CN1560248A - Tiantan remocined vaccine virus of IFN-alpha receptor gene (B8R) deletion and application thereof - Google Patents
Tiantan remocined vaccine virus of IFN-alpha receptor gene (B8R) deletion and application thereof Download PDFInfo
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Abstract
The invention relates to an IFN-gama receptor gene (B8R) deleted attenuating carrier of Tiantan recombinant t vaccinia virus as well as a Tiantan recombinant t vaccinia virus AIDS vaccinum deleting IFN-gama receptor gene (B8R) and expressing various HIV-1 antigens, which is constructed based on this carrier, a Tiantan recombinant t vaccinia virus VTKgpe, recombinant t vaccinia virus VTKgpe CGMCC.No.1099 and another a hepatitis-B virus HBSAg antigen, and a hepatitis-B vaccinum of Tiantan recombinant t vaccinia virus of IL-2.
Description
Technical field
The present invention relates to the Temple of Heaven strain vaccinia virus recombinant attenuated carrier of IFN-gamma receptor gene (B8R) disappearance and based on the Temple of Heaven strain vaccinia virus recombinant AIDS vaccine of IFN-gamma receptor gene (B8R) disappearance of the various antigens of expression HIV-1 (unit price and multivalence) of this vector construction.The Temple of Heaven strain vaccinia virus recombinant attenuated carrier of the present invention can significantly reduce the virulence of vaccinia virus vector, can be used for the multiple antigen of the same cause of disease of construction expression, the multiple antigenic polyvalent recombinant vaccinia virus of Different Kinds of Pathogens.Vaccinia virus recombinant AIDS vaccine of the present invention can be induced high-caliber anti-human immunodeficiency virus (Human Immunodeficiency Virus, HIV) body fluid and cellullar immunologic response, and effectively improve the security and the immunogenicity of vaccinia virus recombinant AIDS vaccine.
Background technology
Vaccinia virus Tiantan strain (vaccine virus TianTan strain, VTT) for exterminating smallpox, China makes huge contribution, use also very active in live vector vaccine research, that the vaccinia virus recombinant vaccine has is effective, to Heat stability is good, inoculation is convenient, do not need advantages such as adjuvant, yet produce heavier local reaction behind the vaccinia virus inoculation human body, particularly in a certain proportion of immune deficiency patient, can cause severe complications.Express some immunogenic vaccinia virus recombinant and fail to reach the ideal immune effect, the security that how can further improve vaccinia virus vector simultaneously in the immunogenicity that strengthens vaccinia virus recombinant, scholars make a lot of deep researchs.Along with new virus disease constantly occurs, to understanding in depth of host anti-virus immune mechanism, the multiple antigen of the same cause of disease of construction expression, the multiple antigenic polyvalent recombinant vaccinia virus vector of Different Kinds of Pathogens, it is significant to seek more multistable fixed effective foreign gene insertion district.
Some special albumen of vaccinia virus gene group coding suppress host immune systems, and according to the literature and genetic analysis, vaccinia virus Tiantan strain B8R gene and IFN-γ receptor membrane exterior domain have high homology as can be known
[1]IFN-γ has important effect aspect antiviral and the immunomodulatory.The present invention makes up the Temple of Heaven strain vaccinia virus recombinant carrier of disappearance IFN-gamma receptor gene, studies the stability that its virulence change and B8R disappearance district insert exogenous gene expression.Further improve the security of Tiantan strain vaccinia virus carrier, development expression polyvalent antigen vaccinia virus recombinant carrier.
Popular high pathogenicity rate and the case fatality rate of having caused in the HIV-1 whole world, infection for control HIV-1, traditional chemotherapy, immunotherapy and new method are able to continuous research, development, most HIV the infecteds are carried out HAART, though can effectively suppress viremia, delay the course of disease, but can not remove the virus of hiding.The vaccine inoculation of protectiveness is to solve control HIV-1 to infect the optimal path of global problem.Scientists has been carried out the research of attenuated live vaccine, inactivated vaccine, recombinant subunit vaccine, various live vector vaccine (carriers such as adenovirus, canary pox virus, influenza virus, vaccinia virus, rabies virus, VSV, Corynebacterium diphtheriae, suis), and there are some problems in traditional attenuated live vaccine and inactivated vaccine in the HIV control.Though attenuated live vaccine is effective at the SIV model validation, in growing up macaque, immunity just has certain lethality rate.The orangutan that is infected by HIV-1 still can be infected by second kind of incoherent HIV-1.The attenuated strain risk and evidence suggests no extensive protectiveness owing to live, the dispute of attenuated live vaccine application waits further research.There are some researches show that at the orangutan model inactivated vaccine can not protect HIV-1 to attack, and can not induce ctl response.Virus live vector vaccine and dna vaccination all both can the inducing cell immunity, can induce humoral immune reaction again, and the combined immunization of expressing HIV-1 antigen vaccinia virus recombinant and HIV-1DNA vaccine becomes new vaccine research strategy.
Summary of the invention
In order further to improve the security and the validity of vaccinia virus recombinant vaccine, the object of the invention be to provide a kind of homologous recombination construction B8R genetically deficient district insert foreign gene the Temple of Heaven strain vaccinia virus recombinant VTT Δ B8RlacZ and the application in preparation vaccinia virus recombinant vaccine thereof of IFN-gamma receptor gene (B8R) disappearance.
Be specifically related to a kind of B8R genetically deficient, (B8R genetically deficient is not with the lacZ selective marker to express the antigenic VTKgpe Δ of HIV-1 B8R, TK district insertion B '/C type CN54 strain HIV-1 gagpolenv gene) AIDS vaccine of the Temple of Heaven strain vaccinia virus recombinant recombinant vaccinia virius VTKgpe, this vaccinia virus recombinant recombinant vaccinia virius VTKgpe on February 24th, 2004 in Beijing preservation, depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center, and its preserving number is CGMCC No.1099; Relate to a kind of B8R genetically deficient and express the AIDS vaccine of the Temple of Heaven strain vaccinia virus recombinant of the antigenic VTKgpe Δ of HIV-1 B8RlacZ (B8R genetically deficient is replaced by lacZ, and the TK district inserts B '/C type CN54 strain HIV-1 gagpol env gene); Also relate to a kind of B8R genetically deficient, express hepatitis B virus HBSAg antigen, the hepatitis B disease vaccine of the Temple of Heaven strain vaccinia virus recombinant of IL-2 (B8R genetically deficient is inserted HBSAg antigen, and the TK district inserts the IL-2 gene).
