PZL-GFP-LMP2 recombinant plasmids
The acquisition of 1.1EBV LMP2 genetic fragments
Pst1 and Not1 double digestion T-LMP2 plasmids (Lixia ZHANG .EBV-LMP2 vaccinia virus recombinants immune effect research north
Capital polytechnical university), digestion system is 20ul (the μ L of DNA plasmid 2；NEBuffer3.1 1μL；BSA：2μL；Pst1 0.5μL；Not1
0.5μL；ddH2The μ L of O 14), 37 DEG C of digestion 2h.The μ L loadings of digestion products 10,1% agarose gel electrophoresis under 110V voltages are purple
See whether purpose band occur under outer light irradiation, purpose fragment, tool are reclaimed using the gel extraction kit of Qiagen companies
Gymnastics is made as follows referring to method：300 μ L QG ratio, 50 DEG C of 10min are added according to 0.1g glue, interruption mixes 2-3 times；Supernatant
It is transferred to QIA quick spin column, 12000rpm centrifugations 1min；Add μ L, the 12000rpm centrifugations of PE Buffer 700
1min；12000rpm centrifuges 1min again；Add 10 μ L ddH2O stands 1min on purification column medium；12000rpm is centrifuged
1min, collect DNA solution.
The structure of 1.2PZL-GFP-LMP2 recombinant plasmids
Restriction enzyme Pst1 and Not1 double digestion plasmid PZL-EGFP (Li Lili, Wang Zhan, Zhou Yubai etc., with mark
Remember structure and Function Identification virus journal .2015.31 (5) of the gene from the shuttle vector of deletion system：507-514.), digestion
System is 20 μ L (the μ L of DNA plasmid 2；NEBuffer3.1 1μL；BSA：2μL；Pst1 0.5μL；Not1 0.5μL；ddH2O 14μ
L), 37 DEG C of digestion 2h.The μ L loadings of digestion products 10,1% agarose gel electrophoresis under 110V voltages, in uviol lamp after being sufficiently separated
See whether purpose band occur under irradiation, cut glue and using gel extraction kit recovery purpose fragment, concrete operations referring to
1.1。
Recovery product surveys concentration, and connection ratio is according to carrier：Purpose fragment=1:The ratio of 3~6 (mol ratios) is connected
Connect.The μ L of linked system 20, concrete operations are as follows：PZL-GFP 9μL；EBV LMP2 4μL；The μ L of T4 ligases 1；Buffer 2μL；
H2O4 μ L, 16 DEG C of connections are overnight.
200 μ L DH5 α competent cells are added in connection product, gently rotation mixes, ice bath 30min, 42 DEG C of water baths
Middle water-bath 90s, 2~3min of ice bath.The LB culture mediums that 800 μ L are free of antibiotic are added, 30min (rotating speeds are incubated on 37 DEG C of shaking tables：
200rpm/min).The competent cell that 50 μ L have been converted, gently it is coated onto the agar plate surface of the resistance of benzyl containing ammonia, 37 DEG C of cultures
12~16h (while doing negative control) is cultivated in case.
The identification of 1.3PZL-GFP-LMP2 recombinant plasmids
Single bacterium colony on picking agar plate, respectively in the LB culture mediums of 5mL ammonia benzyl resistances, 37 DEG C, 220rpm/
Min incubator overnights.Qiagen plasmid extraction kits extract plasmid, specific steps reference reagent box specification.To the matter of extraction
Grain carries out concentration mensuration and digestion identification, will identify that correct plasmid is named as PZL-GFP-LMP2, i.e., successfully builds PZL-
GFP-LMP2 plasmids, sequence is as shown in SEQ ID NO.8 (Fig. 2).
Embodiment 2：MVA-GFP-LMP2A recombinant viruses are built
2.1MVA-GFP-LMP2A builds optimum condition
Table 1MVA-GFP-LMP2A builds condition
Note："+" represents GFP expression intensities under fluorescence microscope in table.
By rMVA (Sutter, G.and C.Staib, Vaccinia vectors as candidate vaccines:
the development of modified vaccinia virus Ankara for antigen delivery.Curr
Drug Targets Infect Disord,2003.3(3):P.263-71.) according to 10-1, 10-2, 10-3Concentration, feel respectively
The BHK-21 cells in 6 orifice plates are contaminated, each dilution factor infects two holes, and PBS washs infection cell 2 times after 2h, each dilution factor
(being shown in Table 1) is transfected with 4ug and 6ug PZL-GFP-LMP2 plasmids content respectively, concrete operation step refers to Roche reagent explanation
Book (X-tremeGENE HP DNA Transfection Reagent specifications).Infection cell is positioned over into 37 DEG C of cells to incubate
In case, fluorescence microscopy Microscopic observation GFP is expressed after 48h, obtains MVA-GFP-LMP2A recombinant viruses (such as Fig. 3) and its optimal sense
Dye condition.As a result illustrate, MVA is with 10-1Diluted concentration infection BHK-21 cells, after 2h after PBS 2 times, 4ug PZL-
GFP-LMP2 Transfected Recombinant Plasmids are optimal infection strategy, and reference is provided for the recombinant virus structure using MVA as carrier.
2.2 acquisitions and identification of M VA-GFP-LMP2A monoclonals
Virus is diluted using the method for extreme dilution, it is glimmering to having in the minimum dilution factor for obtaining expression GFP albumen
The recombinant virus of light expression carries out collection virus, after being screened repeatedly after extreme dilution, obtains the restructuring of MVA-GFP-LMP2A monoclonals
Virus, and preservation is carried out, micro- Microscopic observation infection cell form, see Fig. 4.As a result illustrate, fluorescence microscopy Microscopic observation
The MVA-GFP-LMP2A recombinant viruses obtained through a few wheel screenings, can be very good to express GFP marker gene, prompt MVA-GFP-
LMP2A recombinant viruses can be very good expression alien gene.Expand and collect MVA-GFP-LMP2A recombinant viruses, multigelation 3
Secondary, 20ul recombinant viruses add 4ul Proteinase Ks, 55 DEG C of incubation 1h, boiling water bath 10min, place 5min on ice, are identified as PCR
Template.
