表达绿色荧光蛋白的MVA重组毒株的构建及筛选
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摘要
为了构建及筛选表达绿色荧光蛋白(GFP)的改良型痘苗病毒安卡拉(MVA)重组毒株,并对其进行鉴定,本研究基于同源重组原理设计引物,通过PCR扩增获取含有MVA两侧同源臂的外源基因GFP片段,将GFP基因片段转染到感染了MVA的CEF细胞中使之同源重组到MVA ORF086-087位点(基因组中70303-70304 bp之间),利用倒置荧光显微镜观察并标记表达GFP的单个噬斑,筛选获取重组毒株,应用倒置荧光技术、PCR及Western blotting对该重组毒株进行鉴定。结果显示,经过3轮噬斑筛选,在倒置荧光显微镜下可观察到大量表达GFP的单个噬斑,PCR扩增检测结果表明目的基因已成功整合到重组毒株MVA-GFP中。Western blotting结果表明,GFP在感染的细胞内成功表达。本研究利用基因工程技术成功获得表达GFP的重组毒株MVA-GFP,可为进一步将其他抗原基因插入GFP位点中筛选无标记的重组毒株及疫苗研究提供材料。
To construct and screen the recombinant modified vaccinia virus Ankara(MVA) strain expressing green fluorescent protein(GFP),primers were designed and synthesized.GFP gene was amplified by PCR and inserted into the ORF086-087 site(70303-70304 bp) of MVA based on the principle of homologous recombination.Fluorescence microscope was used to select single plaque with GFP as the reporter gene,and the recombinant strain was identified by fluorescence technique,PCR and Western blotting.The results showed that a large number of single plaques expressing GFP could be observed under fluorescence microscope,GFP gene successfully intergrated into recombinant strain MVA-GFP genome.Western blotting result showed that GFP was successfully expressed in infected cell.In this study,the recombinant strain MVA-GFP expressing GFP was constructed by genetic engineering technology,which provided materials for further screening unmarked recombinant strain and vaccines by inserting foreign target genes into GFP site.
To construct and screen the recombinant modified vaccinia virus Ankara(MVA) strain expressing green fluorescent protein(GFP),primers were designed and synthesized.GFP gene was amplified by PCR and inserted into the ORF086-087 site(70303-70304 bp) of MVA based on the principle of homologous recombination.Fluorescence microscope was used to select single plaque with GFP as the reporter gene,and the recombinant strain was identified by fluorescence technique,PCR and Western blotting.The results showed that a large number of single plaques expressing GFP could be observed under fluorescence microscope,GFP gene successfully intergrated into recombinant strain MVA-GFP genome.Western blotting result showed that GFP was successfully expressed in infected cell.In this study,the recombinant strain MVA-GFP expressing GFP was constructed by genetic engineering technology,which provided materials for further screening unmarked recombinant strain and vaccines by inserting foreign target genes into GFP site.
引文
[1] HENDERSON D A.The eradication of smallpox——An overview of the past,present,and future[J].Vaccine,2011,29(12):D7-D9.
[2] HALL A.The global eradication of smallpox.Final report of the global commission for the certification of smallpoxeradication[J].Postgraduate Medical Journal,1980,58(685):1.
[3] ANTOINE G,SCHEIFLINGER F,DORNER F,et al.The complete genomic sequence of the modified vaccinia Ankara strain:Comparison with other orthopoxviruses[J].Virology,1998,244(2):365-396.
[4] MEYER H,SUTTER G,MAYR A.Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence[J].Journal of General Virology,1991,72(5):1031-1038.
[5] VOLZ A,SUTTER G.Modified vaccinia virus Ankara:History,value in basic research,and current perspectives for vaccine development[J].Advances in Virus Research,2017,97:187-243.
[6] EADY C C,WELD R J,LISTER C E.Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.)[J].Plant Cell Reports,2000,19(4):376-381.