The object of the present invention is to provide that to be used for vaccinia virus VTT be maternal virus, make up B8R district disappearance, insert vaccinia virus recombinant transferring plasmid pSKB8R, pSKB8RLacZ, pSKB8RNeo, pSKB8RPNeo and their construction process of various foreign genes simultaneously in this district.
The invention provides a kind of plasmid pSC65 that transferring plasmid pSKB8RLacZ makes up that is used for, the colon bacillus Esherichia coli that contains this plasmid on February 24th, 2004 in Beijing preservation, depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center, and its preserving number is CGMCC No.1097.
The invention provides a kind of plasmid pVI75 that transferring plasmid pSKB8Rneo makes up that is used for, the colon bacillus Esherichia coli that contains this plasmid on February 24th, 2004 in Beijing preservation, depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center, and its preserving number is CGMCC No.1098.
A kind of B8R genetically deficient involved in the present invention is that the recombinant virus VTT Δ B8R1acZ that lacZ replaced can be used as parent's strain that homologous recombination construction B8R genetically deficient district inserts the vaccine strain of foreign gene.VTT Δ B8RlacZ construction process is about to vaccinia virus Tiantan strain VTT (Beijing institute of Biological Products preservation, Tiantan strain 752-1) carries out homologous recombination with transferring plasmid pSKB8RLacZ cotransfection CEF cell, screen in vain, continuous single spot purifying, identify that obtaining B8R genetically deficient is the recombinant virus VTT Δ B8RlacZ that lacZ replaced through indigo plant.Extract DNA, carry out the stability that pcr amplification and DOTBLOT identify, detect recombinant virus B8R genetically deficient, show that VTT Δ B8RlacZ is stable in the CEF cell cultured continuously 15 generation B8R genetically deficients that go down to posterity.VTT Δ B8RlacZ inserts foreign gene lacZ stably express in CEF cell cultured continuously 15 generations of going down to posterity.The breeding growth curve of recombination deficient virus VTT Δ B8RlacZ in CEF, TK-143, CV-1 cell measured, and the result shows that the growth curve of VTT Δ B8RlacZ is similar to the VTT growth curve.B8R genetically deficient to recombination deficient virus the breeding in cell do not duplicate not influence.Virulence experiment shows that VTT752-1 forms red and swollen pock area and significantly forms pock greater than VTT Δ B8RlacZ in the rabbit, VTT752-1 forms red and swollen pock and diabrosis then occurs and connect scab, VTT Δ B8RlacZ forms pock not to be had diabrosis and connects scab, and VTT Δ B8RlacZ forms pock and disappears to take off and form pock than VTT752-1 and disappear and take off early.B8R genetically deficient significantly reduces the vaccinia virus Tiantan strain virus virulence.
A kind of B8R genetically deficient that the present invention relates to, (B8R genetically deficient is not with the lacZ selective marker to express the antigenic VTKgpe Δ of HIV-1 B8R, the AIDS vaccine of the Temple of Heaven strain vaccinia virus recombinant TK district insertion B '/C type CN54 strain HIV-1 gagpolenv gene) and a kind of B8R genetically deficient are expressed the AIDS vaccine of the Temple of Heaven strain vaccinia virus recombinant of the antigenic VTKgpe/ Δ of HIV-1 B8RlacZ (B8R genetically deficient is replaced by lacZ, and the TK district of parent's strain VTT Δ B8RlacZ inserts B '/C type CN54 strain HIV-1 gagpol env gene).Be about to VTKgpe (the TK district inserts the Temple of Heaven strain vaccinia virus recombinant of B '/C type CN54 strain HIV-1 gagpol env gene) and carry out homologous recombination with transferring plasmid pSKB8RLacZ cotransfection CEF cell, screen in vain through indigo plant, continuous single spot purifying, identify that obtaining B8R genetically deficient is the recombinant virus VTKgpe Δ B8RlacZ that lacZ replaced, be that parent plant and pSKB8RNeo cotransfection CEF cell carry out homologous recombination with VTKgpe Δ B8RlacZ again, screen through G418 pressure, blue hickie screening, continuous single spot purifying, identify to obtain B8R genetically deficient not with the recombinant virus VTKgpe Δ B8R of lacZ selective marker.The nude mice virulence experiment shows that VTKgpe Δ B8R virulence significantly reduces than VTKgpe, abdominal cavity inoculation VTT (10
4Pfu) and VTKgpe (10
6Pfu) nude mice death in 15 days, inoculation VTKgpe Δ B8R (10
7Pfu) nude mice survival.IFN-γ in the born of the same parents. dyeing flow cytometry analysis, T lymphopoiesis, CTL detect and show that B8R genetically deficient significantly improves the Temple of Heaven strain vaccinia virus recombinant specific cellular immunity of expressing the HIV-1 gene.The above-mentioned related TK district of the present invention
Be meant Tiantan strain Genebank, one section nucleotide sequence of the genomic 79799bp-81271bp of gi6969640.
The present invention relates to the acquired immune deficiency syndrome (AIDS) and the hepatitis B disease vaccine of the Temple of Heaven strain vaccinia virus recombinant of B8R genetically deficient, expression HIV-1 and various unit prices of hepatitis B virus or polyvalent antigen.
The Temple of Heaven strain vaccinia virus recombinant that the present invention relates to B8R genetically deficient, expression alien gene unit price or polyvalent antigen comprises: recombiant vaccine, expression alien gene vaccinia virus recombinant and their application in the vaccinia virus recombinant vaccine medicine of preparation treatment virus disease.
More than technical characterictic described in the invention do not see any report at present both at home and abroad, so the present invention is creative, novelty and practicality.Vaccinia virus recombinant vaccine pharmacological agent to human virus's disease will bring crucial meaning.
Description of drawings
Fig. 1 shows the structure route of transferring plasmid pSKB8R, pSKB8RLacZ, pSKB8RNeo
Fig. 2 shows that the pSKB8R enzyme cuts the affirmation analytical results, and swimming lane 2,3 among the figure: molecular weight marker DL2000, DL15000 (available from the precious biotechnology in Dalian company limited), swimming lane 1:pSKB8R (NdeI and BgLII).
Fig. 3 shows that the pSKB8RlacZ enzyme cuts the affirmation analytical results, and swimming lane 1 among the figure: molecular weight marker DL15000, swimming lane 2:pSKB8RLacZ (SpeI), swimming lane 3:pSKB8RLacZ (SacI), swimming lane 4:pSKB8RLacZ (NdeI).
Fig. 4 shows that the pSKB8RNeo enzyme cuts the affirmation analytical results, and swimming lane 1 among the figure: molecular weight marker DL15000, swimming lane 2:pSKB8RNeo (BamHI and BgLII).