PCR primer is respectively
TKF：CGGGACTATGGACGCATG, (SEQ ID NO.1)
TKR：CGGTTTCCTCACCCAATCG；(SEQ ID NO.2)
GFPF：CTCCAGCAGGACCATGTG, (SEQ ID NO.3)
GFPR：GGACGACGGCAACTACAAG；(SEQ ID NO.4)
LMP2F：TCAACCACTAGGAACCCAAGA；(SEQ ID NO.5)
LMP2R：ACGTAAATGCCTTGTAGTCCG.(SEQ ID NO.6)
The μ L of reaction system 50 (the μ L of rTaq enzymes 0.5, primer each μ L of 1 μ L, 10 × buffer 5, sample 1 μ L, ddH2O 32.5μ
L), after reaction condition is 94 DEG C of 2min, 94 DEG C of 10s, 51 DEG C of 10s, 72 DEG C of 1min, 72 DEG C of 4min after 30 circulations, 4 DEG C of preservations.4
μ L PCR primers add 1 μ L5 × sample-loading buffer, 110V constant pressure electrophoresis, observe result under uviol lamp, see Fig. 5.
As a result illustrating, MVA Ye Du TK areas PCR primer is that positive fragment size is 324bp, and GFP areas PCR primer is feminine gender,
LMP2 areas PCR primer is feminine gender；Recombinant virus is that negative TK areas PCR primer is negative, and GFP areas PCR primer is that positive fragment is big
Small is 364bp, and LMP2 areas PCR primer is that positive fragment size is 450bp.The MVA-GFP-LMP2A obtained by a few wheel screenings
Recombinant virus, PCR qualification results show that TK areas qualification result is feminine gender, GFP and LMP2 foreign genes in the recombinant virus built
Qualification result is the positive.The MVA-GFP-LMP2A for illustrating to obtain is the pure recombinant virus for carrying GFP and LMP2 foreign genes；
Embodiment 3：The acquisition and identification of MVA-LMP2A recombinant viruses
3.1MVA-LMP2A recombinant viruses harvest and morphologic observation
Homologous recombination obtains MVA-GFP-LMP2A, because autologous restructuring probability is relatively low, through being same as above extreme dilution method to disease
Poison carries out screening and obtains MVA-LMP2A recombinant viruses, that is, takes off GFP genes, only carries the white weight of EBV LMP2 foreign genes
Group virus, -80 DEG C of preservations are carried out to virus.Fluorescence microscopy Microscopic observation, though infection cell lesion, redgreen fluorescence table
Up to (Fig. 6).In -80 DEG C and 37 DEG C of multigelations 3 times (making clasmatosis, releasing virus), 3 000rpm/min centrifuge 10min,
Draw supernatant, i.e. MVA-LMP2A recombinant viruses, -80 DEG C of preservations.
3.2PCR identifies EBV LMP2 gene expressions
4 μ L Proteinase Ks (20mg/mL) are added in 20 μ L recombinant viruses, 55 DEG C of water-bath 1h, 10min is boiled, places on ice
5min, 10 000rpm centrifuge 5min, as viral DNA template.PCR is anti-using PCR in primer, reaction system and program same 2.2
Should.4 μ L PCR primers add 1 μ L5 × sample-loading buffer, 110V constant pressure electrophoresis, result (Fig. 7) are observed under uviol lamp.
3.3 indirect immunofluorescences identify EBV LMP2 protein expressions
MVA-LMP2A infects BHK-21 as experimental group, and MVA infects BHK-21 as negative control group, Ad5-LMP2 senses
293 cells are contaminated as positive controls, after the complete lesion of cell, are applied to respectively on cell sheet, are stored at room temperature 1~2h, 4 DEG C of acetone
Fixed 30min, room temperature are dried.With 1：40 ratio, PBS dilution EBV LMP2-2A monoclonal antibodies, dilution is added drop-wise to solid
On fixed cell, 37 DEG C of incubation 1h, pure water is cleaned, and room temperature is dried.With 1：50 ratio, PBS dilute the anti-big of biotin labeling
Mouse IgG secondary antibodies, dilution is added drop-wise on cell sheet, 37 DEG C of incubation 30min, pure water cleaning, and room temperature is dried.Equally with 1:40
Ratio, the strepto- avidin of the FITC marks of Azo-Blue dilution, 37 DEG C of incubation 30min is added dropwise, pure water is cleaned, and room temperature is dried.
50% glycerine mounting, fluorescence microscopy Microscopic observation simultaneously take a picture (Fig. 8).IIF testing result shows, acquisition
MVA-LMP2A recombinant viruses can be very good to express LMP2, consistent with expected results subsequently to be expressed；
Embodiment 4：MVA-LMP2A electronic microscope photos
MVA-LMP2A recombinant viruses and 2% phosphotungstic acid dye liquor are added dropwise on the plastic film respectively.Copper mesh is placed in recombinant virus
Liquid, copper mesh is transferred to 2% phosphotungstic acid dye liquor after adsorbing 1min, adsorbs 1min, room temperature is put dry.Observed under electron microscope MVA-
LMP2A recombinant viruses form (Fig. 9).MVA-LMP2A recombinant viruses after negative staining, electron microscope observation result show, surface
There is tubular element projection, size is homogeneous, is the characteristic feature of vaccinia viral particle.
