猪戊型肝炎病毒swCH189株衣壳蛋白基因CP239片段的表达、纯化及抗原性分析

Expression,purification and antigenicity analysis of capsid protein gene CP239 fragment of swCH189 strain of swine hepatitis E virus

根据已克隆的戊型肝炎病毒株swCH189株结构蛋白基因(ORF2)的抗原性及水溶性分析,在其序列上设计套式引物,采用RT-PCR技术扩增猪戊型肝炎病毒(HEV)结构蛋白基因ORF2主要抗原基因片段,长度为728bp,将其克隆于PMD18-T载体,测序结果表明插入的片段属于猪戊型肝炎病毒结构蛋白基因ORF2部分;将该片段插入pet32a表达载体,经酶切、测序鉴定证实获得了带有目的基因的重组表达质粒。将重组质粒转化Rosetta菌,经0.3mmol/L IPTG诱导得到融合表达,得到相对分子质量为45 000的重组蛋白,以包涵体形式存在。Western blot分析表明,该蛋白可以与猪戊型肝炎阳性血清反应,表明该蛋白具有良好的反应原性。并用SDS电泳切胶纯化和透析浓缩方法进行纯化,用纯化的蛋白免疫兔子,经每2周免疫1次,连续3次免疫后,对免疫组和对照组,分别在45,60,90d采血,每只兔子采集1份。用夹心ELISA法测定免疫血清抗体,结果表明免疫组在45d后均产生具有HEV抗原结合活性的抗体。本试验对猪swCH189株CP239基因的克隆和表达研究,为进一步研究结构蛋白基因ORF2的生物学功能奠定了基础。

Based on the antibody and water-soluble analysis of swCH189 strain of swine hepatitis E virus,a pair of primers as designed according to the ORF2 of swine hepatitis E virus swCH189 strain,and the ORF2 about 2025 bp was amplified by PCR.For the establishment of a fast pig HEV antibody detection methods,based on cloning of hepatitis E virus structural protein gene swCH189 strains（ORF2） and water-soluble antigen of the analysis,design,in its sequence a pair of primers,the upper reaches of primer and downstream primers,respectively,with NcoⅠ and NotⅠ restriction sites,using PCR amplification methods to obtain the corresponding size of 728 bp fragment,located in ORF2 128-497 bp Department.Its reorganization in the pET32a（＋） prokaryotic expression vector,after digestion,to identify the correct sequence,the use of the recombinant plasmid into E.coli rosetta,by isopropyl BD-thiogalactoside（IPTG） inducible.expression,and SDS-PAGE electrophoresis test,and optimize the best conditions after IPTG induction for 4 h,the concentration of 0.3 mmol /L,and mainly in the form of inclusion bodies.Western blot detected in about 45 000 there a specific band,consistent with the projected size,results showed that the expression of the protein with biological activity.And using SDS gel electrophoresis method of purification and concentration was purified by dialysis,Immune rabbits with purified protein,Once every two weeks after immunization,After three consecutive immunization,Immune group and control group rabbits were 45,60,90,blood samples,each collected a rabbit.Using sandwich ELISA,immune serum antibody,The results showed that immunized group were produced in 45 d with HEV antigen binding activity of antibodies.CP239 of porcine swCH189 strains cloning and expression studies,in order to further study the biological function of genes laid the foundation.