CSFV E2蛋白主要抗原区域在马克斯克鲁维酵母中的表达及小鼠免疫
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摘要
猪瘟是一种由猪瘟病毒(Classical swine fever virus,CSFV)引起的高度接触传染性和致死性疾病.疫苗预防接种是控制猪瘟病毒传播的重要措施,目前我国使用的弱毒疫苗均为1.1亚型猪瘟病毒兔化弱毒株.随着病毒的不断变异,2.1亚型猪瘟病毒已经逐渐成为我国的主要流行毒株.本研究利用马克斯克鲁维酵母研究了2.1b型CSFV E2蛋白及其截短体mE2(690-916aa)在马克斯克鲁维酵母中的重组表达,发现去除E2蛋白跨膜区域可以显著提高E2蛋白表达水平,mE2的表达量在5L发酵罐中达1.25～2.5g/L.经分子筛纯化,mE2蛋白纯度达90%.纯化的mE2作为抗原蛋白,注射免疫小鼠28d后,可诱导小鼠产生抗CSFV的IgG特异性抗体,表明马克斯克鲁维酵母重组表达的mE2蛋白具有较好的免疫原性.
Classical swine fever,caused by classical swine fever virus(CSFV),is a highly contagious disease and leads to severe fatality of swine.Infection and spread of CSFV can be prevented by prophylactic vaccinations.CSFV vaccines used in China are mainly against CSFV 1.1subtype.However,CSFV 2.1bsubtype has become the predominant epidemic strain in China,due to the rapid evolution of CSFV.In this study,recombinant expression of E2 protein and its truncated form from CSFV 2.1bstrain were obtained in a novel host,Kluyveromyces marxianus.We found that the amount of expression of E2 protein could be significantly inhibited by transmembrane region of E2 protein.High-level expression of mE2,a truncated form of major antigenic protein E2(aa 690—916),was achieved.The protein level of mE2 in the cell lysate reached 1.25—2.5g/L after growing in a5 Lfermentor.After purification by gel-filtration,the purity of the mE2 protein was more than 90%.Purified mE2 was used as antigen protein and injected into mice.Antibodies against E2 could be detected in the serum of mice 28 days after injection.The results suggest purified mE2 displays good immunogenicity and can induce humoral immunity.
Classical swine fever,caused by classical swine fever virus(CSFV),is a highly contagious disease and leads to severe fatality of swine.Infection and spread of CSFV can be prevented by prophylactic vaccinations.CSFV vaccines used in China are mainly against CSFV 1.1subtype.However,CSFV 2.1bsubtype has become the predominant epidemic strain in China,due to the rapid evolution of CSFV.In this study,recombinant expression of E2 protein and its truncated form from CSFV 2.1bstrain were obtained in a novel host,Kluyveromyces marxianus.We found that the amount of expression of E2 protein could be significantly inhibited by transmembrane region of E2 protein.High-level expression of mE2,a truncated form of major antigenic protein E2(aa 690—916),was achieved.The protein level of mE2 in the cell lysate reached 1.25—2.5g/L after growing in a5 Lfermentor.After purification by gel-filtration,the purity of the mE2 protein was more than 90%.Purified mE2 was used as antigen protein and injected into mice.Antibodies against E2 could be detected in the serum of mice 28 days after injection.The results suggest purified mE2 displays good immunogenicity and can induce humoral immunity.
引文
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[2] THIEL H J,MOENNING V.Pestiviruses//Field's Virology[M].Philadelphia,USA:Lippincott Raven,1996,33:1059-1073.
[3] MEYERS G,THIEL H J.Molecular characterization of pestiviruses[J].Advances in Virus Research,1996,47:53-118.
[4] WEILAND F,WEILAND E,UNGER G,et al.Localization of pestiviral envelope proteins E-rns and E2at the cell surface and on isolated particles[J].Journal of General Virology,1999,80:1157-1165.
[5] KONIG M,LENGSFELD T,PAULY T,et al.Classical swine fever virus:Independent induction of protective immunity by two structural glycoproteins[J].Journal of Virology,1995,69:6479-6486.
