呼吸道合胞病毒F蛋白基因片段原核表达和初步应用
Expression of RSV F protein fragment and its application
芦起 1申昆玲 2许文波 3刘中华 3朱贞 3毛乃颖 3杨锡强 1余加林 1王家蓉 1王颖1
作者信息
- 1. 重庆儿童医院,重庆,400014
- 2. 北京儿童医院,北京,100050
- 3. 中国疾病预防控制中心,病毒病预防控制所,北京,100015
- 折叠
摘要
目的:建立呼吸道合胞病毒快速、敏感、特异、廉价的早期血清学检测方法.方法:从北京儿童医院患者鼻咽分泌物分离的呼吸道合胞病毒中,用RT-PCR的方法扩增出RSV F蛋白的F1区部分基因片段F1-1(AA137～363),此区域包含F蛋白的主要中和抗原位点.将该基因片段克隆到pMD18-T载体,将连接质粒经PCR鉴定,证实插入目的片段.酶切后将该片段与pET-32a表达质粒连接,用限制性内切酶酶切及序列测定,证实插入片段的正确性.利用pET-32a表达质粒在大肠杆菌BL21表达正确的RSV F1-1蛋白.重组蛋白N端包含有6个连续的组氨酸标签,通过镍离子金属鏊合亲和层析纯化,获得了高纯度的目的重组蛋白RSV F1-1,然后将重组RSV F1-1包被建立间接ELISA法检测人血清中的RSVF蛋白IgG抗体.结果:RT-PCR产物的片段大小为678 bp.经酶切和测序鉴定后,插入序列无误.Western Blot分析显示:重组蛋白具有较好的抗原性.用该蛋白建立的间接ELISA方法检测结果与进口Euroimmun和维润试剂盒检测结果比较,差异无显著性.结论:F蛋白基因片段的成功表达为进一步开发诊断试剂盒提供了实验依据.
Abstract
AIM:To establish rapid,sensitive,specific,cheap serological detection methods of human respiratory syncytial virus.METHODS:The viruses were isolated from nasopharyngeal secretions which collected from patient in BeiJing Children's hospital.One fragment of F gene(F1-1,AA 137-363)was amplified by RT-PCR,since this region contains the major neutralization sites.The PCR product was cloned into pMD18-T vector and was identified by PCR,then the target gene was subcloned into pET-32a expression vector and was identified by restriction analysis and sequencing.F1-1 was efficiently expressed in E.coli BL21 and purified by nickel HiTrap chelating metal affinity column.The serum levels of IgG antibodies to F antigen were measured in 91 serum samples of suspected RSV from BeiJing Children's hospital using indirect ELISA against F1-1 proteins of HRSV.RESULTS:The size of the RT-PCR product was 678 bp.The sequence of insert was correct after examined by restriction enzyme digestion and sequencing.Western Blotting indicated that the RSV F1-1 protein had specific antigenicity.No significant difference was found when compared our method with commercial detection kits.CONCLUSION:The recombinant RSV F protein fragment offers an efficient way for serological diagnosis.
关键词
人呼吸道合胞病毒/F蛋白/原核表达Key words
human respiratory syncytial virus/fusion(F)gene/prokaryotic expression引用本文复制引用
出版年
2009
NETL
NSTL
维普
万方数据