猪血凝性脑脊髓炎病毒HE基因的克隆及原核表达  

Cloning and Prokaryotic Expression of HE Gene of Swine Hemagglutinating Encephalomyelitis Virus

作　　者：常灵竹[1] 宋德光[1] 贺文琦[1] 陆慧君[1] 李志萍[1] 陈克研[1] 高丰[1] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062

出　　处：《中国生物制品学杂志》2008年第4期273-276,280,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30671551);吉林省科技发展重点项目(20060206-2)

摘　　要：目的构建猪血凝性脑脊髓炎病毒株(67N)血凝素酯酶蛋白(HE蛋白)原核表达系统,为建立特异性免疫学诊断方法提供备选材料。方法克隆猪血凝性脑脊髓炎病毒(67N)HE蛋白抗原表位富集区基因,构建重组表达质粒pET-HE,并在大肠杆菌中诱导表达。经包涵体的初步纯化,金属螯合层析进一步纯化HE蛋白,SDS-PAGE和Western blot进行检测。结果大肠杆菌可高效表达HE蛋白,目的蛋白表达量可占菌体总蛋白的42.2%。纯化后蛋白浓度为0.6 mg/ml,纯度为85.4%。所表达的蛋白可被兔抗猪血凝性脑脊髓炎阳性血清所识别。结论已成功构建猪血凝性脑脊髓炎病毒HE蛋白原核表达载体,重组HE蛋白抗原具有良好的特异性,可作为检测猪血凝性脑脊髓炎病毒血清抗体的候选抗原。Objective To construct the prokaryotic expression system for hemagglufinin esterase （HE） protein of swine hemagglutinating encephalomyelitis virus （HEV） 67N strain and provide a candidate gene for the development of specific immtmodiagnostic method. Methods Clone the gene encoding epitope enrich regions of HE protein of swine HEV 67N strain to construct recombinant plasmid pET-HE and express in E. coli. The expressed product,in a form of inclusion body, was washed,lyzed and further purified by metal chelate chromatography,then identified by SDS-PAGE and Western blot. Results The expressed HE protein contained 42.2 % of total somatic protein. It reached a protein content of 0.6 mg/ml and a purity of 85.4% after purification and was recognized by rabbit serum against swine HEV. Conclusion The prokaryotic expression vector for HE gene of swine HEV was successfully constructed. The expressed recombinant HE protein showed good specificity and might be used as a candidate antigen for determination of serum antibody against swine HEV.

关 键 词：猪血凝性脑脊髓炎病毒 血凝素酯酶蛋白 克隆 原核表达 

分 类 号：S852.659.6[农业科学—基础兽医学] Q786[农业科学—兽医学]