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[7] PATON D J,MCGOLDRICK A,GREISER-WILKE I,et al.Genetic typing of classical swine fever virus[J].Veterinary Microbiology,2000,73(2/3):137-157.
[8] POSTEL A,MOENNIG V,BECHER P.Classical Swine Fever in Europe-the current Situation[J].Berliner Und Munchener Tierarztliche Wochenschrift,2013,126(11/12):468-475.
[9] POSTEL A,SCHMEISER S,PERERA C L,et al.Classical swine fever virus isolates from Cuba form a new subgenotype 1.4[J].Veterinary Microbiology,2013,161(3/4):334-338.
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[12] TU C,LU Z,LI H,et al.Phylogenetic comparison of classical swine fever virus in China[J].Virus Research,2001,81(1/2):29-37.
[13] LIN G J,LIU T Y,TSENG Y Y,et al.Yeast-expressed classical swine fever virus glycoprotein E2induces a protective immune response[J].Veterinary Microbiology,2009,139(3/4):369-374.
[14] HUA R H,HUO H,LI Y N,et al.Generation and efficacy evaluation of recombinant classical swine fever virus E2glycoprotein expressed in stable transgenic mammalian cell line[J].PLoS One,2014,9(9).
[15] SANCHEZ O,BARRERA M,FARNOS O,et al.Effectiveness of the E2-classical swine fever virus recombinant vaccine produced and formulated within whey from genetically transformed goats[J].Clinical and Vaccine Immunology,2014,21(12):1628-1634.
[16] LANE M M,BURKE N,KARREMAN R,et al.Physiological and metabolic diversity in the yeast Kluyveromyces marxianus[J].Antonie Van Leeuwenhoek,2011,100(4):507-519.
[17] RODICIO R,HEINISCH J J.Yeast on the milky way:Genetics,physiology and biotechnology of Kluyveromyces lactis[J].Yeast,2013,30(5):165-177.
[18] KONDO K,MIURA Y,SONE H,et al.High-level expression of a sweet protein,monellin,in the food yeast Candida utilis[J].Nature Biotechnology,1997,15(5):453-457.
[19] CAI X P,ZHANG J,YUAN H Y,et al.Secretory expression of heterologous protein in Kluyveromyces cicerisporus[J].Applied Microbiology and Biotechnology,2005,67(3):364-369.
[20] ZHANG J,YUAN H,WEN T,et al.Cloning of the KcURA3gene and development of a transformation system for Kluyveromyces cicerisporus[J].Appl Microbiol Biotechnol,2003,62(4):387-391.
[21] SHAO W,LIU S,WU F,et al.Complete genome sequence of a field isolate of classical swine fever virus belonging to subgenotype 2.1bfrom hunan province,China[J].Genome Announc,2015,3(4).
[22] GIBSON D G,YOUNG L,CHUANG R Y,et al.Enzymatic assembly of DNA molecules up to several hundred kilobases[J].Nature Methods,2009,6(5):343-U341.
[23] ANTUNES D F,DE SOUZA C G,DE MORAIS M A.A simple and rapid method for lithium acetatemediated transformation of Kluyveromyces marxianus cells[J].World Journal of Microbiology&Biotechnology,2000,16(7):653-654.
[24] XU Y G,GUAN X T,LIU Z M,et al.Immunogenicity in swine of orally administered recombinant Lactobacillus plantarum expressing classical swine fever virus E2protein in conjunction with thymosin alpha-1as an adjuvant[J].Applied and Environmental Microbiology,2015,81(11):3745-3752.
[25] LUO L,NISHI K,MACLEOD E,et al.Baculovirus expression and antigenic characterization of classical swine fever virus E2proteins[J].Transboundary and Emerging Diseases,2013,60(2):143-151.
[26] CHANG C Y,HUANG C C,LIN Y J,et al.Antigenic domains analysis of classical swine fever virus E2glycoprotein by mutagenesis and conformation-dependent monoclonal antibodies[J].Virus Research,2010,149(2):183-189.
[27] RUMENAPF T,UNGER G,STRAUSS J H,et al.Processing of the envelope glycoproteins of pestiviruses[J].Journal of Virology,1993,67(6):3288-3294.
