猪血凝性脑脊髓炎病毒HE基因的克隆及原核表达

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。

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

Objective To construct the prokaryotic expression system for hemagglutinin esterase(HE) protein of swine hemagglutinating encephalomyelitis virus(HEV) 67N strain and provide a candidate gene for the development of specific immunodiagnostic method.MethodsClone 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.ConclusionThe 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.