[7] GUBIN A,REDDY B,MILLER J.Long-term,stable expression of green fluorescent protein in mammalian cells[J].Biochemical & Biophysical Research Communications,1997,236(2):347-350.
[8] RADE J J,CHEUNG M,MIYAMOTO S,et al.Retroviral vector-mediated expression of hirudin by human vascular endothelial cells:Implications for the design of retroviral vectors expressing biologically active proteins[J].Gene Therapy,1999,6(3):385-392.
[9] CHALFIE M,TU Y,EUSKIRCHEN G,et al.Green fluorescent protein as a marker for gene expression[J].Science,1994,263(5148):802-805.
[10] ARNIM A G V,DENG X W,STACEY M G.Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants[J].Gene,1998,221(1):35-43.
[11] VAN DER GEEST A H M,PETOLINO J F.Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny[J].Plant Cell Reports,1998,17(10):760-764.
[12] ZHANG H X,ZEEVAART J A D.An efficient Agrobacterium tumefaciens-mediated transformation and regeneration system for cotyledons of spinach (Spinacia oleracea L.)[J].Plant Cell Reports,1999,18(7-8):640-645.
[13] PONAPPA T,BRZOZOWSKI A E,FINER J J.Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein[J].Plant Cell Reports,1999,19(1):6-12.
[14] YOSHIMIZU T,SUGIYAMA N,DE F M,et al.Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice[J].Development Growth & Differentiation,2010,41(6):675-684.
[15] JONES C A,HURLEY M I,BLACK T A,et al.Expression of a renin/GFP transgene in mouse embryonic,extra-embryonic,and adult tissues[J].Physiological Genomics,2000,4(1):75-81.
[16] GONG Z,JU B,WAN H.Green fluorescent protein (GFP) transgenic fish and their applications[J].Genetica,2001,111(1-3):213-225.
[17] SEITA Y,TSUKIYAMA T,IWATANI C,et al.Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body[J].Scientific Reports,2016,6:24868.
[18] BORRáS T,GABELT B T,KLINTWORTH G K,et al.Non-invasive observation of repeated adenoviral GFP gene delivery to the anterior segment of the monkey eye in vivo[J].Journal of Gene Medicine,2001,3(5):437-449.
[19] ISHIDA Y,SAITO H S,HIEI Y,et al.High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens[J].Nature Biotechnology,1996,14(6):745-750.
[20] CASPER S J,HOLT C A.Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vector[J].Gene,1996,173(1):69-73.
[21] CRUZ S S,CHAPMAN S,ROBERTS A G,et al.Assembly and movement of a plant virus carrying a green fluorescent protein overcoat[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(13):6286-6290.
[22] STRIEPEN B,HE C,SOLDATI M,et al.Expression,selection,and organellar targeting of the green fluorescent protein in Toxoplasma gondii[J].Molecular and Biochemical Parasitology,1998,92(2):325-338.
[23] HA D S,SCHWARZ J K,TURCO S J,et al.Use of the green fluorescent protein as a marker in transfected Leishmania[J].Molecular and Biochemical Parasitology,1996,77(1):57-64.
[24] ORTIN-MARTINEZ A,TSAI E L,NICKERSON P E,et al.A reinterpretation of cell transplantation:GFP transfer from donor to host photoreceptors[J].Stem Cells,2017,35:932-939.
[25] CAMPBELL A S,PLOETZ R C,ROLLINS J A.Comparing avocado,swamp bay,and camphortree as hosts of Raffaelea lauricola using a green fluorescent protein (GFP)-labeled strain of the pathogen[J].Phytopathology,2017,107(1):70-74.
