犬圆环病毒Rep蛋白的原核表达
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摘要
犬圆环病毒(CanineCV)是一种新发现的圆环病毒。制备针对CaineCV Rep的抗体,为建立CaineCV ELISA检测方法及研究Rep蛋白的功能奠定基础。以CaineCV基因组作为模板,扩增出Rep基因序列。PCR产物克隆至pET-28a原核表达载体上,获得了重组质粒pET(28a)-Rep。将重组质粒转入大肠埃希菌BL21感受态细胞后进行诱导表达,SDS-PAGE分析表明重组Rep蛋白在大肠埃希菌BL21中获得表达。Western blot分析表明,重组蛋白能与Anti-His标签抗体发生特异性反应。该研究构建了pET(28a)-Rep重组原核表达质粒,并在大肠埃希菌中获得表达,为进一步制备Rep的抗体奠定了基础。
Canine circovirus is a newly discovered circovirus in recent years.In order to establish the CaineCV ELISA method,the gene sequence of Rep protein was amplified by using the CaineCV genome as template.The PCR product was cloned into the pET-28 aprokaryotic expression vector and the recombinant plasmid was named pET(28 a)-Rep.The recombinant plasmid was transferred into competent cells BL21,and then the recombinant bacteria were induced to express.SDS-PAGE analysis showed that recombinant Rep protein was correctly expressed in Escherichia coli.Western blot analysis showed that the recombinant protein could react specifically with His tag antibody.In this study,pET(28 a)-Rep recombinant prokaryotic expression plasmid was successfully constructed,andd high levels of expression in Escherichia coli were obtaine.This work laid the foundation for the preparation of antibodies against the Rep protein.
Canine circovirus is a newly discovered circovirus in recent years.In order to establish the CaineCV ELISA method,the gene sequence of Rep protein was amplified by using the CaineCV genome as template.The PCR product was cloned into the pET-28 aprokaryotic expression vector and the recombinant plasmid was named pET(28 a)-Rep.The recombinant plasmid was transferred into competent cells BL21,and then the recombinant bacteria were induced to express.SDS-PAGE analysis showed that recombinant Rep protein was correctly expressed in Escherichia coli.Western blot analysis showed that the recombinant protein could react specifically with His tag antibody.In this study,pET(28 a)-Rep recombinant prokaryotic expression plasmid was successfully constructed,andd high levels of expression in Escherichia coli were obtaine.This work laid the foundation for the preparation of antibodies against the Rep protein.
引文
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[4]孙文超,解长占,赵冠宇,等.犬圆环病毒实时荧光定量PCR检测方法的建立[J].动物医学进展,2017,38(5):6-10.
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[7]Steinfeldt T,Finsterbusch T,Mankertz A.Rep and Rep'protein of porcine circovirus type 1bind to the origin of replication in vitro[J].Virology,2001,291(1):152.
[8]Mankertz A,Hillenbrand B.Replication of porcine circovirus type 1requires two proteins encoded by the viral rep gene[J].Virology,2001,279(2):429.
[9]Finsterbusch T,Steinfeldt T,Caliskan R,et al.Analysis of the subcellular localization of the proteins Rep,Rep'and Cap of porcine circovirus type 1[J].Virology,2005,343(1):36-46.
[10]Cheung A K.Porcine circovirus:transcription and DNA replication[J].Virus Res,2012,164(1-2):46-53.
[11]顾金燕,曹瑞兵,张羽,等.PCV2Rep基因启动子区vISRE突变株的生物学特性[J].微生物学报,2009(9):1217-1222.
[12]张鑫.猪圆环病毒2型感染PK-15细胞差异蛋白质组研究[D].浙江杭州:浙江大学,2009.
[13]Nawagitgul P,Morozov I,Bolin S R,et al.Open reading frame2of porcine circovirus type 2encodes a major capsid protein[J].J Gene Virol,2000,81(9):2281-2287.
[14]刘丹,王一平,危艳武,等.猪圆环病毒2型Rep和Rep'蛋白免疫原性及其抗体中和活性的测定[J].中国兽医科学,2012(12):1216-1223.
[2]Decaro N,Martella V,Desario C,et al.Genomic characterization of a circovirus associated with fatal hemorrhagic enteritis in dog,Italy[J].PLoS One,2014,9(8):e105909.
[3]孙文超,曹慧慧,郑敏,等.犬圆环病毒全基因组克隆与序列分析[J].病毒学报,2016(4):429-435.
[4]孙文超,解长占,赵冠宇,等.犬圆环病毒实时荧光定量PCR检测方法的建立[J].动物医学进展,2017,38(5):6-10.
[5]Li L,Sabrina M G,Zhu K,et al.Circovirus in tissues of dogs with vasculitis and hemorrhage[J].Emerg Infect Dis,2013,19(4):534.
[6]Liu J,Chen I,Du Q,et al.The ORF3protein of porcine circovirus type 2is involved in viral pathogenesis in vivo[J].J Virol,2006,80(10):5065-5073.
[7]Steinfeldt T,Finsterbusch T,Mankertz A.Rep and Rep'protein of porcine circovirus type 1bind to the origin of replication in vitro[J].Virology,2001,291(1):152.
[8]Mankertz A,Hillenbrand B.Replication of porcine circovirus type 1requires two proteins encoded by the viral rep gene[J].Virology,2001,279(2):429.
[9]Finsterbusch T,Steinfeldt T,Caliskan R,et al.Analysis of the subcellular localization of the proteins Rep,Rep'and Cap of porcine circovirus type 1[J].Virology,2005,343(1):36-46.
[10]Cheung A K.Porcine circovirus:transcription and DNA replication[J].Virus Res,2012,164(1-2):46-53.
[11]顾金燕,曹瑞兵,张羽,等.PCV2Rep基因启动子区vISRE突变株的生物学特性[J].微生物学报,2009(9):1217-1222.
[12]张鑫.猪圆环病毒2型感染PK-15细胞差异蛋白质组研究[D].浙江杭州:浙江大学,2009.
[13]Nawagitgul P,Morozov I,Bolin S R,et al.Open reading frame2of porcine circovirus type 2encodes a major capsid protein[J].J Gene Virol,2000,81(9):2281-2287.
[14]刘丹,王一平,危艳武,等.猪圆环病毒2型Rep和Rep'蛋白免疫原性及其抗体中和活性的测定[J].中国兽医科学,2012(12):1216-1223.