猪瘟病毒E0蛋白的原核表达及多克隆抗体的制备
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摘要
为表达猪瘟病毒E0蛋白并制备其多克隆抗体,本研究构建E0蛋白的原核表达载体,转化至BL21(DE3)菌株,IPTG诱导表达,亲和层析及切胶回收纯化重组蛋白。SDS-PAGE和Western blot分析显示E0重组蛋白主要以包涵体形式表达,亲和层析纯化获得了E0重组蛋白,用其免疫Balb/c小鼠4次制备E0重组蛋白的多克隆抗体。间接ELISA显示,免疫小鼠血清中E0蛋白抗体效价为1∶50 000。获得的E0蛋白多抗能与病毒感染细胞及E0-EGFP融合表达细胞中天然结构的E0蛋白发生特异性反应。本研究制备的E0重组蛋白及其多克隆抗体为进一步研究E0蛋白的功能和免疫原性奠定了基础。
To express E0 protein of CSFV and prepare its polyclonal antibodies(pAb),the recombinant E0 protein of CSFV Shimen strain was expressed in E.coli BL21(DE3)with IPTG induction and successively purified by affinity chromatography and gel slice.Balb/c mice were immunized four times with the purified recombinant E0 protein.SDS-PAGE and Western blot analysis showed that recombinant protein was expressed mainly in the inclusion body.The indirect ELISA showed that the antibody titers in the serum of immunized mice was at 1:50000.Moreover,the obtained anti-E0 pAb can specifically react with the E0 protein expressed in virus-infected cells and E0-EGFP-expressing cells.The obtained E0 protein and its polyclonal antibodies in this study will be useful for future studies on the function and immunogenicity of CSFV E0 protein.
To express E0 protein of CSFV and prepare its polyclonal antibodies(pAb),the recombinant E0 protein of CSFV Shimen strain was expressed in E.coli BL21(DE3)with IPTG induction and successively purified by affinity chromatography and gel slice.Balb/c mice were immunized four times with the purified recombinant E0 protein.SDS-PAGE and Western blot analysis showed that recombinant protein was expressed mainly in the inclusion body.The indirect ELISA showed that the antibody titers in the serum of immunized mice was at 1:50000.Moreover,the obtained anti-E0 pAb can specifically react with the E0 protein expressed in virus-infected cells and E0-EGFP-expressing cells.The obtained E0 protein and its polyclonal antibodies in this study will be useful for future studies on the function and immunogenicity of CSFV E0 protein.
引文
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[7]高慎阳,查恩辉,王珅,等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.
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[13]Luo Y,Li L,Austermann-Busch S,et al.Enhanced expression of the E0protein of classical swine fever virus in yeast and its application in an indirect enzyme-linked immunosorbent assay for antibody differentiation of infected from vaccinated animals[J].J Virol Meth,2015,222(2):22-27.
[14]Schroeder S,Rosen T V,Blome S,et al.Evaluation of classical swine fever virus antibody detection assays with an emphasis on the differentiation of infected from vaccinated animals[J].Revue Scientifique Et Technique,2012,31(3):997-1010.
[15]Zhu Y,Shi Z,Drew T W,et al.Antigenic differentiation of classical swine fever viruses in China by monoclonal antibodies[J].Virus Res,2009,142(1-2):169-174.
[16]郭东光,朱艳平,孙国鹏,等.猪瘟病毒E0蛋白的原核表达及鉴定[J].动物医学进展,2014,35(5):31-35.
[17]李鹏,张彦明,张志,等.猪瘟流行毒株E0蛋白的原核表达及其间接ELISA方法的建立[J].西北农林科技大学学报:自然科学版,2008,36(10):24-28.
[2]Matzener P,Magkouras I T,Peterhans E,et al.The viral RNase E0prevents IFN type-I triggering by pestiviral singleand double-stranded RNAs[J].Virus Res,2009,140(1-2):15-23.
[3]Bruschke C J,Hulst M M,Moormann R J,et al.Glycoprotein E0of pestiviruses induces apoptosis in lymphocytes of several species[J].J Virol,1997,71(9):6692-6696.
[4]Chen J,He W R,Shen L,et al.The laminin receptor is a cellular attachment receptor for classical swine fever virus[J].J Virol,2015,89(9):4894-4906.
[5]Dong X Y,Yan H C,Tang S Q,et al.Structural glycoproteins of classical swine fever virus:implication for vaccine development[J].Front Cover:Devon Rex,2013,68(2):78-86.
[6]Aebischer A,Müller M,Hofmann M A.Two newly developed E0-based ELISAs allow the differentiation of classical swine fever virus-infected from marker-vaccinated animals and the discrimination of pestivirus antibodies[J].Vet Microbiol,2013,161(3):274-285.
[7]高慎阳,查恩辉,王珅,等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.
[8]Chen L,Xia Y H,Pan Z S,et al.Expression and functional characterization of classical swine fever virus E0protein[J].Protein Expression&Purification,2007,55(2):379-387.
[9]Fetzer C,Tews B A,Meyers G.The carboxy-terminal sequence of the pestivirus glycoprotein E0represents an unusual type of membrane anchor[J].J Virol,2005,79(18):11901-11913.
[10]Python S,Gerber M,Suter R,et al.Efficient sensing of infected cells in absence of virus particles by blasmacytoid dendritic cells is blocked by the viral ribonuclease E0[J].PLoS Pathog,2013,9:1-13.
[11]Luo Y,Su L,Yuan S,et al.Classical swine fever in China:a minireview[J].Vet Microbiol,2014,172(1-2):1-6.
[12]Huang Y L,Deng M C,Wang F I,et al.The challenges of classical swine fever control:modified live and E2subunit vaccines[J].Virus Res,2014,179(3):1-11.
[13]Luo Y,Li L,Austermann-Busch S,et al.Enhanced expression of the E0protein of classical swine fever virus in yeast and its application in an indirect enzyme-linked immunosorbent assay for antibody differentiation of infected from vaccinated animals[J].J Virol Meth,2015,222(2):22-27.
[14]Schroeder S,Rosen T V,Blome S,et al.Evaluation of classical swine fever virus antibody detection assays with an emphasis on the differentiation of infected from vaccinated animals[J].Revue Scientifique Et Technique,2012,31(3):997-1010.
[15]Zhu Y,Shi Z,Drew T W,et al.Antigenic differentiation of classical swine fever viruses in China by monoclonal antibodies[J].Virus Res,2009,142(1-2):169-174.
[16]郭东光,朱艳平,孙国鹏,等.猪瘟病毒E0蛋白的原核表达及鉴定[J].动物医学进展,2014,35(5):31-35.
[17]李鹏,张彦明,张志,等.猪瘟流行毒株E0蛋白的原核表达及其间接ELISA方法的建立[J].西北农林科技大学学报:自然科学版,2008,36(10):24-28.