[26] 李吉霞,葛秀国,王轶敏,等.绿色荧光蛋白标记牛乳腺上皮细胞生长特征和超微结构观察[J].中国畜牧兽医,2013,40(5):115-119.LI J X,GE X G,WANG Y M,et al.Observation of growth characteristics and ultrastructure in GFP-labeled bovine mammary epithelialcells[J].China Animal Husbandry & Veterinary Medicine,2013,40(5):115-119.(in Chinese)
[27] 刘翠,殷相平.携带GFP基因的重组狂犬病病毒的拯救及其生物学特性研究[J].中国畜牧兽医,2018,45(7):1965-1971.LIU C,YIN X P.Rescue and biological characteristics analysis of recombinant rabies virus containing GFP gene[J].China Animal Husbandry & Veterinary Medicine,2018,45(7):1965-1971.(in Chinese)
[28] WALSH E P,BARON M D,ANDERSON J,et al.Development of a genetically marked recombinant rinderpest vaccine expressing green fluorescent protein[J].Journal of General Virology,2000,81(3):709-718.
[29] WALSH E P,BARON M D,RENNIE L F,et al.Recombinant rinderpest vaccines expressing membrane-anchored proteins as genetic markers:Evidence of exclusion of marker protein from the virus envelope[J].Journal of Virology,2000,74(21):10165-10175.
[30] JAHRLING P B,FRITZ E A,HENSLEY L E.Countermeasures to the bioterrorist threat of smallpox[J].Current Molecular Medicine,2005,5(8):817-826.
[31] SLIVA K,SCHNIERLE B.From actually toxic to highly specific——Novel drugs against poxviruses[J].Virology Journal,2007,4:8.
[32] VOIGT E A,KENNEDY R B,POLAND G A.Defending against smallpox:A focus on vaccines[J].Expert Review of Vaccines,2016,15(9):1197-1211.
[33] REYNOLDS M G,DAVIDSON W B,CURNS A T,et al.Spectrum of infection and risk factors for human Monkeypox,United States,2003[J].Emerging Infectious Diseases,2007,13(9):1332-1339.
[34] JACKSON R,RAMSHAW I.The mousepox experience[J].EMBO Reports,2010,11(1):18-24.
[35] PAOLETTI E,LIPINSKAS B R,SAMSONOFF C,et al.Construction of live vaccines using genetically engineered poxviruses:Biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D[J].Proceedings of the National Academy of Sciences of the United States of America,1984,81(1):193-197.
[36] SMITH G L,MACKETT M,MOSS B.Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen[J].Nature,1983,302(5908):490-495.
[37] BENNINK J R,YEWDELL J W,SMITH G L,et al.Recombinant vaccinia virus primes and stimulates influenza haemagglutinin-specific cytotoxic T cells[J].Nature,1984,311(5986):578-579.
[38] WINSLOW R L,MILLIGAN I D,VOYSEY M,et al.Immune responses to novel adenovirus type 26 and modified vaccinia virus Ankara-vectored ebola vaccines at 1 year[J].The Journal of American Medical Association,2017,317(10):1075-1077.
[39] DOMI A,FELDMANN F,BASU R,et al.A single dose of modified vaccinia Ankara expressing Ebola virus like particles protects nonhuman primates from lethal Ebola virus challenge[J].Scientific Reports,2018,8(1):864.
[40] JULANDER J G,TESTORI M,CHEMINAY C,et al.Immunogenicity and protection after vaccination with a modified vaccinia virus Ankara-vectored yellow fever vaccine in the hamster model[J].Frontiers in Immunology,2018,9:1756.
[41] MACKETT M,SMITH G L,MOSS B.Vaccinia virus:A selectable eukaryotic cloning and expression vector[J].Proceedings of the National Academy of Sciences,1982,79(23):7415-7419.
[42] PANICALI D,PAOLETTI E.Construction of poxviruses as cloning vectors:Insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus[J].Proceedings of the National Academy of Sciences of the United States of America,1982,79(16):4927-4931.
[43] BLANCOU J,KIENY M P,LATHE R,et al.Oral vaccination of the fox against rabies using a live recombinant vaccinia virus[J].Nature,1986,322(6077):373-375.
[44] STAIB C,DREXLER I,SUTTER G.Construction and isolation of recombinant MVA[J].Methods in Molecular Biology,2004,269:77-100.
