基因重组戊肝病毒多价复合抗原的表达及初步应用

EXPRESSION AND DIAGNOSTIC APPLICATION OF RECOMBINANT HEV MULTIVALENT ANTIGEN

目的 :利用基因工程技术体外表达高效 HEV多价复合抗原 ,建立一套敏感和特异的抗 HEV检测体系。方法 :将前期构建的基因重组戊肝病毒多价复合抗原表达质粒转化大肠埃希菌 JM10 9,筛选鉴定阳性克隆并进行诱导表达 ,表达产物经分子筛层析纯化后 ,利用 western blot作抗原特异性鉴定。以此抗原建立 EL ISA检测卫生部生物药品检定所 HEV血清参比品及临床血清标本 ,同时以 Genelab公司抗 HEVEL ISA试剂盒作为对照进行对比研究。结果 :重组质粒转化大肠埃希菌 JM10 9株后的阳性克隆 ,经诱导在SDS- PAGE中出现一条分子量大小约为 31.5 k D的蛋白条带 ,该蛋白纯化后经 Western Blotting检测证实为 HEV特异性抗原。以此抗原包被酶联板建立 EL ISA检测 5 3份国家卫生部标准 HEV参比血清 ,灵敏度达 10 0 % (38/38) ,特异度达 93.3% (14 /15 )。用此方法检测 6 8份临床标本 ,并与 Genelab EL ISA试剂盒结果比较 ,两者阴阳性相符的有 6 4份 ,总符合率为 94 .1%。结论 :本实验克隆表达的基因重组戊肝病毒多价复合抗原具有良好抗原性 ,以此抗原建立的抗 HEV EL ISA具有灵敏度高、特异性强的特点 。

Objective:To obtain the effective recombinant HEV multivalent antigen through gene engineering and develop a specific and sensitive serological diagnostic assay for detection of anti HEV.Methods:Recombinant expression plasmid was transformed into E.coli JM109.Positive clones were screened out and induced with IPTG.The expressed recombinant protein was purified through Sephadex G100 column and its specificity was tested by western blotting.Based on the recombinant HEV multivalent antigen,an enzyme linked immunosorbent assay(ELISA) for antibodies to HEV was developed and the sensitivity and specificity of the ELISA were evaluated by the test results of the standard test sera designated by NICPBP and the sera collected clinically.Results:Screening revealed transformed E.coli.Clone with the constructed plasmid encoding the recombinant HEV antigen sequence.After IPTG induction,SDS PAGE of the total protein extracts revealed a new protein of about 31.5 kD,correlating with the predicted molecular weight of the recombinant protein.The soluble recombinant protein was purified from sonified E.coli homogenate by gel chromatography.Positive signals of the western blotting demonstrated the recombinant protein in the study is highly specific.High specificity(100%) and sensitivity(93 3%) were achieved in the anti HEV ELISA system based on the recombinant protein,and the test results were highly consistent with those of the commercial diagnostic test kit.Conclusion:The highly specific recombinant HEV multivalent antigen in the study is a promising agent for the development of an effective diagnostic test of anti HEV antibodies for the serological diagnosis and epidemiological study of hepatitis E.