Fig. 5 shows that the pSKB8RPNeo enzyme cuts the affirmation analytical results, and swimming lane 12 among the figure: molecular weight marker DL2000,15000, swimming lane 3:pSKB8RPNeo (SaLI/SmaLI).
Fig. 6 shows that recombinant virus VTT Δ B8RlacZ, VTKgpe Δ B8RlacZ, VTKgpe Δ B8R make up synoptic diagram.
Fig. 7 shows that pcr amplification detects recombinant virus VTT Δ B8RlacZ B8R genetically deficient 1,6:DNA standard 2:VTTDNA pcr amplification product (primer B8RLF, B8RRR) 3:VTTDNA amplified production (primer B8RF, B8RRR)
Fig. 8 Dot Blot detects B8R gene in the viral genome
Extract vaccinia virus VTT752-1DNA and the recombinant virus VTT Δ B8RlacZ 15 generation DNA that go down to posterity, carry out Dot Blot with B8R gene digoxin labelled probe and detect.VTT752-1Dot Blot result shows hybridization trace spot, and the VTT Δ B8RlacZ 15 generation Dot Blot results of going down to posterity do not show hybridization trace spot, shows that VTT Δ B8RlacZ is in the CEF cell cultured continuously 15 generation B8R stable genes disappearance that goes down to posterity
Fig. 9 shows that the reproduction curve in the recombination deficient virus VTT Δ B8RlacZ cell is measured and breeds in CEF with VTT Δ B8RlacZ recombinant virus, the viral PFU value of its different time results is drawn viral proliferation curve (similar to reproduction curve on TK-143, Wish, the CV-1 cell).The result shows that the reproduction curve of VTT Δ B8RlacZ is similar to contrast VTT reproduction curve.B8R genetically deficient to recombination deficient virus the breeding in cell do not duplicate not influence.48h viral proliferation peak reaches 10
7PFU/ml.(the growth curve longitudinal axis is the PFU logarithmic value)
Figure 10 shows virulence experimental result in the rabbit: left side inoculation 1 * 10 in the rabbit butt
6The VTT Δ B8RlacZ of PFU, right side inoculation 1 * 10
6The VTT752-1 of PFU/ml is with 0.1ml 1 * 10
7Pfu/ml VTT Δ B8RlacZ recombinant virus after injection in the rabbit butt, is observed the red swelling of the skin area day by day, and the 5th day visible red and swollen pock is significantly less than the contrast vaccinia virus, and do not have diabrosis, disappears and takes off early.Show that the comparison of recombinant virus VTT Δ B8RlacZ virulence significantly weakens according to VTT752-1.
Figure 11 shows that it is template that pcr amplification detects recombinant virus VTKgpe Δ B8R B8R genetically deficient extraction recombinant virus dna, carries out pcr amplification with primer B8RLF, B8RRR.Recombinant virus VTKgpe Δ B8R is owing to B8R genetically deficient, and pcr amplification goes out the 1244bp fragment, comprises the outer left side of B8R gene homologous sequence B8RL fragment 583bp, right side homologous sequence B8RR fragment 661bp.And contrast VTTDNA is that template amplification goes out the 2Kb fragment, comprises the outer left side of B8R gene homologous sequence B8RL fragment 583bp, B8R gene 818bp, right side homologous sequence B8RR fragment 661bp.The result shows that VTKgpe Δ B8R is in go down to posterity 15 generation B8R stable genes disappearance of CEF cell cultured continuously.
Figure 12, Dot Blot detect B8R gene in the viral VTKgpe Δ B8R genome
1、VTT752-1?2、VTKgpeΔB8R
Extract vaccinia virus VTT752-1DNA and the recombinant virus VTKgpe Δ B8R 15 generation DNA that go down to posterity, carry out Dot Blot with B8R gene digoxin labelled probe and detect.VTT752-1Dot Blot result shows hybridization trace spot, and the VTKgpe Δ B8R 15 generation Dot Blot results of going down to posterity do not show hybridization trace spot, shows that VTKgpe Δ B8R is in the CEF cell cultured continuously 15 generation B8R stable genes disappearance that goes down to posterity
Figure 13 shows IFN-γ in the born of the same parents. dyeing flow cytometry analysis result.DNA+VTKgpe Δ B8R immune group mouse boosting cell T lymphocyte, external after epitope peptide stimulates, the CD8 of secretion of gamma-IFN
+The T lymphocyte accounts for the lymphocytic per-cent digital display of the total CD8+T of spleen work and is higher than the DNA+VTKgpe immune group.VTKgpe Δ B8R immune group mouse boosting cell secretion of gamma-IFN .CD8 per-cent digital display work is higher than the VTKgpe immune group.
Figure 14 shows T lymphopoiesis 3H-TdR method detected result.DNA+VTKgpe Δ B8R immune group mouse boosting cell T lymphocyte, after epitope peptide stimulated, stimulation index (SI) was significantly higher than the DNA+VTKgpe immune group external.
Figure 15 shows CTL lactic dehydrogenase enzyme process detected result.DNA+VTKgpe Δ B8R immune group mouse boosting cell T lymphocyte, specific killing is significantly higher than the DNA+VTKgpe immune group.VTKgpe Δ B8R immune group is higher than the VTKgpe immune group
Figure 16 Western Blot analyzes VTKgpe Δ B8R destination gene expression
1 VTT infects CEF cell 2,3, and 4VTKgpe Δ B8R infects CEF cell 5 and dyes albumen Marker in advance
Specific embodiments
The structure of embodiment one, transferring plasmid
1. the structure of plasmid pBR-SK
5’PBR322-SACI-FOR
G
GAGCTCCGACCGATGCCCTTGAGAGCC
3’PBR322-KPNI-RE
GG
GGTACCAGGTGGCACTTTTCGGGGAAATG
The commercial plasmid pBR322 of pcr amplification comprises the sequence of all functional elements, is designated as pBR.
Reaction system
10×Pyrobest?Buffer?????????????????????5μl
DNTPMisture (each 2.5mM) 5 μ l
3’PBR322-KPNI-RE(50μM)????????????????0.5μl
5’PBR322-SACI-FOR(50μM)???????????????0.5μl
pBR322??????????????????????????????????0.5μl
Pyrobest?DNA?Polymerase(5U/ml)??????????0.5μl
ddH2O????????????????????????????????????38μl
94 ℃ of pre-sex change 2min of reaction conditions; 94 ℃ of 30s, 68 ℃ of 5min, totally 35 circulations; 72 ℃ of 7min; 4 ℃.
Purifying
Precious biological KpnI in Dalian and SacI enzyme altogether cut PCR product pBR, run glue and reclaim.
