猪输血传播病毒2型 ORF1基因的原核表达及条件优化

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 revealed that the recombinant protein reacted positively with TTV2 positive serum and had a good immunogenicity .The ORF1 gene of TTV2 was successfully cloned and ex-pressed in prokaryotic cells , which provides the immunogen for ELISA .Adding IPTG to a final concentration of 0 .2 mmol/L and shaking the culture at 15 ℃,and then the optimal expression of ORF1 fusion protein were accu-mulated after 24 h.