柯萨奇病毒B组4型VP1蛋白原核表达及间接ELISA检测方法的建立  

Expression of the VP1 protein and establishment of indirect ELISA detection method of Coxsackievirus B4

作　　者：张伟伟 周宏[1] 李娟[1] 张振杰[1] 史卫峰[1] Zhang Weiwei;Zhou Hong;Li Juan;Zhang Zhenjie;Shi Weifeng(Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases in Shandong Universities, Taishan Medical University, Taian 271000, China)

机构地区：[1]泰山医学院山东省高等学校新发传染病病因流行病学实验室,泰安市 271000

出　　处：《中华实验和临床病毒学杂志》2019年第3期303-308,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金(81601773);山东省自然科学基金(ZR2015JL026);山东省泰山学者工程专项经费(ts201511056.

摘　　要：目的 以柯萨奇病毒B组4型(Coxsackievirus B4,CV-B4)VP1基因的原核表达蛋白作为包被抗原,建立并优化间接ELISA方法,用于CV-B4的抗体检测.方法 采用RT-PCR技术扩增CV-B4病毒VP1基因,构建重组表达载体pET-32VP1,并转化至表达菌株大肠埃希菌Rosetta(DE3),进行IPTG诱导表达,经SDS-PAGE和蛋白质谱鉴定目的蛋白成功表达.以纯化的重组蛋白为包被抗原,进行间接ELISA方法反应体系的建立和条件优化.结果 CV-B4的VP1基因可在大肠埃希菌中以包涵体形式稳定表达,确定抗体间接ELISA检测方法的抗原最佳包被浓度为7.5μg/孔,血清最佳稀释浓度为1:100,临界值为OD450值≥0.376,重组蛋白可与CV-B4阳性血清特异识别,与CV-B3阳性血清存在一定交叉反应,但与柯萨奇A组病毒以及CV-B1和CV-B5均无反应,表明蛋白具有良好的免疫原性.结论 大肠埃希菌表达的VP1重组蛋白为抗原建立的间接ELISA方法具有较好的敏感性、特异性和重复性,可用于CV-B4血清抗体的检测.Objective To detect antibodies to Coxsackievirus B4 ( CV-B4) , the indirect enzyme-linked immunosorbent assay ( ELISA) method was established and optimized using the recombinant VP1 protein expressed in the prokaryote system as the envelope antigen. Methods The VP1 gene of CV-B4 was amplified using reverse transcriptase-polymerase chain reaction ( RT-PCR) . It was ligated into the expression vector pET32a(+) to obtain the recombinant plasmid pET32(+)-VP1 and was then transformed into E. coli expression strain Rosetta ( DE3) . The recombinant VP1 protein was induced by IPTG, which was verified using SDS-PAGE electrophoresis and mass spectrometry. The establishment and optimization of the indirect ELISA reaction system was based on the purified recombinant protein mentioned above as the coating antigen. Results The CV-B4 VP1 gene was stably expressed in E. coli in the form of inclusion body. The optimal coating concentration of antigen was 7. 5 μg per well and the optimal serum dilution was 1 :100. The threshold for determining the negative and positive result of the serum samples was the optical density value of ≥0. 376 at 450 nm. The purified recombinant protein could be specifically recognized by CV-B4 positive serum without cross-reaction with Coxsackievirus ( CV)-A, CV-B1 and CV-B5, indicating that it has good immunogenicity. However, it can cross-react with CV-B3 serum samples. Conclusions The indirect ELISA detection method based on the CV-B4 VP1 protein could be used in the detection of serum antibody to CV-B4 infection with good sensitivity, specificity and repeatability.

关 键 词：柯萨奇病毒B组4型 VP1 原核表达 间接ELISA 

分 类 号：R725.1[医药卫生—儿科]