猪源戊型肝炎病毒基因IV型ORF3编码蛋白的表达及鉴定  

Prokaryotic Expression and Identification of Protein Encoded by Open Reading Frame 3 of Genotype IV Swine Hepatitis E Virus

作　　者：屈勇刚[1] 贾鹏[1] 金宁一[2] 陆民[1] 孙中锋[2] 朱光泽[3] 于芳[2] 刘玉生[2] 夏志平[2] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]军事医学科学院军事兽医研究所,长春130062 [3]长春中医药大学第一临床附属医院,长春130021

出　　处：《中国生物工程杂志》2008年第1期8-12,共5页China Biotechnology

基　　金：国家“973”计划资助项目(2005CB523005);国家自然科学基金资助项目(30771609)

摘　　要：通过PCR从已构建的猪源戊型肝炎病毒全基因克隆扩增ORF3全基因,将扩增产物插入到pMD18-T载体中,亚克隆至原核表达载体pET28a(+),构建pET28a-ORF3表达载体,转入E.coliBL21(DE3),IPTG诱导表达。Ni-NTA层析柱纯化表达蛋白,用SDS-PAGE、免疫印迹、ELISA等方法分析鉴定表达产物。结果成功扩增到345bp的目的基因;构建了重组表达载体pET28a-ORF3;转化宿主菌E.coliBL21(DE3)后表达产物的相对分子质量在6.50～16.5kDa之间,与预期表达的目的蛋白相对分子质量相符;表达的目的蛋白能与阳性猪源和人源血清发生特异性反应,证实其具有较好的反应原性。The ORF3 gene from a full-length swine HEV genomic clone was amplified by polymerase chain reaction（PCR） and inserted into vector pMD18-T,then subcloned to prokaryotic expression vector pET28a（ ＋ ）. The recombinant plasmid pET28a-ORF3 was transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed product was identified by SDS-PAGE, Western blotting, then purified by Ni-NTA affinity chromatography and used as an antigen for the determination of HEV antibody in swine and human serum by ELISA. It indicated that the expressed product, with a relative molecular weight 6500 - 16500, was consistent with the designed. Western blotting showed specific reaction of the expressed fusion protein with mouse anti-His Tag serum. ELISA indicated that the expressed fusion protein can reacted with the HEV positive swine and human serum. The expressed product showed good reactionogenicity.

关 键 词：戊型肝炎病毒 结构蛋白 原核表达 

分 类 号：Q786[生物学—分子生物学]