人呼吸道合胞病毒截短F1蛋白的原核表达及其免疫原性评价  

Prokaryotic expression and immunogenicity of human respiratory syncytial virus truncated F1 protein

作　　者：李华[1] 杨婷[1] 岳磊[1] 何晓娟[1] 朱凡丽 谢天宏[1] 龙润乡[1] 杨蓉[1] 罗芳宇 谢忠平[1] LI Hua YANG Ting YUE Lei HE Xiao-juan ZHU Fan-li XIE Tian-hong LONG Run-xiang YANG Rong LUO Fang-yu XIE Zhong-ping(Institute of Medical Biology, Peking Union Medical College, Chinese Academy of Medical Science, Yunnan Provincial Engineering and Technological Research Center for Development of Vaccines against Major Infectious Diseases, Yunnan Provincial Key Laboratory for Development of Vaccines against Major Infections Disease, Kunming 650118, Yunnan Province, China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗工程技术研究中心云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2017年第1期19-24,共6页Chinese Journal of Biologicals

基　　金：国家高技术研究发展计划(863计划项目)(2012AA02-A404);云南省应用基础研究面上项目(2011FZ210)

摘　　要：目的原核表达重组人呼吸道合胞病毒(human respiratory syncytial virus,HRSV)A型Long株截短F1融合性蛋白,并评价其免疫原性。方法采用RT-PCR法扩增HRSV截短F1蛋白基因序列,克隆至pET-28b表达载体,构建重组原核表达质粒RSV F1/pET-28b,转化大肠埃希菌Shuffle T7菌株,IPTG诱导表达,表达产物经SDS-PAGE分析;用Ni亲和层析柱纯化,纯化产物经SDS-PAGE及Western blot分析。将纯化的重组截短F1蛋白免疫ICR小鼠,ELISA法检测小鼠血清抗体效价。结果重组原核表达质粒经双酶切及测序鉴定证明构建正确;表达的截短F1蛋白相对分子质量约44 000,主要以包涵体形式存在,纯度达90.6%。重组截短F1蛋白免疫小鼠血清抗体效价最高可达1∶4 096。结论原核表达的重组HRSV截短F1蛋白免疫原性好,为单克隆抗体的制备及检测试剂盒的研究奠定了基础。Objective To express the truncated F1 protein of Long strain of human respiratory syncytial virus（HRSV）type A in prokaryotic cells and evaluate its immunogenicity. Methods The truncated F1 fragment of HRSV was amplified by RT-PCR and cloned into expression vector pET-28 b. The constructed recombinant plasmid RSV F1/pET-28 b was transformed to E. coli Shuffle T7 for expression under induction of IPTG. The expressed product was identified by SDSPAGE, then purified by nickel ion affinity chromatography and further identified by SDS-PAGE and Western blot. ICR mice were immunized with the purified protein, of which the serum antibody titer was determined by ELISA. Results Restriction analysis and sequencing proved that recombinant plasmid RSV F1/pET-28 b was constructed correctly. The expressed truncated F1 proved, with a relative molecular mass of about 44 000, mainly existed in a form of inclusion body,and reached a purity of 90. 6%, with which the serum antibody titer induced in mice were 1 ∶ 4 096 at most. Conclusion The truncated F1 protein of HRSV showed good immunogenicity, which laid a foundation of preparation of monoclonal antibody and development of detection kit.

关 键 词：呼吸道合胞病毒 截短F1蛋白 原核表达 免疫原性 

分 类 号：R373.1[医药卫生—病原生物学] Q786[医药卫生—基础医学]