Embodiment 5：The preliminary of MVA-LMP2A inducing mouse immune responses is probed into
4~6 weeks female Balb/c mouse are selected, are randomly divided into 3 groups, respectively MVA-LMP2A experimental groups, rAd-LMP2 sun
Property control group, BHK-21 negative control groups, every group 5.Intramuscular injection comprises the following steps that：Collect the MVA-LMP2A diseases of amplification
The BHK-21 cells of poison and uninfecting virus, multigelation 3 times.MVA-LMP2A is diluted to 109Pfu/mL, rAd-LMP2 dilute
To 1011Vp/mL, 0,1,2 week difference tibialis anterior meat injection three of the above dilute sample, each every μ l of mouse 100.Lasting raising
1 week after observation is immune, mouse spleen lymphocyte is taken to be ELISPOT, detection vaccinia virus recombinant MVA-LMP2A is small in Balb/c
Response situation (Figure 10) in mouse body, Elispot methods detection MVA-LMP2A recombinant viruses are immunized mouse results and shown, MVA-
LMP2A recombinant viruses can produce 614 spot formation cell/106Lymphocyte, you can be produced in good inducing mouse body
EBV LMP2 specific CTL responses.Specifically immune packet is shown in Table 2.
Mouse protocol is immunized in table 2
Table2The design for immunity in mouse
LMP2 is one of virus protein of continuous expression in the Epstein-Barr virus associated tumor tissue such as NPC, no carcinogenesis, is held
Continued is reached on tumour cell, and its antigenic determinant is consistent in different virus strain and is present in cancer pathology section sample
In, and be the albumen that can trigger ctl response well, therefore, LMP2 resists as immunization therapy NPC good target
It is former.
Existing document utilization carries the recombined adhenovirus infection DC of LMP2 antigens and MSC, LMP2 can be very good to express
And inducing specific CLT reaction well, this also be MVA-LMP2A using vaccinia virus as carrier, the application of deduction LMP2 antigens
Provide possibility.
The invention is not limited in foregoing embodiment.The present invention, which expands to, any in this manual to be disclosed
New feature or any new combination, and disclose any new method or process the step of or any new combination.
SEQUENCE LISTING
<110>China Sickness Prevention Control Center Virus Disease Prevention Control Institute
<120>One plant of vaccinia virus recombinant for carrying the latent gene of membranous antigen 2 of Epstein-Barr virus and its application
<160>8
<170>PatentIn version 3.5
<210>1
<211>18
<212>DNA
<213>TKF
<400>CGGGACTATGGACGCATG
<210>2
<211>19
<212>DNA
<213>TKR
<400>CGGTTTCCTCACCCAATCG
<210>3
<211>19
<212>DNA
<213>GFPF
<400>TCTCCAGCAGGACCATGTG
<210>4
<211>19
<212>DNA
<213>GFPR
<400>GGACGACGGCAACTACAAG
<210>5
<211>21
<212>DNA
<213>LMP2F
<400>TCAACCACTAGGAACCCAAGA
<210>6
<211>21
<212>DNA
<213>LMP2R
<400>ACGTAAATGCCTTGTAGTCCG
<210>7
<211>1494
<212>DNA
<213>EBV LMP2
<400>
ATGGGGTCCCTAGAAATGGTGCCAATGGGCGCGGGTCCCCCTAGCCCCGGCGGGGATCCGGATGGGTAC
GATGGCGGAAACAACTCCCAATATCCATCTGCTTCTGGCTCTTCTGGGAACACCCCCACCCCACCGAACGATGAGGA
ACGTGAATCTAATGAAGAGCCCCCACCGCCTTATGAGGACCCATATTGGGGCAATGGCGACCGTCACTCGGACTATC
AACCACTAGGAACCCAAGATCAAAGTCTGTACTTGGGATTGCAACACGACGGGAATGACGGGCTCCCTCCCCCTCCC
TACTCTCCACGGGATGACTCATCTCAACACATATACGAAGAAGCGGGCAGAGGAAGTATGAATCCAGTATGCCTGCC
TGTAATTGTTGCGCCCTACCTCTTTTGGCTGGCGGCTATTGCCGCCTCGTGTTTCACGGCCTCAGTTAGTACCGTTG
TGACCGCCACCGGCTTGGCCCTCTCACTTCTACTCTTGGCAGCAGTGGCCAGCTCATATGCCGCTGCACAAAGGAAA
CTGCTGACACCGGTGACAGTGCTTACTGCGGTTGTCACTTTCTTTGCAATTTGCCTAACATGGAGGATTGAGGACCC
ACCTTTTAATTCTCTTCTGTTTGCATTGCTGGCCGCAGCTGGCGGACTACAAGGCATTTACGTTCTGGTGATGCTTG
TGCTCCTGATACTAGCGTACAGAAGGAGATGGCGCCGTTTGACTGTTTGTGGCGGCATCATGTTTTTGGCATGTGTA
CTTGTCCTCATCGTCGACGCTGTTTTGCAGCTGAGTCCCCTCCTTGGAGCTGTAACTGTGGTTTCCATGACGCTGCT
GCTACTGGCTTTCGTCCTCTGGCTCTCTTCGCCAGGGGGCCTAGGTACTCTTGGTGCAGCCCTTTTAACATTGGCAG
CAGCTCTGGCACTGCTAGCGTCACTGATTTTGGGCACACTTAACTTGACTACAATGTTCCTTCTCATGCTCCTATGG
ACACTTGTGGTTCTCCTGATTTGCTCTTCGTGCTCTTCATGTCCACTGAGCAAGATCCTTCTGGCACGACTGTTCCT
ATATGCTCTCGCACTCTTGTTGCTAGCCTCCGCGCTAATCGCTGGTGGCAGTATTTTGCAAACAAACTTCAAGAGTT
TAAGCAGCACTGAATTTATACCCAATTTGTTCTGCATGTTATTACTGATTGTCGCTGGCATACTCTTCATTCTTGCT
