人细小病毒B19VP1蛋白表达及检测特异性抗体临床应用研究  

Expression of human parvovirus B19 VP1 protein and its clinical application to detection of specific antibody.

作　　者：许东亮[1] 张国成[1] 聂晓晶[1] 李志宏[1] 孙新[1] 张学红[1] 

机构地区：[1]第四军医大学西京医院儿科,陕西西安710032

出　　处：《中国实用儿科杂志》2009年第10期769-771,共3页Chinese Journal of Practical Pediatrics

基　　金：国家自然科学基金资助项目[30571948]

摘　　要：目的探讨检测人细小病毒B19IgM和IgG抗体国产试剂的敏感性、特异性以及诊断准确度,探讨其临床应用价值。方法标本来源为2006年5月至2008年6月第四军医大学西京医院收治的心肌炎、血液病、呼吸道感染等患儿165例,用重组质粒VP1-PQE30,转化大肠埃希菌M15,测序正确后发酵培养,IPIG诱导表达,通过SDS-PAGE及蛋白质印迹分析表达产物,表达蛋白经AKTAexplorer100快速纯化系统纯化。经滴定选择最佳抗原包被浓度及血清最佳稀释度,建立表达蛋白ELISA检测人细小病毒B19抗体方法,探讨其实用性。结果用表达纯化蛋白抗原建立的ELISA方法与腺病毒、呼吸道合胞病毒、流感病毒、副流感病毒、疱疹病毒的抗体阳性血清无交叉反应。其检测B19IgM和IgG的敏感性分别为87.50%、89.28%,特异性分别为90.34%、90.90%,诊断准确度为97.01%、95.54%。两种方法有较好的一致性。结论用国产的表达纯化B19病毒VP1蛋白,所建立的表达蛋白ELISA方法,检测人细小病毒B19IgM、IgG抗体,有较好的敏感性、特异性和诊断准确度。方法方便快捷、成本低,值得进一步深入研究、推广应用。Objective To establish enzyme-linked immunosorbent assay （ELISA）method for detection of human parvo- virus B 19 antigen and compare it with parvovirus B 19 IgG. IgM ELISA Kit （Germany）to determine its sensitivity , speci- ficity and accuracy. Methods Constructed VP1-PQE30 was transformed into E.coli M15 and was analysed by se- quencing. The E.coli M15 with VP1-PQE30 were fermented and induced by IPTG to express confluenced VP1 protein. SDS-PAGE and Western blot were used to evaluate the expressed protein. The expressed confluenced VP1 protein was purified by AKTA explorer 100 system. After determining the optimal concentration of coated antigen and the optimal di- lution of blood serum, establish enzyme-linked immunosorbent assay （ELISA）method for detection of human parvovirus B19 antigen and evaluate its value for clinical application. Results This ELISA system had no cross-reaction with the serum antibodies of adenovirus, respiratory syncytial virus, influenza virus, parainfluenza virus, and human herpes virus. The sensitivity of detection of IgM and IgG was 87.50 % and 89.28%, the specificity 90.34 % and 90.90% and the accura- cy 97.01% and 95.54%. There was a good concordance between the two methods. Conclusions It is sensitive, specific and precise to use domestic-made B19 VP1 unique recombinant protein to detect human parvovirus B19 antigen. It is quick and economical, so it is worth investigating deeply and spreading.

关 键 词：人细小病毒B19 VP1独特区蛋白 ELISA 原核表达 

分 类 号：R72[医药卫生—儿科]