表位依赖型急性期戊型肝炎诊断抗原的制备与抗原性分析

目的 原核表达制备表位依赖型戊型肝炎诊断抗原,并对其抗原性及其在急性戊肝诊断的性能进行评价.方法 以包含密码子优化的GST、HEV优势表位的H15质粒为模板,PCR获取GST-HEV优势表位片段,将其插入表达质粒pET 43.1a中,在大肠杆菌中进行表达;利用亲和层析和阴离子交换层析纯化目的蛋白;利用Western Blotting技术对目的蛋白抗原性进行鉴定;建立HEV-IgM抗体检测ELISA方法,初步评价其对阴、阳血清样本的鉴别能力.结果 获取高度均质的表位依赖型急性戊肝诊断抗原;WB实验表明目的蛋白不但可以被anti-GST单克隆抗体识别,还可以被戊肝急性期血清相应抗体所识别;分别对60份戊肝急性期血清、阴性血清进行检测,发现其与临床和实验室诊断结果一致性较高.结论 原核表达与层析纯化可以获取抗原性优异的表位依赖型急性戊肝诊断抗原,偶联HRP后可应用于HEV-IgM ELISA血清学检测.

The preparation of epitope-based diagnostic antigen of acute-phase Hepatitis E

Objective To prepare epitope-based recombinant diagnostic antigen of acute-phase hepatitis E and to evaluate its antigenicity preliminarily.Methods Using plasmid H15 containing GST and HEV immunodominant epitopes,the codon-optimized GST-HEV genes was obtained by PCR,and was inserted into pET 43.la vector (designated'H16'),then was expressed in Escherichia coli and was purified by affinity and anion exchange chromatography.Based on antigenicity of H16 identified by Western Blotting,we constructed and evaluated a novel ELISA kit for acute-phase Hepatitis E.Results The H16 protein with high homogeneity was confirmed to react with anti-GST monoclonal antibody and acutephase serum.With the ELISA,60 serum of acute-phase Hepatitis E and negative serum of were detected with excellent consistency.Conclusions By prokaryotic expression and chromatography purification,the epitope-based recombinant acute-phase Hepatitis E diagnostic antigen was obtained with excellent antigenicity,which could be applied for serological diagnosis of early HEV infections.