ATCCTGACCGAATGGGGCAGTGGAAATAGAACATACGGTCCAGTTTTTATGTGCCTCGGTGGCCTGCTCACCATGGT
AGCCGGCGCTGTGTGGCTGACGGTGATGTCTAACACGCTTTTGTCTGCCTGGATTCTTACAGCAGGATTCCTGATTT
TCCTCATTGGCTTTGCCCTCTTTGGGGTCATTAGATGCTGCCGCTACTGCTGCTACTACTGCCTTACACTGGAAAGT
GAGGAGCGCCCACCGACCCCATATCGCAACACTGTATAA
<210>8
<211>7364
<212>DNA
<213>PZL-GFP-LMP2
<400>
ATCGATTTATTATTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGCGGTCACGAACTCCAG
CAGGACCATGTGATCGCGCTTCTCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCA
GCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTG
TGGCGGATCTTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTT
GTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGG
TGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACG
TAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCC
GTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCT
TGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACC
AGGATGGGCACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATCCTCGAGGAGTTCATTTATAGCATAGAAAA
AAACAAAATGAAATTCTACTATATTTTTACATACATATATTCTAAATATGAAAGTGGTGATTGTGACTAGCGTAGCA
TCGCTTCTAGACATCTCTTAAGTCTGTTATTATTATTGATCCAATCAAAAAATAAATTAGAAGCCGTGGGTCATTGT
TATGAATCTCTTTCAGAGGAATACAGACAATTGACAAAATTCACAGACTCTCAAGATTTTAAAAAACTGTTTAACAA
GGTCCCTATTGTTACAGATGGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGTTTGATGCGAT
TCAAAAAAGAATCAGCTCTAGCTACCACCGCAATAGATCCTGTTAGATACATAGATCCTCGTCGCGATATCGCATTT
TCTAACGTGATGGATATATTAAAGTCGAATAAAGTGAACAATAATTAATTCTTTATTGTCATCATGAACGGCGGACA
TATTCAGTTGATAATCGGCCCCATGTTTTCAGGTAAAGCTAGCATATACTATATAGTAATACCAATACTCAAGACTA
CGAAACTGATACAATCTCTTATCATGTGGGTAATGTTCTCGATGTCGATAGCCATATGCCCGGTAGTTGCGATATAC
ATAAACTGATCACTAATTCCAAACCCACCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATT
AATTTCTCGTAAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCATAAAGAACAGTGACGGA
TCCCCAACTAGTGTACCTGCAATGGGGTCCCTAGAAATGGTGCCAATGGGCGCGGGTCCCCCTAGCCCCGGCGGGGA
TCCGGATGGGTACGATGGCGGAAACAACTCCCAATATCCATCTGCTTCTGGCTCTTCTGGGAACACCCCCACCCCAC
CGAACGATGAGGAACGTGAATCTAATGAAGAGCCCCCACCGCCTTATGAGGACCCATATTGGGGCAATGGCGACCGT
CACTCGGACTATCAACCACTAGGAACCCAAGATCAAAGTCTGTACTTGGGATTGCAACACGACGGGAATGACGGGCT
CCCTCCCCCTCCCTACTCTCCACGGGATGACTCATCTCAACACATATACGAAGAAGCGGGCAGAGGAAGTATGAATC
CAGTATGCCTGCCTGTAATTGTTGCGCCCTACCTCTTTTGGCTGGCGGCTATTGCCGCCTCGTGTTTCACGGCCTCA
GTTAGTACCGTTGTGACCGCCACCGGCTTGGCCCTCTCACTTCTACTCTTGGCAGCAGTGGCCAGCTCATATGCCGC
TGCACAAAGGAAACTGCTGACACCGGTGACAGTGCTTACTGCGGTTGTCACTTTCTTTGCAATTTGCCTAACATGGA
GGATTGAGGACCCACCTTTTAATTCTCTTCTGTTTGCATTGCTGGCCGCAGCTGGCGGACTACAAGGCATTTACGTT
CTGGTGATGCTTGTGCTCCTGATACTAGCGTACAGAAGGAGATGGCGCCGTTTGACTGTTTGTGGCGGCATCATGTT
TTTGGCATGTGTACTTGTCCTCATCGTCGACGCTGTTTTGCAGCTGAGTCCCCTCCTTGGAGCTGTAACTGTGGTTT
CCATGACGCTGCTGCTACTGGCTTTCGTCCTCTGGCTCTCTTCGCCAGGGGGCCTAGGTACTCTTGGTGCAGCCCTT
TTAACATTGGCAGCAGCTCTGGCACTGCTAGCGTCACTGATTTTGGGCACACTTAACTTGACTACAATGTTCCTTCT
CATGCTCCTATGGACACTTGTGGTTCTCCTGATTTGCTCTTCGTGCTCTTCATGTCCACTGAGCAAGATCCTTCTGG
CACGACTGTTCCTATATGCTCTCGCACTCTTGTTGCTAGCCTCCGCGCTAATCGCTGGTGGCAGTATTTTGCAAACA
AACTTCAAGAGTTTAAGCAGCACTGAATTTATACCCAATTTGTTCTGCATGTTATTACTGATTGTCGCTGGCATACT
CTTCATTCTTGCTATCCTGACCGAATGGGGCAGTGGAAATAGAACATACGGTCCAGTTTTTATGTGCCTCGGTGGCC
TGCTCACCATGGTAGCCGGCGCTGTGTGGCTGACGGTGATGTCTAACACGCTTTTGTCTGCCTGGATTCTTACAGCA
GGATTCCTGATTTTCCTCATTGGCTTTGCCCTCTTTGGGGTCATTAGATGCTGCCGCTACTGCTGCTACTACTGCCT
TACACTGGAAAGTGAGGAGCGCCCACCGACCCCATATCGCAACACTGTATAAGGCCGCTGGGAATTCTGTGAGCGTA
TGGCAAACGAAGGAAAAATAGTTATAGTAGCCGCACTCGATGGGACATTTCAACGTAAACCGTTTAATAATATTTTG
AATCTTATTCCATTATCTGAAATGGTGGTAAAACTAACTGCTGTGTGTATGAAATGCTTTAAGGAGGCTTCCTTTTC
TAAACGATTGGGTGAGGAAACCGAGATAGAGATAATAGGAGGTAATGATATGTATCAATCGGTGTGTAGAAAGTGTT
ACATCGACTCATAATATTATATTTTTTATCTAAAAAACTAAAAATAAACATTGATTAAATTTTAATATAATACTTAA
AAATGGATGTTGTGTCGTTAGATAAACCGTTTATGTATTTTGAGGAAATTGATAATGAGTTAGATTACGAACCAGAA
AGTGCAAATGAGGTCGCAAAAAAACTACCGTATCAAGGACAGTTAAAACTATTACTAGGAGAATTATTTTTTCTTAG
TAAGTTACAGCGACACGGTATATTAGATGGTGCCACCGTAGTGTATATAGGATCTGCTCCCGGTACACATATACGTT
ATTTGAGAGATCATTTCTATAATTTAGGAGTGATCATCAAATGGATGCTAATTGACGGCCGCCATCATGATCCTATT
TTAAATGGATTGCGTGATGTGACTCTAGTGACTCGGTTCGTTGATGAGGAATATCTACGATCCATCAAAAAACAACT