[45] KREIJTZ J H,GOEIJENBIER M,MOESKER F M,et al.Safety and immunogenicity of a modified-vaccinia-virus-Ankara-based influenza A H5N1 vaccine:A randomised,double-blind phase 1/2a clinical trial[J].The Lancet Infectious Diseases,2014,14(12):1196-1207.
[46] VOLZ A,LIM S,KASERER M,et al.Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens[J].Vaccine,2016,34(16):1915-1926.
[47] XIAO Y L,REDMAN J C,MONAGHAN E L,et al.High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns[J].Plant Methods,2010,6(1):18.
[48] HASHIMOTO H,MIZUSHIMA T,CHIJIWA T,et al.Efficient production of recombinant adeno-associated viral vector,serotype DJ/8,carrying the GFP gene[J].Virus Research,2017,238:63-68.
[49] DE JONG J,HOFIUS D,HENNIG L.Salicylic acid interferes with GFP fluorescence in vivo[J].Journal of Experimental Botany,2017,68(7):1689-1696.
[50] CHOI J Y,JANG T H,PARK H H.The mechanism of folding robustness revealed by the crystal structure of extra-superfolder GFP[J].FEBS Letters,2017,591(2):442-447.
[51] GMEZ C E,PERDIGUERO B,GARCíA-ARRIAZA J,et al.Clinical applications of attenuated MVA poxvirus strain[J].Expert Review of Vaccines,2013,12(12):1395-1416.
[2] HALL A.The global eradication of smallpox.Final report of the global commission for the certification of smallpoxeradication[J].Postgraduate Medical Journal,1980,58(685):1.
[3] ANTOINE G,SCHEIFLINGER F,DORNER F,et al.The complete genomic sequence of the modified vaccinia Ankara strain:Comparison with other orthopoxviruses[J].Virology,1998,244(2):365-396.
[4] MEYER H,SUTTER G,MAYR A.Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence[J].Journal of General Virology,1991,72(5):1031-1038.
[5] VOLZ A,SUTTER G.Modified vaccinia virus Ankara:History,value in basic research,and current perspectives for vaccine development[J].Advances in Virus Research,2017,97:187-243.
[6] EADY C C,WELD R J,LISTER C E.Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.)[J].Plant Cell Reports,2000,19(4):376-381.
[7] GUBIN A,REDDY B,MILLER J.Long-term,stable expression of green fluorescent protein in mammalian cells[J].Biochemical & Biophysical Research Communications,1997,236(2):347-350.
[8] RADE J J,CHEUNG M,MIYAMOTO S,et al.Retroviral vector-mediated expression of hirudin by human vascular endothelial cells:Implications for the design of retroviral vectors expressing biologically active proteins[J].Gene Therapy,1999,6(3):385-392.
[9] CHALFIE M,TU Y,EUSKIRCHEN G,et al.Green fluorescent protein as a marker for gene expression[J].Science,1994,263(5148):802-805.
[10] ARNIM A G V,DENG X W,STACEY M G.Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants[J].Gene,1998,221(1):35-43.
[11] VAN DER GEEST A H M,PETOLINO J F.Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny[J].Plant Cell Reports,1998,17(10):760-764.
[12] ZHANG H X,ZEEVAART J A D.An efficient Agrobacterium tumefaciens-mediated transformation and regeneration system for cotyledons of spinach (Spinacia oleracea L.)[J].Plant Cell Reports,1999,18(7-8):640-645.
[13] PONAPPA T,BRZOZOWSKI A E,FINER J J.Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein[J].Plant Cell Reports,1999,19(1):6-12.
[14] YOSHIMIZU T,SUGIYAMA N,DE F M,et al.Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice[J].Development Growth & Differentiation,2010,41(6):675-684.
[15] JONES C A,HURLEY M I,BLACK T A,et al.Expression of a renin/GFP transgene in mouse embryonic,extra-embryonic,and adult tissues[J].Physiological Genomics,2000,4(1):75-81.