[28] CHANG C Y,HUANG C C,DENG M C,et al.Identification of conformational epitopes and antigenspecific residues at the D/A domains and the extramembrane C-terminal region of E2glycoprotein of classical swine fever virus[J].Virus Research,2012,168(1-2):56-63.
[29] HULST M M,WESTRA,D F,WENSVOORT G,et al.Glycoprotein E1of hog cholera virus expressed in insect cells protects swine from hog cholera[J].Journal of Virology,1993,67:5435-5442.
[30]贺番,王靖飞,郝永清,等.猪瘟病毒囊膜蛋白E2的锚定序列部分缺失对其在细胞内表达的影响[J].中国预防兽医学报,2010,5:342-346.
[31] GONG W J,WU J,LU Z J,et al.Genetic diversity of subgenotype 2.1isolates of classical swine fever virus[J].Infection,Genetics and Evolution,2016,41:218-226
[32]黄耀华.猪瘟病毒分子流行病学研究及流行毒株全基因组序列分析[D].海口:海南大学,2015.
[33] JIANG D L,GONG W J,LI R C,et al.Phylogenetic analysis using E2gene of classical swine fever virus reveals a new subgenotype in China[J].Infection,Genetics and Evolution,2013,17:231-238.
[34] ZHANG H L,LENG C,FENG L P,et al.A new subgenotype 2.1disolates of classical swine fever virus in China,2014[J].Infection,Genetics and Evolution,2015,34:94-105.
[35]吕永智.猪瘟E2亚单位疫苗研究进展[J].家畜生态学报,2017,38(5):86-90.
[36] RISATTI G R,HOLINKA L G,FERNANDEZ SAINZ I,et al.N-linked glycosylation status of classical swine fever virus strain Brescia E2glycoprotein influences virulence in swine[J].Journal of Virology,2007,81(2):924-933.
[37] GAVRILOV B K,ROGERS K,FERNANDEZ-SAINZ I J,et al.Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins[J].Virology,2011,420(2):135-145.
[38] SCHWARZ F,AEBI M.Mechanisms and principles of N-linked protein glycosylation[J].Current Opinion in Structural Biology,2011,21(5):576-582.
[39] LEE S J,EVERS S,ROEDER D,et al.Mannose receptor-mediated regulation of serum glycoprotein homeostasis[J].Science,2002,295(5561):1898-1901.
[40] GEMMILL T R,TRIMBLE R B.Overview of N-and O-linked oligosaccharide structures found in various yeast species[J].Biochimica et Biophysica Acta-General Subjects,1999,1426(2):227-237.
[41]吴玥,周峻岗,吕红.信号肽对木聚糖酶在马克斯克鲁维酵母中分泌表达的影响[J].复旦学报(自然科学版),2017,56(4):446-454.
[42]余兴龙,涂长春,徐兴然,等.猪瘟病毒E2基因的定点突变、在大肠杆菌中的高效表达及表达产物的免疫原性[J].生物工程学报,2003,19(4):439-443.
[43] ZHOU B,LIU K,JIANG Y,et al.Multiple linear B-cell epitopes of classical swine fever virus glycoprotein E2expressed in E.coli as multiple epitope vaccine induces a protective immune response[J].Virology Journal,2011,8:378.
[44]董炳梅,孙全文,王爱华,等.N-糖基化猪瘟病毒E2蛋白不同接种剂量对BALB/c小鼠的免疫效果分析[J].中国兽医学报,2014,34(8):1225-1230.
[2] THIEL H J,MOENNING V.Pestiviruses//Field's Virology[M].Philadelphia,USA:Lippincott Raven,1996,33:1059-1073.
[3] MEYERS G,THIEL H J.Molecular characterization of pestiviruses[J].Advances in Virus Research,1996,47:53-118.
[4] WEILAND F,WEILAND E,UNGER G,et al.Localization of pestiviral envelope proteins E-rns and E2at the cell surface and on isolated particles[J].Journal of General Virology,1999,80:1157-1165.
[5] KONIG M,LENGSFELD T,PAULY T,et al.Classical swine fever virus:Independent induction of protective immunity by two structural glycoproteins[J].Journal of Virology,1995,69:6479-6486.
