风疹病毒结构蛋白多免疫显性区域诊断抗原的制备及其诊断效能评价

Design of a chimeric antigen containing multiple immunodominant epitopes and its use in detection of IgM antibodies against Rubella virus

摘要:

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。

Abstract:

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.