Be total to digested plasmid pBS-SK with precious biological KpnI in Dalian and SacI, obtain multiple clone site sequence (small segment), be designated as SK, run 1.5% agarose gel and reclaim.
Ligase enzyme cuts back to close product pBR and SK, transforms Top10 intestinal bacteria competence, and filters out correct clone's, is designated as pBR-SK.
2. the structure of transferring plasmid pSKB8R
Left side homologous sequence B8RL fragment, right side homologous sequence B8RR fragment outside vector plasmid pBR-SK inserts vaccinia virus Tiantan strain B8R gene.See Fig. 1.Adopt round pcr respectively with the outer left side of vaccinia virus DNA cloning vaccinia virus Tiantan strain B8R gene homologous sequence B8RL fragment (primer B8RLF, B8RLR) right side homologous sequence B8RR fragment (primer B8RRF, B8RRR), B8RL fragment, B8RR fragment are cut connection with BgLII respectively, are that the outer left and right sides homologous sequence of template pcr amplification (primer B8RLF, B8RRR) B8R gene connects big fragment B8RLR to connect product.Big fragment B8RLR, pBRSK plasmid respectively with the KpnI/BamHI enzyme cut, T4DNALigase connects and makes up the transferring plasmid pSKB8R that has the outer left and right sides homology arm of the Temple of Heaven strain B8R gene.Primer sequence: B8RLF:gtaataggtaccgtagcatcctattcg B8RLR:tataatagatcttatggtgttgtttgB8RRF:aactaaagatctcggtag cac B8RRR:attcgtggatccattgtaacaagatatc
B8RL fragment (primer B8RLF, B8RLR), B8RR fragment (primer B8RRF, B8RRR),, connecting the test kit that big fragment B8RLR (primer B8RLF, B8RRR) pcr amplification reaction adopts Dalian precious biotechnology company limited, reaction system is as follows:
Each 1 μ l of forward and reverse primer
10 * Pyrobest damping fluid, 5 μ l
DNTP mixture (each 2.5mM) 5 μ l
Pyrobest archaeal dna polymerase (5U/ml) 0.5 μ l
ddH2O????????????????????????37.5μl
PCR reaction conditions: 94 ℃ of pre-sex change 2min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, totally 20 circulations; 72 ℃ of 7min; 4 ℃.
Big fragment B8RLR extension products carries out the purifying recovery with the E.Z.N.A Cycle-Pure Kit of Omega company, and recovery product KpnI/BamHI enzyme altogether cuts (Takara company product).PBRSK plasmid vector KpnI/BamHI enzyme altogether cuts (Takara company product).Carrying out the electrophoresis purifying with the E.Z.N.A.GelExtraction Kit of Omega company reclaims..The two T4DNALigase (Takara company product) connects the transferring plasmid pSKB8R that structure has the outer left and right sides homology arm of the Temple of Heaven strain B8R gene.Connect product transformed competence colibacillus intestinal bacteria Top10 respectively, be coated with the LB flat board that contains penbritin, cultivated 12-16 hour for 37 ℃.Extract plasmid, with respective limits endonuclease digestion Analysis and Identification, enzyme is cut qualification result respectively referring to Fig. 2.
3. the structure of transferring plasmid pSKB8RLacZ
Between pSKB8R left side homologous sequence B8RL fragment, right side homologous sequence B8RR fragment, insert vaccinia virus promoter sequence pE/L, p7.5 and downstream LacZ sequence.With plasmid pSC65 is template amplification vaccinia virus promoter sequence pE/L, p7.5 and downstream LacZ sequence (primer pSC65F, pSC65R).PE/L, p7.5 and downstream LacZPCR amplified fragments (primer pSC65F, pSC65R) with the BamHI enzyme cut, transferring plasmid pSKB8R cuts with the BgLII enzyme, the two T4DNALigase connects and makes up transferring plasmid pSKB8RLacZ.PSKB8RLacZ has the LacZ sequence in vaccinia virus promoter sequence pE/L, p7.5 and p7.5 downstream and the outer homology arm of B8R gene of both sides.pSC65F：tctttcggatccttgttgaattagatcg?pSC65R：gtttgccatacgctcacagaag
PE/L, p7.5 and downstream LacZPCR amplified fragments pcr amplification reaction adopt the test kit of Dalian precious biotechnology company limited, and reaction system is as follows:
Each 1 μ l of forward and reverse primer (pSC65F, pSC65R)
10 * Pyrobest damping fluid, 5 μ l
DNTP mixture (each 2.5mM) 5 μ l
Pyrobest archaeal dna polymerase (5U/ml) 0.5 μ l
ddH20??????????????????????????37.5μl
PCR reaction conditions: 94 ℃ of pre-sex change 2min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, totally 20 circulations; 72 ℃ of 7min; 4 ℃.
PE/L, p7.5 and downstream LacZPCR amplified fragments extension products carry out the purifying recovery with the E.Z.N.A Cycle-Pure Kit of Omega company, reclaim product B amHI enzyme and cut (Takara company product).Transferring plasmid pSKB8R carrier B gLII enzyme is cut (Takara company product).Carrying out the electrophoresis purifying with the E.Z.N.A.Gel Extraction Kit of Omega company reclaims..The two T4DNALigase (Takara company product) connects structure pSKB8RlacZ.Connect product transformed competence colibacillus intestinal bacteria Top10 respectively, be coated with the LB flat board that contains penbritin, cultivated 12-16 hour for 37 ℃.Extract plasmid, with respective limits endonuclease digestion Analysis and Identification, enzyme is cut qualification result respectively referring to Fig. 3.
The structure of 4 transferring plasmid pSKB8Rneo
Promptly insert vaccinia virus promoter sequence pH6 and downstream Neo sequence in pSKB8R left side homologous sequence B8RL fragment, the right side homologous sequence B8RR fragment outside.With plasmid pVI75 is template amplification vaccinia virus promoter sequence pH6 and downstream Neo sequence (primer NeoF, NeoR), BamHI, SacII enzyme are cut pH6+Neo fragment, transferring plasmid pSKB8RBamHI, SacII enzyme and are cut, and the two T4DNALigase connects and makes up transferring plasmid pSKB8RNeo.Primer NeoF:cgcggatcctcgatccccagattacaaacaactagg, primer NeoR:tccccgcggacagatttctccgtgatagggatcg
PH6 and downstream Neo sequence pcr amplification reaction adopt the test kit of Dalian precious biotechnology company limited, and reaction system is as follows:
Forward and reverse primer (NeoF, NeoR) each 1 μ l
10 * Pyrobest damping fluid, 5 μ l
DNTP mixture (each 2.5mM) 5 μ l
Pyrobest archaeal dna polymerase (5U/ml) 0.5 μ l
ddH2O???????????????????????37.5μl
PCR reaction conditions: 94 ℃ of pre-sex change 2min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 60s, totally 20 circulations; 72 ℃ of 7min; 4 ℃.