[6] VAN GENNIP H G P,VAN RIJN P A,WIDJOJOATMODJO M N,et al.Chimeric classical swine fever viruses containing envelope protein E-RNS or E2of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response[J].Vaccine,2000,19(4/5):447-459.
[7] PATON D J,MCGOLDRICK A,GREISER-WILKE I,et al.Genetic typing of classical swine fever virus[J].Veterinary Microbiology,2000,73(2/3):137-157.
[8] POSTEL A,MOENNIG V,BECHER P.Classical Swine Fever in Europe-the current Situation[J].Berliner Und Munchener Tierarztliche Wochenschrift,2013,126(11/12):468-475.
[9] POSTEL A,SCHMEISER S,PERERA C L,et al.Classical swine fever virus isolates from Cuba form a new subgenotype 1.4[J].Veterinary Microbiology,2013,161(3/4):334-338.
[10] DE SMIT A J,BOUMA A,VAN GENNIP H G P,et al.Chimeric(marker)C-strain viruses induce clinical protection against virulent classical swine fever virus(CSFV)and reduce transmission of CSFV between vaccinated pigs[J].Vaccine,2001,19(11/12):1467-1476.
[11] EDWARDS S,FUKUSHO A,LEFEVRE P C,et al.Classical swine fever:The global situation[J].Veterinary Microbiology,2000,73(2/3):103-119.
[12] TU C,LU Z,LI H,et al.Phylogenetic comparison of classical swine fever virus in China[J].Virus Research,2001,81(1/2):29-37.
[13] LIN G J,LIU T Y,TSENG Y Y,et al.Yeast-expressed classical swine fever virus glycoprotein E2induces a protective immune response[J].Veterinary Microbiology,2009,139(3/4):369-374.
[14] HUA R H,HUO H,LI Y N,et al.Generation and efficacy evaluation of recombinant classical swine fever virus E2glycoprotein expressed in stable transgenic mammalian cell line[J].PLoS One,2014,9(9).
[15] SANCHEZ O,BARRERA M,FARNOS O,et al.Effectiveness of the E2-classical swine fever virus recombinant vaccine produced and formulated within whey from genetically transformed goats[J].Clinical and Vaccine Immunology,2014,21(12):1628-1634.
[16] LANE M M,BURKE N,KARREMAN R,et al.Physiological and metabolic diversity in the yeast Kluyveromyces marxianus[J].Antonie Van Leeuwenhoek,2011,100(4):507-519.
[17] RODICIO R,HEINISCH J J.Yeast on the milky way:Genetics,physiology and biotechnology of Kluyveromyces lactis[J].Yeast,2013,30(5):165-177.
[18] KONDO K,MIURA Y,SONE H,et al.High-level expression of a sweet protein,monellin,in the food yeast Candida utilis[J].Nature Biotechnology,1997,15(5):453-457.
[19] CAI X P,ZHANG J,YUAN H Y,et al.Secretory expression of heterologous protein in Kluyveromyces cicerisporus[J].Applied Microbiology and Biotechnology,2005,67(3):364-369.
[20] ZHANG J,YUAN H,WEN T,et al.Cloning of the KcURA3gene and development of a transformation system for Kluyveromyces cicerisporus[J].Appl Microbiol Biotechnol,2003,62(4):387-391.
[21] SHAO W,LIU S,WU F,et al.Complete genome sequence of a field isolate of classical swine fever virus belonging to subgenotype 2.1bfrom hunan province,China[J].Genome Announc,2015,3(4).
[22] GIBSON D G,YOUNG L,CHUANG R Y,et al.Enzymatic assembly of DNA molecules up to several hundred kilobases[J].Nature Methods,2009,6(5):343-U341.
[23] ANTUNES D F,DE SOUZA C G,DE MORAIS M A.A simple and rapid method for lithium acetatemediated transformation of Kluyveromyces marxianus cells[J].World Journal of Microbiology&Biotechnology,2000,16(7):653-654.
[24] XU Y G,GUAN X T,LIU Z M,et al.Immunogenicity in swine of orally administered recombinant Lactobacillus plantarum expressing classical swine fever virus E2protein in conjunction with thymosin alpha-1as an adjuvant[J].Applied and Environmental Microbiology,2015,81(11):3745-3752.