[16] GONG Z,JU B,WAN H.Green fluorescent protein (GFP) transgenic fish and their applications[J].Genetica,2001,111(1-3):213-225.
[17] SEITA Y,TSUKIYAMA T,IWATANI C,et al.Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body[J].Scientific Reports,2016,6:24868.
[18] BORRáS T,GABELT B T,KLINTWORTH G K,et al.Non-invasive observation of repeated adenoviral GFP gene delivery to the anterior segment of the monkey eye in vivo[J].Journal of Gene Medicine,2001,3(5):437-449.
[19] ISHIDA Y,SAITO H S,HIEI Y,et al.High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens[J].Nature Biotechnology,1996,14(6):745-750.
[20] CASPER S J,HOLT C A.Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vector[J].Gene,1996,173(1):69-73.
[21] CRUZ S S,CHAPMAN S,ROBERTS A G,et al.Assembly and movement of a plant virus carrying a green fluorescent protein overcoat[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(13):6286-6290.
[22] STRIEPEN B,HE C,SOLDATI M,et al.Expression,selection,and organellar targeting of the green fluorescent protein in Toxoplasma gondii[J].Molecular and Biochemical Parasitology,1998,92(2):325-338.
[23] HA D S,SCHWARZ J K,TURCO S J,et al.Use of the green fluorescent protein as a marker in transfected Leishmania[J].Molecular and Biochemical Parasitology,1996,77(1):57-64.
[24] ORTIN-MARTINEZ A,TSAI E L,NICKERSON P E,et al.A reinterpretation of cell transplantation:GFP transfer from donor to host photoreceptors[J].Stem Cells,2017,35:932-939.
[25] CAMPBELL A S,PLOETZ R C,ROLLINS J A.Comparing avocado,swamp bay,and camphortree as hosts of Raffaelea lauricola using a green fluorescent protein (GFP)-labeled strain of the pathogen[J].Phytopathology,2017,107(1):70-74.
[26] 李吉霞,葛秀国,王轶敏,等.绿色荧光蛋白标记牛乳腺上皮细胞生长特征和超微结构观察[J].中国畜牧兽医,2013,40(5):115-119.LI J X,GE X G,WANG Y M,et al.Observation of growth characteristics and ultrastructure in GFP-labeled bovine mammary epithelialcells[J].China Animal Husbandry & Veterinary Medicine,2013,40(5):115-119.(in Chinese)
[27] 刘翠,殷相平.携带GFP基因的重组狂犬病病毒的拯救及其生物学特性研究[J].中国畜牧兽医,2018,45(7):1965-1971.LIU C,YIN X P.Rescue and biological characteristics analysis of recombinant rabies virus containing GFP gene[J].China Animal Husbandry & Veterinary Medicine,2018,45(7):1965-1971.(in Chinese)
[28] WALSH E P,BARON M D,ANDERSON J,et al.Development of a genetically marked recombinant rinderpest vaccine expressing green fluorescent protein[J].Journal of General Virology,2000,81(3):709-718.
[29] WALSH E P,BARON M D,RENNIE L F,et al.Recombinant rinderpest vaccines expressing membrane-anchored proteins as genetic markers:Evidence of exclusion of marker protein from the virus envelope[J].Journal of Virology,2000,74(21):10165-10175.
[30] JAHRLING P B,FRITZ E A,HENSLEY L E.Countermeasures to the bioterrorist threat of smallpox[J].Current Molecular Medicine,2005,5(8):817-826.
[31] SLIVA K,SCHNIERLE B.From actually toxic to highly specific——Novel drugs against poxviruses[J].Virology Journal,2007,4:8.
[32] VOIGT E A,KENNEDY R B,POLAND G A.Defending against smallpox:A focus on vaccines[J].Expert Review of Vaccines,2016,15(9):1197-1211.