PH6 and downstream Neo sequence pcr amplified fragment extension products carry out the purifying recovery with the E.Z.N.ACycle-Pure Kit of Omega company, reclaim product S acII/BamHI enzyme and cut (Takara company product).Transferring plasmid pSKB8R carrier S acII/BamHI enzyme is cut (Takara company product).Carrying out the electrophoresis purifying with the E.Z.N.A.Gel Extraction Kit of Omega company reclaims..The two T4DNALigase (Takara company product) connects structure transferring plasmid pSKB8RNeo.Connect product transformed competence colibacillus intestinal bacteria Top10 respectively, be coated with the LB flat board that contains penbritin, cultivated 12-16 hour for 37 ℃.Extract plasmid, with respective limits endonuclease digestion Analysis and Identification, enzyme is cut qualification result respectively referring to Fig. 4.
The structure of 5 transferring plasmid pSKB8RPNeo
Promptly between pSKB8RNeo left side homologous sequence B8RL fragment, right side homologous sequence B8RR fragment, insert two reverse each other vaccinia virus promoter sequence pE/L, p7.5.With plasmid pSC65 is template amplification vaccinia virus promoter sequence pE/L, p7.5 and multiple clone site.PE/L, p7.5 and multiple clone site amplified fragments (primer PMF, PMR) with the BamHI enzyme cut, transferring plasmid pSKB8R cuts with the BgLII enzyme, the two T4DNALigase connects and makes up pSKB8RP.Plasmid pSKB8RPSacII/BamHI enzyme is cut, and pH6 and downstream Neo sequence pcr amplified fragment extension products are cut (Takara company product) with the SacII/BamHI enzyme, and the two T4DNALigase connects structure transferring plasmid pSKB8RPNeo.PMF：cgggatccgtcgacttcgaacttattt?PMR：cgggatcccccgggctcgagttatgatctt
PE/L, p7.5 and multiple clone site amplified fragments pcr amplification reaction adopt the test kit of Dalian precious biotechnology company limited, and reaction system is as follows:
Forward and reverse primer (PMF, PMR) each 1 μ l
10 * Pyrobest damping fluid, 5 μ l
DNTP mixture (each 2.5mM) 5 μ l
Pyrobest archaeal dna polymerase (5U/ml) 0.5 μ l
ddH2O???????????????????????????37.5μl
PCR reaction conditions: 94 ℃ of pre-sex change 2min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, totally 20 circulations; 72 ℃ of 7min; 4 ℃.
PE/L, p7.5 and multiple clone site amplified fragments extension products carry out the purifying recovery with the E.Z.N.ACycle-Pure Kit of Omega company, reclaim product B amHI enzyme and cut (Takara company product).Transferring plasmid pSKB8R carrier B glII enzyme is cut (Takara company product).Carrying out the electrophoresis purifying with the E.Z.N.A.Gel Extraction Kit of Omega company reclaims..The two T4DNALigase (Takara company product) connects structure pSKB8RP.
Plasmid pSKB8RP SacII/BamHI enzyme is cut, and pH6 and downstream Neo sequence pcr amplified fragment (as preceding) are cut (Takara company product) with the SacII/BamHI enzyme, and the two T4DNALigase connects structure transferring plasmid pSKB8RPNeo.Fig. 5.
Embodiment two B8R genetically deficients are the structure of the recombinant virus VTT Δ B8RlacZ that lacZ replaced.Fig. 6.
Vaccinia virus Tiantan strain VTT and transferring plasmid pSKB8RLacZ cotransfection CEF cell are carried out homologous recombination, screen in vain, continuous single spot purifying, identify that obtaining B8R genetically deficient is the recombinant virus VTT Δ B8RlacZ that lacZ replaced through indigo plant.Behind 0.1 ~ 0.01PFU/cell virus quantity VTT752-1 infection, 80% CEF cell absorption 1 ~ 1.5h in blocks, adopt liposome transfection technology (with reference to the American I NVITROGEN Lipofectin of company test kit) with in the recombinant plasmid pSKB8RLacZ transfection CEF cell.Results recombinant virus liquid after 48 hours, with containing 200 μ g/ml X-gal nutrient agar mediums shop spot, picking isolates locus coeruleus virus at random, and 5 generations of continuous single spot purifying are the recombinant virus VTT Δ B8RlacZ that lacZ was replaced with acquisition B8R genetically deficient to be identified.
Extract DNA, carry out the stability that pcr amplification and DOT BLOT identify, detect recombinant virus B8R genetically deficient, show that VTT Δ B8RlacZ is stable in the CEF cell cultured continuously 15 generation B8R genetically deficients that go down to posterity.VTT Δ B8RlacZ inserts foreign gene lacZ stably express in CEF cell cultured continuously 15 generations of going down to posterity.VTT Δ B8RlacZ went down to posterity for 15 generations in CEF cell cultured continuously, and VTT Δ B8RlacZ the 5th, 15 generations of going down to posterity are infected the CEF cell respectively, detect betagalactosidase activity in its sample, set up the contrast of VTT cells infected simultaneously.The 5th generation beta-galactosidase enzymes OD that measures alive
420nmValue is 3.027, the 15 generation OD
420nmValue is 2.819.VTT cells infected contrast OD
420nmValue is 0.04.The result shows that VTT Δ B8RlacZ goes down to posterity in CEF cell cultured continuously and inserts foreign gene lacZ stably express. (Fig. 7, Fig. 8) and table 1.
Table 1
| ??5passegeVTTΔ ????B8RlacZ | ??15passegeVTTΔ ????B8RlacZ | ????VTT | |
| ???0.D420nm | ??3.027 | ??2.819 | ????0.04 |
The breeding growth curve of recombination deficient virus VTT Δ B8RlacZ in CEF, TK-143, CV-1 cell measured, and the result shows the growth curve and the similar (Figure 90 of VTT growth curve of VTT Δ B8RlacZ.B8R genetically deficient to recombination deficient virus the breeding in cell do not duplicate not influence.Virulence experiment shows that VTT752-1 forms red and swollen pock area and significantly forms pock greater than VTT Δ B8RlacZ in the rabbit, VTT752-1 forms red and swollen pock and diabrosis then occurs and connect scab, VTT Δ B8RlacZ forms pock not to be had diabrosis and connects scab, and VTT Δ B8RlacZ forms pock and disappears to take off and form pock than VTT752-1 and disappear and take off early.B8R genetically deficient significantly reduces the vaccinia virus Tiantan strain virus virulence.See Figure 10.
