猪戊型肝炎病毒广西株ORF2三段连续基因编码蛋白的表达及抗原性分析  

Expression and antigenic analysis of three recombinant continuous ORF2 proteins of swine hepatitis E virus Guangxi strain

作　　者：肖爱欢[1,2] 赵武[2] 梁家幸[2] 何颖[2] 李茂宁[1,2] 秦毅斌[2] 李斌[2] 侯韶毅[1,2] 苏乾莲[2] 梁保忠[2] 黄伟坚[1] 

机构地区：[1]广西大学动物科学技术学院预防兽医学广西区重点实验室,广西南宁530005 [2]广西兽医研究所病毒研究室,广西南宁530001

出　　处：《中国兽医科学》2010年第6期556-560,共5页Chinese Veterinary Science

基　　金：广西基本科研业务费专项(桂兽研专项08-3)

摘　　要：为了确定猪戊型肝炎病毒(HEV)ORF2抗原性片段的分布,参考中国T1株序列设计套式引物,从新鲜猪粪中扩增出HEV部分基因片段,长1725bp,位于ORF2第205～1929bp之间,命名为HEVORF2-A。根据HEVORF2-A序列设计了3对引物,分别扩增HEVORF2-A三段连续的基因片段,命名为HEVORF2-B1、HEVORF2-B2、HEVORF2-B3,分别位于HEVORF2-A第1～576bp,577～1149bp,1150～1725bp之间。将这4段扩增的基因片段分别与表达载体pET32a(+)连接,并转入BL21(DE3)pLysS感受态细胞,经IPTG诱导,SDS-PAGE分析。结果显示,经1mmol/LIPTG37℃诱导6h后,除pET32a-A在82.6ku处有微量融合蛋白表达外,pET32a-B1、pET32a-B2、pET32a-B3分别在41.3ku、41.5ku、41.8ku处有高水平的融合蛋白表达。用自然感染猪血清和北京万泰HEVELISA诊断试剂盒阳性对照血清进行Western-blot分析,结果3个短的融合蛋白均能检测到明显的条带,表明构建的3个表达载体具有良好的抗原性。In order to determine the distribution of hepatitis E virus（HEV） ORF2 antigenic fragments,two pairs of primers were designed according to the sequence of China T1 strain and a fragment of ORF2 gene was amplified from fresh porcine feces.The fragment named as HEVORF2 A was 1 725 bp in size,located between nucleotide 205 and 1 929 of ORF2.Subsequently,three pairs of primers were designed based on HEVORF2 A,and three continuous gene fragments were amplified from HEVORF2-A,which were named as HEVORF2-B1,HEVORF2-B2 and HEVORF2-B3,and corresponded with the nucleotide 1 to 576,577 to 1149 and 1150 to 1725 in HEVORF2-A,respectively.The above four gene fragments were cloned into the expression vector pET32a（＋）,and the recombinant expression plasmids were transformed into competent cells BL21（DE3） pLysS and induced with IPTG,respectively.Three fusion proteins of 41.3 ku,41.5 ku and 41.8 ku in molecular weight were highly expressed from pET32a-B1,pET32a-B2 and pET32a-B3,respectively,but only a few of protein of 82.6 ku was expressed from pET32a-A.Western-blot analysis showed that the 3 short fusion proteins could be recognized by the sera from the naturally-infected pigs and the positive control sera from the HEV ELISA Kit of Beijing Wantai Coporation,indicating that the 3 short fusion proteins expressed from the 3 constructed expression vectors had good antigenicity.

关 键 词：猪戊型肝炎病毒 ORF2 表达 抗原性 

分 类 号：S852.659.6[农业科学—基础兽医学]