GCATCCTTCTAAGATTATTTTAATTTCTGATGTGAGATCCAAACGAGGAGGAAATGAACCTAGTACGGCGGATTTAC
TAAGTAATTACGCTCTACAAAATGTCATGATTAGTATTTTAAACCCCGTGGCATCTAGTCTTAAATGGAGATGCCCG
TTTCCAGATCAATGGATCAAGGACTTTTATATCCCACACGGTAATAAAATGTTACAACCTTTTGCTCCTTCATATTC
AGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATC
CCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC
GAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTAC
AATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTC
TGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCA
CCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTA
GACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGT
ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTC
CGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA
AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTT
TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGAC
GCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAA
GCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACT
TACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT
GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAAC
AACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGG
ATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAG
CGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG
GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGT
CAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATC
CTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAT
CAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGG
TGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT
ACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT
AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG
ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTG
AGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG
CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTC
GCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG
GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGA
TAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCG
AGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGAC
AGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGC
TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATG
ACCATGATTACGCCAAGCTTTTGCGATCAATAAATGGATCACAACCAGTATCTCTTAACGATGTTCTTCGCAGATGA
TGATTCATTTTTTAAGTATTTGGCTAGTCAAGATGATGAATCTTCATTATCTGATATATTGCAAATCACTCAATATC
TAGACTTTCTGTTATTATTATTGATCCAATCAAAAAATAAATTAGAAGCCGTGGGTCATTGTTATGAATCTCTTTCA
GAGGAATACAGACAATTGACAAAATTCACAGACTCTCAAGATTTTAAAAAACTGTTTAACAAGGTCCCTATTGTTAC
AGATGGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGTTTGATGCGATTCAAAAAAGAATCAG
CTCTAGCTACCACCGCAATAGATCCTGTTAGATACATAGATCCTCGTCGCGATATCGCATTTTCTAACGTGATGGAT
ATATTAAAGTCGAATAAAGTGAACAATAATTAATTCTTTATTGTCATCATGAACGGCGGACATATTCAGTTGATAAT
CGGCCCCATGTTTTCAGGTAAAAGTACAGAATTAATTAGACGAGTTAGACGTTATCAAATAGCTCAATATAAATGCG
TGACTATAAAATATTCTAACGATAATAGATACGGAACGGGACTATGGACGCATGATAAGAATAATTTTGAAGCATTG
GAAGCAACTAAACTATGTGATGTCTTGGAATCAATTACAGATTTCTCCGTGATAGGT
SEQUENCE LISTING
<110>China Sickness Prevention Control Center Virus Disease Prevention Control Institute
<120>One plant of vaccinia virus recombinant for carrying the latent gene of membranous antigen 2 of Epstein-Barr virus and its application
<160>8
<170> PatentIn version 3.5
<210> 1
<211>18
<212>DNA
<213>TKF
<400>CGGGACTATGGACGCATG
<210>2
<211>19
<212>DNA
<213>TKR
<400>CGGTTTCCTCACCCAATCG
<210>3
<211>19
<212>DNA
<213>GFPF
<400>TCTCCAGCAGGACCATGTG
<210>4
<211>19
<212>DNA
<213>GFPR
<400>GGACGACGGCAACTACAAG
<210>5
<211>21
<212>DNA
<213>LMP2F
<400>TCAACCACTAGGAACCCAAGA
<210>6
<211>21
<212>DNA
<213>LMP2R
<400>ACGTAAATGCCTTGTAGTCCG
<210>7
<211>1494
<212>DNA
<213>EBV LMP2
<400>
ATGGGGTCCCTAGAAATGGTGCCAATGGGCGCGGGTCCCCCTAGCCCCGGCGGGGATCCGGATGGGTACGATG
GCGGAAACAACTCCCAATATCCATCTGCTT
CTGGCTCTTCTGGGAACACCCCCACCCCACCGAACGATGAGGAACGTGAATCTAATGAAGAGCCCCCACCGCC
TTATGAGGACCCATATTGGGGCAATGGCGA
CCGTCACTCGGACTATCAACCACTAGGAACCCAAGATCAAAGTCTGTACTTGGGATTGCAACACGACGGGAAT
GACGGGCTCCCTCCCCCTCCCTACTCTCCA
CGGGATGACTCATCTCAACACATATACGAAGAAGCGGGCAGAGGAAGTATGAATCCAGTATGCCTGCCTGTAA
TTGTTGCGCCCTACCTCTTTTGGCTGGCGG
CTATTGCCGCCTCGTGTTTCACGGCCTCAGTTAGTACCGTTGTGACCGCCACCGGCTTGGCCCTCTCACTTCT