Embodiment three B8R genetically deficients are expressed the AIDS vaccine of the Temple of Heaven strain vaccinia virus recombinant of the antigenic VTKgpe Δ of HIV-1 B8RlacZ (B8R genetically deficient is replaced by lacZ, and the TK district inserts B '/C type CN54 strain HIV-1 gagpol env gene).
Be about to VTKgpe (TK district inserts the Temple of Heaven strain vaccinia virus recombinant of B '/C type CN54 strain HIV-1 gagpol env gene) and carry out homologous recombination, screen in vain, continuous single spot purifying, identify that acquisition B8R genetically deficient is the recombinant virus VTKgpe Δ B8RlacZ that lacZ replaced through indigo plant with transferring plasmid pSKB8RLacZ cotransfection CEF cell.Behind 0.1 ~ 0.01PFU/cell virus quantity VTKgpe infection, 80% CEF cell absorption 1 ~ 1.5h in blocks, adopt liposome transfection technology (with reference to the American I NVITROGEN Lipofectin of company test kit) with in the recombinant plasmid pSKB8RLacZ transfection CEF cell.Results recombinant virus liquid after 48 hours, with containing 200 μ g/ml X-gal nutrient agar mediums shop spot, picking isolates locus coeruleus virus at random, and 5 generations of continuous single spot purifying are the recombinant virus VTKgpe Δ B8RlacZ that lacZ was replaced with acquisition B8R genetically deficient to be identified
Extract DNA, carry out the stability that pcr amplification and DOT BLOT identify, detect recombinant virus B8R genetically deficient, show that VTKgpe Δ B8RlacZ is stable in the CEF cell cultured continuously 15 generation B8R genetically deficients that go down to posterity.VTKgpe Δ B8RlacZ inserts foreign gene gagpol env stably express in CEF cell cultured continuously 15 generations of going down to posterity.
The AIDS vaccine of the Temple of Heaven strain vaccinia virus recombinant of embodiment four B8R genetically deficients, the antigenic VTKgpe Δ of expression HIV-1 B8R (B8R genetically deficient is not with the lacZ selective marker, and the TK district inserts B '/C type CN54 strain HIV-1 gagpol env gene).
Be that parent plant and pSKB8RNeo cotransfection CEF cell carry out homologous recombination with VTKgpe Δ B8RlacZ again, through the screening of G418 pressure, the screening of blue hickie, continuous single spot purifying, identify and obtain B8R genetically deficient not with the recombinant virus VTKgpe Δ B8R of lacZ selective marker.Behind 0.1 ~ 0.01PFU/cell virus quantity VTKgpe Δ B8RlacZ infection, 80% CEF cell absorption 1 ~ 1.5h in blocks, adopt liposome transfection technology (with reference to the American I NVITROGEN Lipofectin of company test kit) with in the recombinant plasmid pSKB8RNeo transfection CEF cell.Results recombinant virus liquid is pressed the biography three generations with 400ug/mlG418 after 48 hours.With containing 200 μ g/ml X-gal nutrient agar mediums shops spot, the isolated white spot virus of picking at random, 5 generations of continuous single spot purifying, with acquisition B8R genetically deficient to be identified not with the recombinant virus VTKgpe Δ B8R of lacZ selective marker.
Extract DNA, carry out the stability that pcr amplification and DOT BLOT identify, detect recombinant virus B8R genetically deficient, show that VTKgpe Δ B8R is stable in the CEF cell cultured continuously 15 generation B8R genetically deficients that go down to posterity.VTKgpe/ Δ B8R inserts foreign gene gagpolenv stably express in CEF cell cultured continuously 15 generations of going down to posterity.
The nude mice virulence experiment shows that VTKgpe/ Δ B8R virulence significantly reduces than VTKgpe, abdominal cavity inoculation VTT (10
4Pfu) and VTKgpe (10
6Pfu) nude mice death in 15 days, inoculation VTKgpe Δ B8R (10
7Pfu) nude mice survival (table 2).IFN-γ in the born of the same parents. dyeing flow cytometry analysis, T lymphopoiesis, CTL detect and show that B8R genetically deficient significantly improves the Temple of Heaven strain vaccinia virus recombinant specific cellular immunity (Figure 13,14,15) of expressing the HIV-1 gene.
Table 2
Virus???????????Dose(pfu)???????Number?of?death
VTT?????????????10
2????????????0/4
10
3????????????1/4
10
4????????????4/4
VTKgpe??????????10
4????????????0/4
10
5????????????0/4
10
6????????????3/4
VTKgpeΔB
8R????10
5????????????0/4
10
6????????????0/4
10
7????????????0/4
Embodiment five pcr amplifications detect recombinant virus VTKgpe Δ B8R B8R genetically deficient.Westernblot detects VTKgpe Δ B8R 15 generations of going down to posterity and inserts foreign gene gagpol env stably express:
The extraction recombinant virus dna is a template, carries out pcr amplification with primer B8RLF, B8RRR.Recombinant virus VTKgpe Δ B8R is owing to B8R genetically deficient, and pcr amplification goes out the 1244bp fragment, comprises the outer left side of B8R gene homologous sequence B8RL fragment 583bp, right side homologous sequence B8RR fragment 661bp.And contrast VTTDNA is that template amplification goes out the 2Kb fragment, comprises the outer left side of B8R gene homologous sequence B8RL fragment 583bp, B8R gene 818bp, right side homologous sequence B8RR fragment 661bp (Figure 11).The result shows that VTKgpe Δ B8R is in go down to posterity 15 generation B8R stable genes disappearance of CEF cell cultured continuously.Dot Blot detects B8R gene in the viral VTKgpe Δ B8R genome, extracts vaccinia virus VTT752-1DNA and the recombinant virus VTKgpe Δ B8R 15 generation DNA that go down to posterity, and carries out Dot Blot with B8R gene digoxin labelled probe and detects.VTT752-1Dot Blot result shows hybridization trace spot, and the VTKgpe Δ B8R 15 generation Dot Blot results of going down to posterity do not show hybridization trace spot, shows that VTKgpe Δ B8R is in the CEF cell cultured continuously 15 generation B8R stable genes disappearance that goes down to posterity.See Figure 12.VTKgpe Δ B8R infects the CEF cell, harvested cell and supernatant after 48 hours, carrying out WesternBlot with people's polyvalent antibody (NIH's acquired immune deficiency syndrome (AIDS) research and reference reagent project provide) analyzes, special reaction band (gp140, p55) the expression goal gene (Figure 16) that the constructed vaccine of explanation can be correct have appearred.
