风疹病毒E1蛋白与DsbA蛋白原核融合表达及应用

目的 利用DsbA蛋白分子伴侣性质,原核系统内可溶性表达风疹病毒E1蛋白并进行纯化,用于检测风疹病毒特异性抗体IgM. 方法 将PCR扩增的风疹病毒E1核酸序列克隆至融合表达载体pET-DsbA中,依次进行表达和亲和纯化,利用产物建立IgM捕获ELISA方法鉴定DsbA-E1融合蛋白的抗原性及实用性. 结果 表达纯化的E1蛋白经酶标记建立IgM捕获ELISA方法,检测60份抗风疹病毒IgM阳性血清和60份健康人血清,其中阳性血清检出例数为45例,检出率为75％;健康人血清检出1例,检出率为1.7％,特异度为98％;用酶标记E1蛋白建立的捕获ELISA法阳性检出率98.3％ (59/60),阴性检出率100％,初步表明E1蛋白抗原表位有较好的抗原特异性. 结论 融合蛋白DsbA-E1在大肠杆菌中以可溶性表达形式存在,该重组蛋白抗原性强,利用其建立的IgM捕获ELISA方法,可用于检测风疹病毒抗体.

Fusion Expression of Rubella Virus E1 Protein with DsbA Protein in Prokaryotic System and its Application

Objective To obtain soluble expression of rubella virus(RV) E1 protein in prokaryotic system with the aid of a molecular chaperone,DsbA protein,and to purify it and use it to detect RV specific antibody IgM. Methods E1 gene was obtained by PCR and cloned into pET-DsbA fusion vector.After soluble expression and purification,the E1 protein was labeled by enzyme to establish IgM capture ELISA assay,which was applied to examine the antigenicity and practicality of the recombinant protein DsbA-E1. Results Totally,60 RV antibody IgM positive sera and 60 RV antibody IgM negative sera were subject to detection.When the serum IgM was detected by purified DsbA-E1 fusion protein,45 out of 60 RV antibody IgM positive sera were found to be positive,with a detection rate of 75%,while 59 out of 60 RV antibody IgM negative sera were detected as negative,with a specificity of 98%.When the serum IgM was determined by IgM capture ELISA,the detection rate for positive sera was 98.3%(59/60). Conclusions DsbA-E1 fusion protein can express in solubility in Escherichia coli and has strong antigenicity.The IgM capture ELISA assay established with recombinant E1 protein can be applied in RV antibody detection.