ACTCTTGGCAGCAGTGGCCAGCTCATATGC
CGCTGCACAAAGGAAACTGCTGACACCGGTGACAGTGCTTACTGCGGTTGTCACTTTCTTTGCAATTTGCCTA
ACATGGAGGATTGAGGACCCACCTTTTAAT
TCTCTTCTGTTTGCATTGCTGGCCGCAGCTGGCGGACTACAAGGCATTTACGTTCTGGTGATGCTTGTGCTCC
TGATACTAGCGTACAGAAGGAGATGGCGCC
GTTTGACTGTTTGTGGCGGCATCATGTTTTTGGCATGTGTACTTGTCCTCATCGTCGACGCTGTTTTGCAGCT
GAGTCCCCTCCTTGGAGCTGTAACTGTGGT
TTCCATGACGCTGCTGCTACTGGCTTTCGTCCTCTGGCTCTCTTCGCCAGGGGGCCTAGGTACTCTTGGTGCA
GCCCTTTTAACATTGGCAGCAGCTCTGGCA
CTGCTAGCGTCACTGATTTTGGGCACACTTAACTTGACTACAATGTTCCTTCTCATGCTCCTATGGACACTTG
TGGTTCTCCTGATTTGCTCTTCGTGCTCTT
CATGTCCACTGAGCAAGATCCTTCTGGCACGACTGTTCCTATATGCTCTCGCACTCTTGTTGCTAGCCTCCGC
GCTAATCGCTGGTGGCAGTATTTTGCAAAC
AAACTTCAAGAGTTTAAGCAGCACTGAATTTATACCCAATTTGTTCTGCATGTTATTACTGATTGTCGCTGGC
ATACTCTTCATTCTTGCTATCCTGACCGAA
TGGGGCAGTGGAAATAGAACATACGGTCCAGTTTTTATGTGCCTCGGTGGCCTGCTCACCATGGTAGCCGGCG
CTGTGTGGCTGACGGTGATGTCTAACACGC
TTTTGTCTGCCTGGATTCTTACAGCAGGATTCCTGATTTTCCTCATTGGCTTTGCCCTCTTTGGGGTCATTAG
ATGCTGCCGCTACTGCTGCTACTACTGCCT
TACACTGGAAAGTGAGGAGCGCCCACCGACCCCATATCGCAACACTGTATAA
<210>8
<211>7364
<212>DNA
<213> PZL-GFP-LMP2
<400>
ATCGATTTATTATTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGCGGTCACGAACTCCAGCAGG
ACCATGTGATCGCGCTTCTCGTTGGGGTCT
TTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGT
TCTGCTGGTAGTGGTCGGCGAGCTGCACGC
TGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATA
GACGTTGTGGCTGTTGTAGTTGTACTCCAG
CTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCG
CCCTCGAACTTCACCTCGGCGCGGGTCTTG
TAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGT
CGTGCTGCTTCATGTGGTCGGGGTAGCGGC
TGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCA
GATGAACTTCAGGGTCAGCTTGCCGTAGGT
GGCATCGCCCTCGCCCTCGCCGGACACGCTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATG
GGCACCACCCCGGTGAACAGCTCCTCGCCC
TTGCTCACCATCCTCGAGGAGTTCATTTATAGCATAGAAAAAAACAAAATGAAATTCTACTATATTTTTACAT
ACATATATTCTAAATATGAAAGTGGTGATT
GTGACTAGCGTAGCATCGCTTCTAGACATCTCTTAAGTCTGTTATTATTATTGATCCAATCAAAAAATAAATT
AGAAGCCGTGGGTCATTGTTATGAATCTCT
TTCAGAGGAATACAGACAATTGACAAAATTCACAGACTCTCAAGATTTTAAAAAACTGTTTAACAAGGTCCCT
ATTGTTACAGATGGAAGGGTCAAACTTAAT
AAAGGATATTTGTTCGACTTTGTGATTAGTTTGATGCGATTCAAAAAAGAATCAGCTCTAGCTACCACCGCAA
TAGATCCTGTTAGATACATAGATCCTCGTC
GCGATATCGCATTTTCTAACGTGATGGATATATTAAAGTCGAATAAAGTGAACAATAATTAATTCTTTATTGT
CATCATGAACGGCGGACATATTCAGTTGAT
AATCGGCCCCATGTTTTCAGGTAAAGCTAGCATATACTATATAGTAATACCAATACTCAAGACTACGAAACTG
ATACAATCTCTTATCATGTGGGTAATGTTC
TCGATGTCGATAGCCATATGCCCGGTAGTTGCGATATACATAAACTGATCACTAATTCCAAACCCACCCGCTT
TTTATAGTAAGTTTTTCACCCATAAATAAT
AAATACAATAATTAATTTCTCGTAAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCA
TAAAGAACAGTGACGGATCCCCAACTAGTG
TACCTGCAATGGGGTCCCTAGAAATGGTGCCAATGGGCGCGGGTCCCCCTAGCCCCGGCGGGGATCCGGATGG
GTACGATGGCGGAAACAACTCCCAATATCC
ATCTGCTTCTGGCTCTTCTGGGAACACCCCCACCCCACCGAACGATGAGGAACGTGAATCTAATGAAGAGCCC
CCACCGCCTTATGAGGACCCATATTGGGGC
AATGGCGACCGTCACTCGGACTATCAACCACTAGGAACCCAAGATCAAAGTCTGTACTTGGGATTGCAACACG
ACGGGAATGACGGGCTCCCTCCCCCTCCCT
ACTCTCCACGGGATGACTCATCTCAACACATATACGAAGAAGCGGGCAGAGGAAGTATGAATCCAGTATGCCT
GCCTGTAATTGTTGCGCCCTACCTCTTTTG
GCTGGCGGCTATTGCCGCCTCGTGTTTCACGGCCTCAGTTAGTACCGTTGTGACCGCCACCGGCTTGGCCCTC
TCACTTCTACTCTTGGCAGCAGTGGCCAGC
TCATATGCCGCTGCACAAAGGAAACTGCTGACACCGGTGACAGTGCTTACTGCGGTTGTCACTTTCTTTGCAA
TTTGCCTAACATGGAGGATTGAGGACCCAC
CTTTTAATTCTCTTCTGTTTGCATTGCTGGCCGCAGCTGGCGGACTACAAGGCATTTACGTTCTGGTGATGCT
TGTGCTCCTGATACTAGCGTACAGAAGGAG
ATGGCGCCGTTTGACTGTTTGTGGCGGCATCATGTTTTTGGCATGTGTACTTGTCCTCATCGTCGACGCTGTT
TTGCAGCTGAGTCCCCTCCTTGGAGCTGTA
ACTGTGGTTTCCATGACGCTGCTGCTACTGGCTTTCGTCCTCTGGCTCTCTTCGCCAGGGGGCCTAGGTACTC
TTGGTGCAGCCCTTTTAACATTGGCAGCAG
CTCTGGCACTGCTAGCGTCACTGATTTTGGGCACACTTAACTTGACTACAATGTTCCTTCTCATGCTCCTATG
GACACTTGTGGTTCTCCTGATTTGCTCTTC
GTGCTCTTCATGTCCACTGAGCAAGATCCTTCTGGCACGACTGTTCCTATATGCTCTCGCACTCTTGTTGCTA
GCCTCCGCGCTAATCGCTGGTGGCAGTATT
TTGCAAACAAACTTCAAGAGTTTAAGCAGCACTGAATTTATACCCAATTTGTTCTGCATGTTATTACTGATTG
TCGCTGGCATACTCTTCATTCTTGCTATCC
TGACCGAATGGGGCAGTGGAAATAGAACATACGGTCCAGTTTTTATGTGCCTCGGTGGCCTGCTCACCATGGT
AGCCGGCGCTGTGTGGCTGACGGTGATGTC
TAACACGCTTTTGTCTGCCTGGATTCTTACAGCAGGATTCCTGATTTTCCTCATTGGCTTTGCCCTCTTTGGG
GTCATTAGATGCTGCCGCTACTGCTGCTAC