The effect experiment of embodiment six, vaccinia virus recombinant AIDS vaccine:
Use the effectiveness of 6-8 week BALB/c in age (H-2d) female mice (body weight 19-25 gram is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute) the detection vaccine of the present invention of aseptic raising in this example.Each dna vaccination is prepared into the injection liquid of 1mg/ml with 1 * PBS.Every kind of immune group contains 10 mouse.Preceding 24 hours every mouse left and right sides hind leg tibialis anterior muscles of immunity are injected 50ul 25% (w/v) sucrose hypertonic solution to increase the permeability of muscle cell, improve picked-up plasmid efficient, and simultaneously, sucrose also has the effect of certain adjuvant.After the saccharose treatment 24 hours, tibialis anterior muscle injection DNA 100ug/ is (every hind leg 50ug) only.2 weeks of every interval are with same dosage booster immunization 3 times.Strengthen 10 with VTKgpe Δ B8R, VTKgpe vaccinia virus recombinant respectively for 1,2 group during the 6th week
7The pfu/ mouse.Use VTKgpe Δ B8R, the immunity of VTKgpe vaccinia virus recombinant, 10 respectively for 3,4 groups during the 4th, 6 week
7The pfu/ mouse.When the 9th week, get serum and splenic lymphocyte detection antibody and cytokine.PCDN A empty carrier, the vaccinia virus Tiantan strain (antismallpox vaccine is so kind as to give by Beijing institute of Biological Products) that does not insert goal gene and blank mouse contrast (table 3) are used in contrast.
Table 3
| ??0?week | ??2?week | ??4?week | 6?week | |
| ?DNA+VTKgpeΔ ?B8R | ??pcDNA-syng ??ag， ??pcDNA-ngp1 ??20 | ??pcDNA-synga ??g， ??pcDNA-ngp12 ??0 | ??pcDNA-synga ??g， ??pcDNA-ngp12 ??0 | VTKgpeΔB8R |
| ?VTKgpeΔB8R | ??VTKgpeΔB8R | VTKgpeΔB8R | ||
| ?DNA+VTKgpe | ??pcDNA-syng ??ag， ??pcDNA-ngpl ??20 | ??pcDNA-synga ??g， ??pcDNA-ngp12 ??0 | ??pcDNA-synga ??g， ??pcDNA-ngp12 ??0 | VTKgpe |
| ?VTKgpe | ??VTKgpe | VTKgpe | ||
| ?VTT | ??VTT | VTT | ||
| ?DNA+VTT | ??pcDNA | ??pcDNA | ??pcDNA | VTT |
Related experiment and result are as follows:
1, the detection of serum Gag specific antibody IgG level
For detecting different vaccinia virus recombinants immunity and booster immunization institute inductive specific humoral immunity level separately, the 4th two weeks of immunity back and get mice serum, with Gag P55 antigen (NIH's acquired immune deficiency syndrome (AIDS) research and reference reagent project are so kind as to give) coated elisa plate, indirect elisa method carries out the detection of Gag specific IgG antibodies level.Each immune group Gag specific IgG antibodies titre is listed in table 4.
Table 4
| ?DNA+ ?VTKgpe | ?DNA+ ?VTKgpΔB8R | ?VTKgΔB8R | ?VTKgpe | |
| ?3?week | ?35481 | ?31622 | ?25600 | ?21134 |
| ?4?week | ?31622 | ?51200 | ?25600 | ?25600 |
2, flow cytometer detection bodies exoantigen epitope peptide stimulates the CD8+T lymphocyte of back secretion of gamma-IFN
CD8
+The T lymphocyte is a kind of most important antiviral effect cell, so detected the HIV-1 specific C D8 of secretion of gamma-IFN in the splenic lymphocyte of immune back
+The T lymphocyte.The splenic lymphocyte restricted HIV of MHC-I (gag p24 (AMQILKDTI), V3 (SIRIGPGQTF YATGD) andpol (NPDIVIYQYMDDLYVGSDL, YMDDLYVGSDLEIGQHRTK, KEPPFLWMGYELHPDKWTV) stimulate after 12 hours, IFN-γ and CD8 in the pair cell, the CD3 molecule dyes, and flow cytometer (Beckman Coulter Inc.'s product) is analyzed.DNA+VTKgpe Δ B8R immune group mouse boosting cell T lymphocyte, the CD8 of secretion of gamma-IFN
+The T lymphocyte accounts for the lymphocytic per-cent digital display of the total CD8+T of spleen work and is higher than the DNA+VTKgpe immune group.VTKgpe Δ B8R immune group mouse boosting cell secretion of gamma-IFN .CD8 per-cent digital display work is higher than the VTKgpe immune group.
Embodiment seven, B8R genetically deficient, expression hepatitis B virus HBSAg antigen, the structure of the hepatitis B disease vaccine of the Temple of Heaven strain vaccinia virus recombinant of IL-2 (B8R genetically deficient is inserted HBSAg antigen, and the TK district inserts the IL-2 gene).
Building process: vaccinia virus recombinant VTKIL-2 (TK district insert IL-2 gene) is carried out homologous recombination with transferring plasmid pSKB8RLacZ cotransfection CEF cell, screen in vain, continuous single spot purifying, identify that obtaining B8R genetically deficient is the recombinant virus VTKIL-2 Δ B8RlacZ that lacZ replaced through indigo plant.The HBSAg gene is inserted (the PSKB8RPNeoSaLI enzyme is cut to mend and put down, and the T4DNA ligase enzyme connects HBSAg gene and PSKB8RPNeo) structure plasmid PSKB8RPSNeo among the transferring plasmid PSKB8RPNeo, and the HBSAg gene places under the transferring plasmid vaccinia virus promotor.VTKIL-2 Δ B8RlacZ and PSKB8RPSNeo cotransfection CEF cell are carried out homologous recombination, screen in vain, continuous single spot purifying, identify to obtain the B8R gene through indigo plant, disappearance lacZ is the recombinant virus VTKIL-2 Δ B8RS that the HBSAg gene is replaced.
Concrete grammar is referring to aforesaid method.
Claims (12)
1, a kind of the Temple of Heaven strain vaccinia virus recombinant that is used for the IFN-gamma receptor gene B8R disappearance of homologous recombination construction B8R genetically deficient district insertion foreign gene
VTT Δ B8RlacZ, its construction step is: the structure of transferring plasmid pSKB8R, pSKB8RLacZ, pSKB8RNeo and pSKB8RPNeo; Vaccinia virus Tiantan strain VTT and transferring plasmid pSKB8RLacZ cotransfection CEF cell are carried out homologous recombination, screen in vain, continuous single spot purifying, identify that obtaining B8R genetically deficient is the Temple of Heaven strain vaccinia virus recombinant attenuated carrier VTT Δ B8RlacZ that lacZ replaced through indigo plant.
