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原核表达与层析纯化获取风疹病毒(Rubella Virus,RV)多表位诊断抗原,并初步评价其抗原性。将RV衣壳蛋白C aa 3~29、aa 96~123以及包膜糖蛋白E1aa208~247三个主要免疫显性表位串联,密码子优化后全基因合成;利用基因重组技术将多表位串联片段插入带有GST标签的表达质粒中,在大肠杆菌中进行表达,并利用亲和层析和离子交换层析纯化目的蛋白;利用Western Blot(WB)技术对目的蛋白抗原性进行鉴定,并建立RV-IgM抗体检测ELISA技术,初步评价此方法对阴阳血清样本的鉴别能力。获取高度均质的RV多表位诊断抗原,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anti-RV多克隆抗体、RV-IgM阳性血清相应抗体所识别。分别对48份RV急性感染病例的阳性血清、阴性血清和健康人血清进行检测,发现一致性优异。原核表达与层析纯化可以获取抗原性良好的RV多表位诊断抗原,偶联HRP后可以作为检测抗原应用于RV-IgM ELISA血清学检测中。
The prokaryotic expression and chromatographic purification of Rubella Virus (RV) multi-epitope diagnostic antigen, and preliminary evaluation of antigenicity. The three major immunodominant epitopes of RV capsid proteins C aa 3 ~ 29, aa 96 ~ 123, and envelope glycoprotein E1aa208 ~ 247 were tandemly codon-optimized and all genes were synthesized. Using the gene recombination technique, The fragment was inserted into the expression plasmid with GST tag, expressed in E. coli, and the target protein was purified by affinity chromatography and ion exchange chromatography. The antigenicity of the target protein was identified by Western Blot (WB) RV-IgM antibody detection ELISA technology, a preliminary evaluation of this method of identification of serum samples of yin and Yang. Obtained high homogeneity of RV polytope diagnostic antigen, WB experiments showed that the target protein can not only be anti-GST monoclonal antibody can also be anti-RV polyclonal antibodies, RV-IgM positive serum corresponding antibodies recognized. 48 cases of acute infection of RV positive serum, negative serum and healthy human serum were detected and found to be excellent. Prokaryotic expression and chromatographic purification can be used to obtain antigenicity of RV polytope diagnostic antigen, coupled HRP can be used as detection antigen in RV-IgM ELISA serological test.