TACTGCCTTACACTGGAAAGTGAGGAGCGCCCACCGACCCCATATCGCAACACTGTATAAGGCCGCTGGGAAT
TCTGTGAGCGTATGGCAAACGAAGGAAAAA
TAGTTATAGTAGCCGCACTCGATGGGACATTTCAACGTAAACCGTTTAATAATATTTTGAATCTTATTCCATT
ATCTGAAATGGTGGTAAAACTAACTGCTGT
GTGTATGAAATGCTTTAAGGAGGCTTCCTTTTCTAAACGATTGGGTGAGGAAACCGAGATAGAGATAATAGGA
GGTAATGATATGTATCAATCGGTGTGTAGA
AAGTGTTACATCGACTCATAATATTATATTTTTTATCTAAAAAACTAAAAATAAACATTGATTAAATTTTAAT
ATAATACTTAAAAATGGATGTTGTGTCGTT
AGATAAACCGTTTATGTATTTTGAGGAAATTGATAATGAGTTAGATTACGAACCAGAAAGTGCAAATGAGGTC
GCAAAAAAACTACCGTATCAAGGACAGTTA
AAACTATTACTAGGAGAATTATTTTTTCTTAGTAAGTTACAGCGACACGGTATATTAGATGGTGCCACCGTAG
TGTATATAGGATCTGCTCCCGGTACACATA
TACGTTATTTGAGAGATCATTTCTATAATTTAGGAGTGATCATCAAATGGATGCTAATTGACGGCCGCCATCA
TGATCCTATTTTAAATGGATTGCGTGATGT
GACTCTAGTGACTCGGTTCGTTGATGAGGAATATCTACGATCCATCAAAAAACAACTGCATCCTTCTAAGATT
ATTTTAATTTCTGATGTGAGATCCAAACGA
GGAGGAAATGAACCTAGTACGGCGGATTTACTAAGTAATTACGCTCTACAAAATGTCATGATTAGTATTTTAA
ACCCCGTGGCATCTAGTCTTAAATGGAGAT
GCCCGTTTCCAGATCAATGGATCAAGGACTTTTATATCCCACACGGTAATAAAATGTTACAACCTTTTGCTCC
TTCATATTCAGCACTGGCCGTCGTTTTACA
ACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGG
CGTAATAGCGAAGAGGCCCGCACCGATCGC
CCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCG
GTATTTCACACCGCATATGGTGCACTCTCA
GTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACG
GGCTTGTCTGCTCCCGGCATCCGCTTACAG
ACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGA
AAGGGCCTCGTGATACGCCTATTTTTATAG
GTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTA
TTTGTTTATTTTTCTAAATACATTCAAATA
TGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCA
ACATTTCCGTGTCGCCCTTATTCCCTTTTT
TGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTG
GGTGCACGAGTGGGTTACATCGAACTGGAT
CTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTC
TGCTATGTGGCGCGGTATTATCCCGTATTG
ACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCAC
AGAAAAGCATCTTACGGATGGCATGACAGT
AAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGA
GGACCGAAGGAGCTAACCGCTTTTTTGCAC
AACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGC
GTGACACCACGATGCCTGTAGCAATGGCAA
CAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGA
GGCGGATAAAGTTGCAGGACCACTTCTGCG
CTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATT
GCAGCACTGGGGCCAGATGGTAAGCCCTCC
CGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAG
GTGCCTCACTGATTAAGCATTGGTAACTGT
CAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAA
GATCCTTTTTGATAATCTCATGACCAAAAT
CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCT
TTTTTTCTGCGCGTAATCTGCTGCTTGCAA
ACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAA
CTGGCTTCAGCAGAGCGCAGATACCAAATA
CTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCT
GCTAATCCTGTTACCAGTGGCTGCTGCCAG
TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGA
ACGGGGGGTTCGTGCACACAGCCCAGCTTG
GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGA
GAAAGGCGGACAGGTATCCGGTAAGCGGCA
GGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTT
TCGCCACCTCTGACTTGAGCGTCGATTTTT
GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT
TGCTGGCCTTTTGCTCACATGTTCTTTCCT
GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAA
CGACCGAGCGCAGCGAGTCAGTGAGCGAGG
AAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGA
CAGGTTTCCCGACTGGAAAGCGGGCAGTGA
GCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGT
ATGTTGTGTGGAATTGTGAGCGGATAACAA
TTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTTTGCGATCAATAAATGGATCACAACCAGTAT
CTCTTAACGATGTTCTTCGCAGATGATGAT
TCATTTTTTAAGTATTTGGCTAGTCAAGATGATGAATCTTCATTATCTGATATATTGCAAATCACTCAATATC
TAGACTTTCTGTTATTATTATTGATCCAAT
CAAAAAATAAATTAGAAGCCGTGGGTCATTGTTATGAATCTCTTTCAGAGGAATACAGACAATTGACAAAATT
CACAGACTCTCAAGATTTTAAAAAACTGTT
TAACAAGGTCCCTATTGTTACAGATGGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGT
TTGATGCGATTCAAAAAAGAATCAGCTCTA
GCTACCACCGCAATAGATCCTGTTAGATACATAGATCCTCGTCGCGATATCGCATTTTCTAACGTGATGGATA
TATTAAAGTCGAATAAAGTGAACAATAATT
AATTCTTTATTGTCATCATGAACGGCGGACATATTCAGTTGATAATCGGCCCCATGTTTTCAGGTAAAAGTAC
AGAATTAATTAGACGAGTTAGACGTTATCA
AATAGCTCAATATAAATGCGTGACTATAAAATATTCTAACGATAATAGATACGGAACGGGACTATGGACGCAT
GATAAGAATAATTTTGAAGCATTGGAAGCA
ACTAAACTATGTGATGTCTTGGAATCAATTACAGATTTCTCCGTGATAGGT
Claims (9)
