猪日本脑炎病毒NS3蛋白的基因克隆、原核表达及纯化

Cloning,Expression and Purification of Nonstructural Protein-3 of Japanese Encephalitis Virus from Pig

提取猪日本脑炎病毒(JEV)上海分离株的基因组RNA,反转录合成cDNA,RT-PCR扩增JEV的NS3基因片段,亚克隆到原核表达载体pET-28(a)上,酶切鉴定和PCR鉴定重组原核表达质粒pET-28(a)-JEV-NS3,转化大肠埃希菌BL21(DE3)菌株,IPTG诱导表达重组His-JEV-NS3蛋白,SDS-PAGE分析重组蛋白的表达,his-band试剂盒纯化重组蛋白。获得了1.8kb NS3基因片段,成功构建重组原核表达质粒pET-28(a)-JEV-NS3。IPTG诱导获得分子质量为64ku的His-JEV-NS3蛋白,该蛋白主要以包涵体形式表达,表达量占总菌体蛋白的30%以上,纯化后获得高纯度重组蛋白,其占总蛋白比例达70%以上。

NS3 is associated with viral RNA packaging and replication,viral pathogen.In this study,the cDNA of JEV were synthenized from viral genome by RT-PCR.The NS3 gene was cloned from cDNA by PCR and subcloned into pET-28（a） plasmid.The recombinant plasmid pET-28（a）-JEV NS3 was verified by PCR and enzyme digestion.pET-28（a）-JEV NS3 was transformed into E.coli.BL21（DE3）,and the recombinant JEV NS3 protein was expressed by IPTG induction and purified by his-band kit.Expression and purification of His-JEV NS3 were analyzed by SDS-PAGE.In this paper,JEV NS3 product is 1.8 kb,the recombinant plasmid pET-28（a）-JEV-NS3 was constructed.His-JEV NS3 molecular weight is 64 ku.The protein was mainly expressed in the form of inclusion bodies and was more than 30% of the total cell protein.A large number of high-purity recombinant proteins were obtained by purification and the proportion reached more than 70% after purification.This study laid a foundation for JEV NS3 function and virus pathogen mechanism.