[33] REYNOLDS M G,DAVIDSON W B,CURNS A T,et al.Spectrum of infection and risk factors for human Monkeypox,United States,2003[J].Emerging Infectious Diseases,2007,13(9):1332-1339.
[34] JACKSON R,RAMSHAW I.The mousepox experience[J].EMBO Reports,2010,11(1):18-24.
[35] PAOLETTI E,LIPINSKAS B R,SAMSONOFF C,et al.Construction of live vaccines using genetically engineered poxviruses:Biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D[J].Proceedings of the National Academy of Sciences of the United States of America,1984,81(1):193-197.
[36] SMITH G L,MACKETT M,MOSS B.Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen[J].Nature,1983,302(5908):490-495.
[37] BENNINK J R,YEWDELL J W,SMITH G L,et al.Recombinant vaccinia virus primes and stimulates influenza haemagglutinin-specific cytotoxic T cells[J].Nature,1984,311(5986):578-579.
[38] WINSLOW R L,MILLIGAN I D,VOYSEY M,et al.Immune responses to novel adenovirus type 26 and modified vaccinia virus Ankara-vectored ebola vaccines at 1 year[J].The Journal of American Medical Association,2017,317(10):1075-1077.
[39] DOMI A,FELDMANN F,BASU R,et al.A single dose of modified vaccinia Ankara expressing Ebola virus like particles protects nonhuman primates from lethal Ebola virus challenge[J].Scientific Reports,2018,8(1):864.
[40] JULANDER J G,TESTORI M,CHEMINAY C,et al.Immunogenicity and protection after vaccination with a modified vaccinia virus Ankara-vectored yellow fever vaccine in the hamster model[J].Frontiers in Immunology,2018,9:1756.
[41] MACKETT M,SMITH G L,MOSS B.Vaccinia virus:A selectable eukaryotic cloning and expression vector[J].Proceedings of the National Academy of Sciences,1982,79(23):7415-7419.
[42] PANICALI D,PAOLETTI E.Construction of poxviruses as cloning vectors:Insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus[J].Proceedings of the National Academy of Sciences of the United States of America,1982,79(16):4927-4931.
[43] BLANCOU J,KIENY M P,LATHE R,et al.Oral vaccination of the fox against rabies using a live recombinant vaccinia virus[J].Nature,1986,322(6077):373-375.
[44] STAIB C,DREXLER I,SUTTER G.Construction and isolation of recombinant MVA[J].Methods in Molecular Biology,2004,269:77-100.
[45] KREIJTZ J H,GOEIJENBIER M,MOESKER F M,et al.Safety and immunogenicity of a modified-vaccinia-virus-Ankara-based influenza A H5N1 vaccine:A randomised,double-blind phase 1/2a clinical trial[J].The Lancet Infectious Diseases,2014,14(12):1196-1207.
[46] VOLZ A,LIM S,KASERER M,et al.Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens[J].Vaccine,2016,34(16):1915-1926.
[47] XIAO Y L,REDMAN J C,MONAGHAN E L,et al.High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns[J].Plant Methods,2010,6(1):18.
[48] HASHIMOTO H,MIZUSHIMA T,CHIJIWA T,et al.Efficient production of recombinant adeno-associated viral vector,serotype DJ/8,carrying the GFP gene[J].Virus Research,2017,238:63-68.
[49] DE JONG J,HOFIUS D,HENNIG L.Salicylic acid interferes with GFP fluorescence in vivo[J].Journal of Experimental Botany,2017,68(7):1689-1696.
[50] CHOI J Y,JANG T H,PARK H H.The mechanism of folding robustness revealed by the crystal structure of extra-superfolder GFP[J].FEBS Letters,2017,591(2):442-447.
[51] GMEZ C E,PERDIGUERO B,GARCíA-ARRIAZA J,et al.Clinical applications of attenuated MVA poxvirus strain[J].Expert Review of Vaccines,2013,12(12):1395-1416.