DEN-2分离株E基因部分序列原核蛋白表达  

Prokaryotic expression of E partial nucleotide sequence of clinical isolated strain of dengue virus type 2

作　　者：刘世国[1] 左丽[1] 王娇[1] 

机构地区：[1]贵阳医学院免疫学教研室,贵阳550004

出　　处：《中国公共卫生》2009年第6期660-662,共3页Chinese Journal of Public Health

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

摘　　要：目的通过构建登革2型病毒(DEN-2)临床分离株(B株)E基因区1-476bp的原核表达载体,进行原核表达,为DEN-2E蛋白功能的研究提供基础依据。方法将DEN-2B株E基因区部分序列克隆入原核表达载体pET28a(+),命名为pET28a(+)-Eb;经酶切、PCR及测序鉴定后转化BL21(DE3)菌株,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过十二烷基硫酸钠-聚丙烯胺凝胶电泳(SDS-PAGE)、蛋白印迹(Western blot)鉴定表达蛋白;对表达蛋白进行纯化,并滴定对C6/36细胞的细胞半数致死量(TCID50)。结果成功构建了pET28a(+)-Eb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,其相对分子量约为23kDa,表达量约占菌体总蛋白的29%;Western blot表明,该目的蛋白可与DEN-2鼠单克隆抗体结合。用Ni柱亲和层析法纯化原核表达蛋白,纯度达90%。DEN-2B株E基因部分序列原核表达蛋白对C6/36细胞的TCID50为10-5.31。结论pET28a(+)-Eb可在BL21(DE3)菌株中高效表达;DEN-2B株E基因部分序列原核表达蛋白对C6/36细胞有明显的细胞毒作用。Objective To construct prokaryotic expression vector of E gene partial sequence of B strain dengue virus type 2 for prokaryotic expression for further study of dengue virus. Methods E gene partial sequence of B strain dengue vi- rus type 2 was amplified by RT-PCR,and then inserted into prokaryotic vector pET28a（ ＋ ） and transformed to E. coli BL21 cells. The E gene partial sequence was expressed with IPTG induction. Results The expressed products was identified by SDS-PAGE and Western-Blot and purified. Meanwhile, the cytotoxicity of the purified protein on C6/36 was detected. The experimental results showed that prokaryotic expression recombinant vectors pET28a（ ＋ ） - Eb was successfully construc- ted. SDS-PAGE assay suggested that the recombinant proteins with a relative molecular weight of 23KDa could be highly ex- pressed in BL21 and the yields were 29% of total bacterial proteins, the result of Western-Blot indicated that the expression products could specifically react with monoantibody against dengue virus type. Vitro cytotoxic experiments suggested that target proteins had relatively cytotoxic function for C6/36. Conclusion pET28a（ ＋ ）-Eb with E gene partial sequence can be highly expressed in BL21. The antigenicity of target protein provide a potential source for further developing dengue virus diagnosis reagent. Cytotoxic experiments suggest that target proteins had relatively cytotoxic function for C6/36.

关 键 词：登革2型病毒(DEN-2) E基因序列 原核表达 

分 类 号：Q754[生物学—分子生物学]