[25] LUO L,NISHI K,MACLEOD E,et al.Baculovirus expression and antigenic characterization of classical swine fever virus E2proteins[J].Transboundary and Emerging Diseases,2013,60(2):143-151.
[26] CHANG C Y,HUANG C C,LIN Y J,et al.Antigenic domains analysis of classical swine fever virus E2glycoprotein by mutagenesis and conformation-dependent monoclonal antibodies[J].Virus Research,2010,149(2):183-189.
[27] RUMENAPF T,UNGER G,STRAUSS J H,et al.Processing of the envelope glycoproteins of pestiviruses[J].Journal of Virology,1993,67(6):3288-3294.
[28] CHANG C Y,HUANG C C,DENG M C,et al.Identification of conformational epitopes and antigenspecific residues at the D/A domains and the extramembrane C-terminal region of E2glycoprotein of classical swine fever virus[J].Virus Research,2012,168(1-2):56-63.
[29] HULST M M,WESTRA,D F,WENSVOORT G,et al.Glycoprotein E1of hog cholera virus expressed in insect cells protects swine from hog cholera[J].Journal of Virology,1993,67:5435-5442.
[30]贺番,王靖飞,郝永清,等.猪瘟病毒囊膜蛋白E2的锚定序列部分缺失对其在细胞内表达的影响[J].中国预防兽医学报,2010,5:342-346.
[31] GONG W J,WU J,LU Z J,et al.Genetic diversity of subgenotype 2.1isolates of classical swine fever virus[J].Infection,Genetics and Evolution,2016,41:218-226
[32]黄耀华.猪瘟病毒分子流行病学研究及流行毒株全基因组序列分析[D].海口:海南大学,2015.
[33] JIANG D L,GONG W J,LI R C,et al.Phylogenetic analysis using E2gene of classical swine fever virus reveals a new subgenotype in China[J].Infection,Genetics and Evolution,2013,17:231-238.
[34] ZHANG H L,LENG C,FENG L P,et al.A new subgenotype 2.1disolates of classical swine fever virus in China,2014[J].Infection,Genetics and Evolution,2015,34:94-105.
[35]吕永智.猪瘟E2亚单位疫苗研究进展[J].家畜生态学报,2017,38(5):86-90.
[36] RISATTI G R,HOLINKA L G,FERNANDEZ SAINZ I,et al.N-linked glycosylation status of classical swine fever virus strain Brescia E2glycoprotein influences virulence in swine[J].Journal of Virology,2007,81(2):924-933.
[37] GAVRILOV B K,ROGERS K,FERNANDEZ-SAINZ I J,et al.Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins[J].Virology,2011,420(2):135-145.
[38] SCHWARZ F,AEBI M.Mechanisms and principles of N-linked protein glycosylation[J].Current Opinion in Structural Biology,2011,21(5):576-582.
[39] LEE S J,EVERS S,ROEDER D,et al.Mannose receptor-mediated regulation of serum glycoprotein homeostasis[J].Science,2002,295(5561):1898-1901.
[40] GEMMILL T R,TRIMBLE R B.Overview of N-and O-linked oligosaccharide structures found in various yeast species[J].Biochimica et Biophysica Acta-General Subjects,1999,1426(2):227-237.
[41]吴玥,周峻岗,吕红.信号肽对木聚糖酶在马克斯克鲁维酵母中分泌表达的影响[J].复旦学报(自然科学版),2017,56(4):446-454.
[42]余兴龙,涂长春,徐兴然,等.猪瘟病毒E2基因的定点突变、在大肠杆菌中的高效表达及表达产物的免疫原性[J].生物工程学报,2003,19(4):439-443.
[43] ZHOU B,LIU K,JIANG Y,et al.Multiple linear B-cell epitopes of classical swine fever virus glycoprotein E2expressed in E.coli as multiple epitope vaccine induces a protective immune response[J].Virology Journal,2011,8:378.
[44]董炳梅,孙全文,王爱华,等.N-糖基化猪瘟病毒E2蛋白不同接种剂量对BALB/c小鼠的免疫效果分析[J].中国兽医学报,2014,34(8):1225-1230.
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