猪输血传播病毒2型ORF1基因的原核表达及条件优化  

Prokaryotic Expression and Optimization of Expression Conditions of Open Reading Frame 1 Gene of Porcine Torque Teno Virus Type 2

作　　者：王小敏[1] 何孔旺[1] 张文文[1,2] 王东田 周忠涛[1,2] 张雪寒[1] 周俊明[1] 汪伟[1] 倪艳秀[1] 温立斌[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 [3]山东省蓬莱市村里集镇畜牧兽医工作站,山东蓬莱265604

出　　处：《华北农学报》2013年第5期38-43,共6页Acta Agriculturae Boreali-Sinica

基　　金：国家基金面上项目(31272574);江苏省自主创新专项(CX(11)2060)

摘　　要：为了克隆猪输血传播病毒2型(TTV2)ORF1基因,构建原核表达载体,在大肠杆菌BL21(DE3)中诱导表达猪TTV2 ORF1蛋白,并对其表达条件进行优化。利用普通PCR从猪TTV2阳性样本中扩增出TTV2 ORF1基因,利用分子克隆的方法将猪TTV2 ORF1基因克隆到原核表达载体pcoldI上,构建表达载体pcold-ORF1,在大肠杆菌中诱导表达带有6个组氨酸标签的ORF1融合蛋白。并对影响重组蛋白表达的3个因素,即诱导时间、诱导温度和IPTG浓度进行优化,确定了pcold-ORF1重组蛋白表达的最佳表达条件。SDS-PAGE分析结果表明,重组蛋白在BL21中高效表达,以包涵体形式表达为主,分子质量约为39 kDa。蛋白表达量随诱导时间增加而有所增加,在15℃时表达量最高,而IPTG浓度对蛋白的表达量没有显著影响。Western Blotting结果表明,重组蛋白与猪TTV2阳性血清特异性反应,具有很好的免疫原性。成功获得TTV2-ORF1基因,构建了原核表达载体并获得高效表达,为TTV的ELISA检测方法的建立提供抗原。且重组蛋白在15℃条件下,加入0.2 mmol/L浓度的IPTG,诱导24 h,表达条件最佳。To clone the open reading frame 1（ORF1）gene of porcine torque teno virus type 2 and construct a prokaryotic expression vector .TTV2 ORF1 recombinant protein was expressed in Escherichia coli and optimal ex-pression was performed.The ORF1 gene of TTV2 was amplified from the TTV2 positive samples and sub-cloned in a prokaryotic expression vector pcoldI .The constructed recombinant plasmid pcold-ORF1 was transformed to E.coli BL21 for expression the ORF1 fusion protein with six histidine-tag under induction of IPTG .The induction time ,in-duction in different temperature and IPTG concentrations were optimized .The recombinant fusion protein had high expression level in BL21（DE3）,and SDS-PAGE analysis showed the exogenous gene was mainly expressed as in-clusion bodies and its molecular weight was about 39 kDa.The induction time were positively related trends with re-combinant protein expression ,the best induction temperature was 15 ℃,whereas IPTG concentration had no signifi-To clone the open reading frame 1（ORF1）gene of porcine torque teno virus type 2 and construct a prokaryotic expression vector .TTV2 ORF1 recombinant protein was expressed in Escherichia coli and optimal ex-pression was performed.The ORF1 gene of TTV2 was amplified from the TTV2 positive samples and sub-cloned in a prokaryotic expression vector pcoldI .The constructed recombinant plasmid pcold-ORF1 was transformed to E.coli BL21 for expression the ORF1 fusion protein with six histidine-tag under induction of IPTG .The induction time ,in-duction in different temperature and IPTG concentrations were optimized .The recombinant fusion protein had high expression level in BL21（DE3）,and SDS-PAGE analysis showed the exogenous gene was mainly expressed as in-clusion bodies and its molecular weight was about 39 kDa.The induction time were positively related trends with re-combinant protein expression ,the best induction temperature was 15 ℃,whereas IPTG concentration had no signifi- cant impact on protein expression .Western Blotting

关 键 词：猪输血传播病毒2型 基因克隆 原核表达 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]