风疹病毒复合多表位肽的构建、表达、纯化及其诊断效能的初步评价

Construction,expression,purification and evaluation of diagnostic effects of rubella virus compound epitope peptide

目的采用基因工程技术构建风疹病毒复合多表位肽,探讨其对风疹病毒感染的血清学诊断价值。方法根据风疹病毒主要抗原分子E1、E2,设计并构建风疹病毒复合多表位肽的重组表达载体,用异丙基-β-D-硫代半乳糖苷(IPTG)在大肠埃希菌Rosetta中进行诱导表达,并用Ni2+螯合柱亲和纯化获得复合多表位肽;经western blot鉴定其对风疹病毒感染后的Ig M阳性血清的免疫反应性;建立基于风疹病毒复合多表位肽的Ig M-ELISA检测方法,用于风疹病毒感染患者中血清Ig M抗体的检测。结果成功构建风疹病毒复合多表位肽,在大肠埃希菌Rosetta中获得了高效表达;纯化后的风疹病毒复合多表位肽经western blot分析显示,复合多表位肽对风疹病毒Ig M阳性血清具有较好的免疫反应性;Ig M-ELISA法检测结果表明,重组的复合多表位肽能够检测出风疹病毒感染后Ig M阳性血清,与Ig M阴性血清不发生反应。结论制备的风疹病毒复合多表位肽具有良好的抗原性,可用于风疹病毒感染患者Ig M抗体的检测,提高风疹病毒感染的诊断效能。

Objective To construct rubella virus compound epitope peptide by gene engineering technology and explore its serological diagnosis value for rubella virus infection. Methods The recombinant expression vector of rubella virus compound epitope peptide was constructed based on the analysis for the major antigen molecules of rubella virus E1 and E2, and the compound epitope peptide was expressed in E. coli Rosetta with induction of isopropyhhio-β-D-galactoside（IPTG）. The expressed compound epitope peptide was purified with Ni2＋ chelating column. The immune reactivity of the expressed product to the positive serum of RV IgM antibody was identified with westeru blot. An ELISA was developed to detect the immune reactivity of the expressed product with RV IgM antibody in the serum of patients. Results The vector of rubella virus compound epitope peptide was successfully constructed and the high efficient expression was realized in E. coli Rosetta. Western blot results showed that the compound epitope peptide after purification presented strong immune reactivity to RV IgM antibody. ELISA showed that the rubella virus compound epitope peptide reacted with IgM antibody in positive serum of patients infected with rubella virus, nut not with the IgM antibody-negative serum. Conclusion The rubella virus compound epitope peptide presented good antigenicity and could be applied in the clinical detection of RV IgM antibody and improve the efficiency of diagnosis for rubella virus infection.