猪TTV2 ORF1基因的原核表达及多克隆抗体的制备  

Prokaryotic expression of ORF1 gene of torque teno virus type 2 and preparation of polyclonal antibody

作　　者：张文文[1,2] 王小敏[1] 肖琦[1] 周忠涛[1,2] 汪伟[1] 温立斌[1] 李彬[1] 郭容利[1] 张雪寒[1] 周俊明[1] 倪艳秀[1] 吕立新[1] 俞正玉[1] 茅爱华[1] 胡屹屹[1] 刘永杰[2] 何孔旺[1] 

机构地区：[1]江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《江苏农业学报》2014年第1期119-124,共6页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金面上项目(31272574);江苏省农业自主创新基金项目[CX(11)2060]

摘　　要：为了构建猪输血传播病毒2型(TTV2)ORF1基因部分片段的原核表达载体,进行原核表达,并利用重组蛋白质制备猪TTV2的多克隆抗体,采用PCR方法扩增出猪TTV2 ORF1基因的部分片段,将其克隆至原核表达载体pcoldI中,构建原核表达质粒pcold-g1,然后转化大肠杆菌BL21(DE3),用IPTG进行诱导表达。表达产物通过镍离子亲和层析柱进行纯化,用Western blot进行鉴定。将纯化的重组蛋白质免疫新西兰大白兔制备猪TTV2多克隆抗体,Western blot检测抗体特异性,间接ELISA检测抗体效价。PCR扩增获得大小为1 227 bp的片段,重组表达质粒经双酶切及测序证实构建正确;重组蛋白质分子量为5.2×104,主要以包涵体形式存在,纯化后纯度可达95%以上,具有较好的抗原性;制备的多克隆抗体特异性高,抗体效价达1∶12 800。重组蛋白质在大肠杆菌中得到了高效表达,为建立TTV2间接ELISA检测方法提供了优质的包被抗原。To construct prokaryotic expression vector of a fragment of ORF1 gene of porcine torque teno virus type 2（TTV2）, and to prepare polyclonal antibody for porcine TTV2, a fragment of ORF1 of porcine TTV2 was amplified andcloned into prokaryotic expression vector pcoldI. Theconstructed recombinant plasmid pcold-g1 was transformedinto Escherichia. coli BL21 （DE3） and was induced byIPTG. The expression product was purified by nickel ionaffinity chromatography and identified by Western blot.NEW Zealand white rabbits were immunized with purified recombinant protein to generate polyclonal anti-porcine TTV2 antibody. The specificity of the polyclonal antibodies was con-firmed by Western blot, and the titer was determined by indirect ELISA. DNA fragment with a length of 1 227 bp was am-plified by PCR. Restriction enzyme digestion and sequencing proved that recombinant plasmids pcold-g1 was constructedcorrectly. The molecular weight of recombinant protein was 5. 2×10^4 and most of the recombinant protein was inclusionbody. The purified recombinant protein reached a purity of more than 95%, and had good antigenicity. The specificity ofthe polyclonal antibodies was high and the titer was 1 ：12 800. The recombinant protein could be used as the coating anti-gen for detecting the antibody against TTV2 by indirect ELISA.

关 键 词：猪TTV2 ORF1基因 原核表达 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]