2, vaccinia virus recombinant VTT Δ B8RlacZ according to claim 1, its transferring plasmid pSKB8RLacZ, it is to insert vaccinia virus promoter sequence pE/L, p7.5 and downstream LacZ sequence between transferring plasmid pSKB8R left side homologous sequence B8RL fragment, right side homologous sequence B8RR fragment, is that template amplification vaccinia virus promoter sequence pE/L, p7.5 and downstream LacZ aligning primer are that pSC65F, pSC65R obtain with plasmid pSC65.
3, vaccinia virus recombinant VTT Δ B8RlacZ according to claim 2, the preserving number of its plasmid pSC65 is: CGMCC No.1097.
4, the described vaccinia virus recombinant VTT of claim 2 Δ B8RlacZ, its transferring plasmid pSKB8R, i.e. left side homologous sequence B8RL fragment, the segmental a kind of transferring plasmid of right side homologous sequence B8RR outside vector plasmid pBR-SK inserts vaccinia virus Tiantan strain B8R gene.
5, vaccinia virus recombinant VTT Δ B8RlacZ according to claim 1, its transferring plasmid pSKB8RNeo are a kind of transferring plasmids that inserts vaccinia virus promoter sequence pH6 and downstream Neo in pSKB8R left side homologous sequence B8RL fragment, the right side homologous sequence B8RR fragment outside.
6, vaccinia virus recombinant VTT Δ B8RlacZ according to claim 1, its transferring plasmid pSKB8RPNeo insert two reverse each other vaccinia virus promoter sequence pE/L, p7.5 and a kind of transferring plasmid of multiple clone site between pSKB8RNeo left side homologous sequence B8RL fragment, right side homologous sequence B8RR fragment.
7, recombinant vaccinia VTT Δ B8RlacZ according to claim 1, relates to the Temple of Heaven strain vaccinia virus recombinant vaccine construction that genetically deficient TK district inserts exogenous gene expression various unit prices of virus or polyvalent antigen gene at its B8R genetically deficient.
8, recombinant vaccinia VTT Δ B8RlacZ according to claim 7, its the Temple of Heaven strain vaccinia virus recombinant vaccine is that a kind of B8R genetically deficient is expressed the antigenic VTKgpe Δ of HIV-1 B8RlacZ, B8R genetically deficient is replaced by lacZ, the TK district inserts the AIDS vaccine of the Temple of Heaven strain vaccinia virus recombinant of B '/C type CN54 strain HIV-1 gagpol env gene, with VTKgpe, the Temple of Heaven strain vaccinia virus recombinant and the transferring plasmid pSKB8RLacZ cotransfection CEF cell that are TK district insertion B '/C type CN54 strain HIV-1 gagpol env gene carry out homologous recombination, screen in vain through indigo plant, continuous single spot purifying, identify that obtaining B8R genetically deficient is the recombinant virus VTKgpe Δ B8RlacZ that lacZ replaced.
9, according to the described recombinant vaccinia VTT of claim 7 Δ B8RlacZ, its the Temple of Heaven strain vaccinia virus recombinant vaccine is a kind of B8R genetically deficient, the antigenic VTKgpe Δ of expression HIV-1 B8R, be that B8R genetically deficient is not with the lacZ selective marker, the TK district inserts the AIDS vaccine of the Temple of Heaven strain vaccinia virus recombinant VTKgpe Δ B8R of B '/C type CN54 strain HIV-1 gagpolenv gene.
10, recombinant vaccinia VTT Δ B8RlacZ according to claim 9, the AIDS vaccine of its vaccinia virus recombinant VTKgpe Δ B8R is the Temple of Heaven strain vaccinia virus recombinant recombinant vaccinia virus VTKgpe CGMCC.NO.1099 of IFN-gamma receptor gene B8R disappearance.
11, recombinant vaccinia VTT Δ B8RlacZ according to claim 7, its B8R genetically deficient, relate to genetically deficient and insert HBSAg antigen, the TK district inserts IL-2 genetic expression hepatitis B virus HBSAg antigen, the hepatitis B disease vaccine of the Temple of Heaven strain vaccinia virus recombinant of IL-2.
12, according to the described recombinant vaccinia VTT of claim 1-11 Δ B8RlacZ, optional wherein application in the vaccinia virus recombinant vaccine medicine of preparation treatment virus disease.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1295339C (en) * | 2005-01-11 | 2007-01-17 | 武汉大学 | Attenuated vaccinia virus Tiantan strain vector and its preparation and application |
| WO2007093134A1 (en) * | 2006-02-16 | 2007-08-23 | Chinese Center For Disease Control And Prevention Center For Aids/Std Control And Prevention | Aids vaccine based on replicative vaccinia virus vector |
| CN100393872C (en) * | 2006-03-09 | 2008-06-11 | 中国疾病预防控制中心病毒病预防控制所 | Reproduction of defect type Tiantan strain vaccinia virus |
| CN102988966A (en) * | 2011-09-09 | 2013-03-27 | 中国疾病预防控制中心性病艾滋病预防控制中心 | Method, vaccine composition and kit for exciting specific mucosal immunoreaction |
| CN106795527A (en) * | 2014-04-01 | 2017-05-31 | 玛丽女王伦敦大学 | Oncolytic virus |
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2004
- 2004-03-10 CN CNA2004100282790A patent/CN1560248A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1295339C (en) * | 2005-01-11 | 2007-01-17 | 武汉大学 | Attenuated vaccinia virus Tiantan strain vector and its preparation and application |
| WO2007093134A1 (en) * | 2006-02-16 | 2007-08-23 | Chinese Center For Disease Control And Prevention Center For Aids/Std Control And Prevention | Aids vaccine based on replicative vaccinia virus vector |
| CN100393872C (en) * | 2006-03-09 | 2008-06-11 | 中国疾病预防控制中心病毒病预防控制所 | Reproduction of defect type Tiantan strain vaccinia virus |
| CN102988966A (en) * | 2011-09-09 | 2013-03-27 | 中国疾病预防控制中心性病艾滋病预防控制中心 | Method, vaccine composition and kit for exciting specific mucosal immunoreaction |
| CN106795527A (en) * | 2014-04-01 | 2017-05-31 | 玛丽女王伦敦大学 | Oncolytic virus |
| CN106795527B (en) * | 2014-04-01 | 2020-10-30 | 玛丽女王伦敦大学 | Oncolytic virus |
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