1. one plant of vaccinia virus recombinant for carrying the latent gene of membranous antigen 2 of Epstein-Barr virus, it is characterised in that described recombinant vaccinia disease
Poison carries Epstein-Barr virus latent membrane protein2 (EBVLMP2) foreign gene, and does not carry any foreign gene in addition to EBVLMP2.
2. vaccinia virus recombinant as claimed in claim 1, it is characterised in that described vaccinia virus recombinant from it is highly attenuated,
Ankara strain vaccinia virus strain structure of replication defect type.
3. vaccinia virus recombinant as claimed in claim 1, it is characterised in that the EBV LMP2 of described carrying are EBV LMP2
Full-length gene, length scale are 1.5kb gene order (SEQ ID NO.7).
4. a kind of method for preparing the vaccinia virus recombinant for carrying the latent gene of membranous antigen 2 of Epstein-Barr virus, utilizes PZL-GFP-LMP2
GFP genes, the MVA-LMP2A recombinant viruses of redgreen fluorescent protein expression are sloughed in recombinant plasmid system, acquisition.
5. the application of one plant of vaccinia virus recombinant for carrying the latent gene of membranous antigen 2 of Epstein-Barr virus, the application are treatment and prevention
Purposes in the vaccine and medicine of EBV related neoplasms.
6. the purposes described in claim 5, wherein described EBV related neoplasms are nasopharyngeal carcinoma (NPC).
A kind of 7. vaccine combination, wherein containing vaccinia virus recombinant and one or more described in claim any one of 1-3
Pharmaceutically acceptable carrier, adjuvant or excipient.
8. a kind of cytotoxic T lymphocyte of activation, the cell is with the restructuring acne any one of claim 1-4
Seedling diseases telson, which swashs cytotoxic T lymphocyte, makes what its activation obtained.
9. purposes of the cytotoxic T of the activation described in claim 8 in preparing for adoptive medicine.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710826442.5A CN107488677A (en) | 2017-09-14 | 2017-09-14 | One plant of vaccinia virus recombinant for carrying the latent gene of membranous antigen 2 of Epstein-Barr virus and its application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358805A (en) * | 2018-04-10 | 2019-10-22 | 北京工业大学 | A kind of appraisal procedure of the specific killing function suitable for specificity cell toxicity T lymphocyte |
| WO2021244653A1 (en) * | 2020-06-05 | 2021-12-09 | Guangdong Tcrcure Biopharma Technology Co., Ltd. | Tcr-t cell therapy targeting epstein-barr virus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007126163A1 (en) * | 2006-04-27 | 2007-11-08 | Seoul National University Industry Foundation | B cell-based vaccine loaded with the ligand of natural killer t cell and antigen |
| CN101861167A (en) * | 2007-11-19 | 2010-10-13 | 财团法人首尔大学校产学协力财团 | Vaccine comprising monocyte or immature myeloid cells(IMC) which were loaded with the ligand of natural killer t cell and antigen |
-
2017
- 2017-09-14 CN CN201710826442.5A patent/CN107488677A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007126163A1 (en) * | 2006-04-27 | 2007-11-08 | Seoul National University Industry Foundation | B cell-based vaccine loaded with the ligand of natural killer t cell and antigen |
| CN101861167A (en) * | 2007-11-19 | 2010-10-13 | 财团法人首尔大学校产学协力财团 | Vaccine comprising monocyte or immature myeloid cells(IMC) which were loaded with the ligand of natural killer t cell and antigen |
Non-Patent Citations (2)
| Title |
|---|
| WANG Z等: "Specific Cellular Immune Responses in Mice Immunized with DNA, Adeno-associated Virus and Adenviral Vaccines of Epstein-Barr Virus-LMP2 Alone or in Combination", 《SCIENCE CHINA》 * |
| 张丽霞: "EBV-LMP2重组痘苗病毒免疫效果研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358805A (en) * | 2018-04-10 | 2019-10-22 | 北京工业大学 | A kind of appraisal procedure of the specific killing function suitable for specificity cell toxicity T lymphocyte |
| WO2021244653A1 (en) * | 2020-06-05 | 2021-12-09 | Guangdong Tcrcure Biopharma Technology Co., Ltd. | Tcr-t cell therapy targeting epstein-barr virus |
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