登革2型病毒NGC株E基因部分序列原核蛋白表达  

Prokaryotic Expression of Gene E Partial Nucleotide Sequence of Dengue Virus Type 2 Strain NGC

作　　者：刘世国[1,2] 左丽[1] 王娇[1,3] 

机构地区：[1]贵阳医学院免疫学教研室,贵州贵阳550004 [2]华中理工大学医学院 [3]贵州省中心血站

出　　处：《贵阳医学院学报》2008年第6期581-584,588,共5页Journal of Guiyang Medical College

基　　金：国家973计划前期研究专项项目(2008CB517408);国家自然科学基金资助项目(30360101)

摘　　要：目的:构建登革2型病毒NGC株E基因区1～476bp的原核表达载体并进行原核表达。方法:将登革2型病毒NGC株E基因区部分序列克隆入原核表达载体pET28a(+),命名为pET28a(+)-En;经酶切、PCR及测序鉴定后转化BL21(DE3)菌株,用IPTG诱导表达,通过SDS-PAGE、Western印迹鉴定表达蛋白;对表达蛋白进行纯化,并滴定对C6/36细胞的TCID50。结果:(1)成功构建了pET28a(+)-En原核表达重组质粒,SDS-PAGE分析表明,E基因区部分序列获得高效表达,相对分子量约为23kD,表达量约占菌体总蛋白的29%;Western印迹表明该目的蛋白可与登革2型病毒鼠单克隆抗体结合;(2)用Ni柱亲和层析法纯化原核表达蛋白,纯度达90%;(3)DEN-2NGC株E基因部分序列原核表达蛋白对C6/36细胞的TCID50为10-4.88/0.1ml。结论:pET28a(+)-En可在BL21(DE3)菌株中高效表达,DEN-2NGC株E基因部分序列原核表达蛋白对C6/36细胞有一定的细胞毒作用。Objective： To construct expression vector of gene E partial sequence of dengue virus type 2 strain NGC for prokaryotic expression. Methods： Gene E partial sequence of dengue virus type 2 strain NGC was amplified by RT-PCR, inserted into prokaryotic vector pET28a（＋）, and transformed E. coli BL21 cells. The gene partial sequence was expressed under the induction of IPTG. The expressed products was identified by SDS-PAGE and Western-Blot and purified. The cytotoxicity （indicated as TCID50） of the purified protein on C6/36 was detected. Results： Prokaryotic expression re-combinant vector, pET28a（＋）-Eb, was successfully constructed. SDS-PAGE assay showed that the gene E partial sequence could be highly expressed in BI21 ,and the yields were 29% of total bacterial proteins. The expressed protein had a relative molecular weight of 23KDa, and Western-Blot indicated that the expression products could specifically react with monoantibody against dengue virus type 2. Cytotoxic experiments in vitro suggested that the protein had relatively cytotoxicity to C6/36, and the TCID50 was 10-4.88/0. l ml. Conclusion： pET28a（＋）-Eb containing gene E partial sequence can be highly expressed in BL21. The antigenicity of the produced proteins provide a potential source of reagent for dengue virus diagnosis ;The expressed proteins have some cytotoxicity to C6/36.

关 键 词：登革热病毒 基因 E 基因顺序 基因表达 

分 类 号：R373.33[医药卫生—病原生物学]