A chimeric antigen designated B103 containing six immunodominant regions derived from three structural proteins of Rubella virus (RV) was designed and its utility in serological diagnosis was assessed. Protein B103 is comprised of aa 1-30 & aa 96-123 of C protein, aa 31-105 of E2 protein, as well as aa 11-39, aa 154-277 & aa 389-412 of E1 protein. In addition, it contains thioredoxin (TRX) at the N-terminal and His tag at the C-terminal. B103 was expressed in Escherichia coli BL21(DE3) and purified by Streamline Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B103 as verified by Western blotting analysis, we constructed and evaluated a novel capture ELISA for RV-IgM detection. B103 was expressed in a soluble form, accounting for 18.57% of the total bacterial proteins. After purification, the concentration and purity of protein B103 were 3.026 mg/mL and 95.35%, respectively. Western blotting analysis demonstrated that protein B103 could react with acute-phase serum of RV. By ELISA, 40 negative sera and 40 RV-acute phase sera were detected. The sensitivity, specificity, positive predictive value, negative predictive value and coincidence rate of the ELISA were 92.50%, 95.00%, 94.87%, 92.68% and 93.75%, respectively. The McNemer analysis suggested that there was no statistical difference between the 'Gold standard' and the novel ELISA with a kappa coefficient of 0.900, indicating excellent consistency. B103 chimeric protein with excellent antigenicity obtained from prokaryotic expression followed by chromatography purification could prove useful for early diagnosis of RV infection.
对串联风疹病毒 (Rubella virus，RV) 3 个结构蛋白6 个免疫显性区域进行制备 (命名为B103)，并将其应用于血清学诊断中。选取RV C aa 1–30 & aa 96–123、E2 aa 31–105 和E1 aa 11–39 & aa 154–277 & aa 389–412等6 个免疫显性区域，将其串联并基因合成；将此基因片段插入TRX 和His 标签之间构建表达质粒；蛋白B103在大肠杆菌BL21(DE3) 中诱导表达，并利用Streamline Chelating 亲和层析和DEAE 阴离子交换层析纯化目的蛋白，Sephadex G-25 分子筛分析其折叠情况及均一性；利用免疫印迹技术对蛋白B103 的抗原性进行鉴定，并建立RV-IgM 抗体捕获法ELISA 检测技术，初步评价此方法对阴阳血清样本的鉴别能力。蛋白B103 以可溶性形式表达，其表达量约占菌体总蛋白的18.57%，经纯化后蛋白B103 浓度为3.026 mg/mL，纯度为95.35%；免疫印迹实验表明蛋白B103 能与RV 急性期血清发生反应；对40 份RV 急性期血清及40 份RV 阴性血清进行检测发现可以很好地鉴别阴阳性血清标本；其灵敏度为92.50%，特异性为95.00%，阳性预测值为94.87%，阴性预测值为92.68%，符合率为93.75%，McNemer 检验的结果提示与“金标准”诊断结果无差异，kappa=0.900，提示两种方法诊断结果一致性优异。原核表达与层析纯化可以获取抗原性优异的RV 血清学诊断抗原，可以应用于RV 早期诊断中。.
Keywords: Rubella virus; chromatography purification; immunodominant region; prokaryotic expression; serological diagnosis.