重组Ⅱ型登革病毒NS1的表达及其免疫原性的研究  

Clone,Expression and Immunological Analysis of Type ⅡDengue Virus NS1 Protein

作　　者：李多[1] 杨丽娟[2] 赵玉娇[1] 潘玥[1] 陈俊英[1] 付娟娟[1] 黄新伟[1] 邱丽娟[1] 孙强明[1] 

机构地区：[1]北京协和医学院/中国医学科学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,昆明650118 [2]昆明医科大学第二附属医院中心实验,昆明650101

出　　处：《中国生物工程杂志》2014年第9期4-8,共5页China Biotechnology

基　　金：国家自然科学基金面上项目(81171946);云南省科技厅社会发展科技计划项目(2011CA016);云南省自然科学基金(2009ZC187M,2012FB188)资助项目

摘　　要：目的:表达制备重组Ⅱ型登革病毒非结构蛋白NS1,检测该重组表达蛋白的免疫原性,为检测试剂盒的研制提供基础。方法:根据Ⅱ型登革病毒NS1蛋白基因序列(GenBank登录号:NC_001474),对基因序列进行编码密码子优化后进行全基因合成,随后,经双酶切将目的片段克隆到表达载体pET28a上,通过IPTG诱导表达;表达产物经Western blot鉴定后,进一步进行蛋白纯化和透析复性,制备获得重组Ⅱ型登革病毒NS1蛋白。收获的NS1蛋白免疫小鼠,制备多克隆抗体,通过间接酶联免疫学方法测定其效价,检测其免疫原性。结果:成功构建了Ⅱ型登革病毒NS1蛋白的表达载体。Western blot显示表达的重组蛋白能够被NS1单抗特异性的结合,纯化复性后的重组蛋白在小鼠体内表现出良好的免疫原性。结论:重组表达的NS1蛋白有良好的免疫原性,为以后的NS1单克隆抗体的制备和登革病毒检测试剂盒的研制提供了良好的基础。Objective： To express and prepare the recombinant type Ⅱ dengue virus nonstructural protein NS1, and study its immunogenicity. Methods： Type Ⅱ Dengue virus NS1 gene were cloned into plasmid pUC57 after codon-optimizing and gene synthesizing. Then the NS1 gene were subcloned into expression vector pET28a. The NS1 protein were inducing expressed by adding IPTG followed by immunoblot identification. Recombinant NS1 protein were renaturated through dialysis and then purified. NS1 polyclonal antibody were preparation by animal immunization. The titer and immunogenicity were detected by indirect enzyme-linked immunological method. Results： The recombinant type II dengue virus nonstructural protein NS1 were successfully expressed, renaturated and purified. It＇ s proved that the recombinant NS1 protein showed good immunogenicity by Western blotting and mice immunization. Conclusion ：The recombinant NS1 protein has good immunogenicity, and it＇s a good beginning for NS1 monoclonal antibody preparation and the development of dengue virus detection kit.

关 键 词：登革病毒 重组蛋白 NS1蛋白 免疫原性 ELISA 

分 类 号：Q939[生物学—微生物学]