刘世国,左丽,王娇
LIU Shiguo,ZUO Li,WANG Jiao(Department of Immunology

• 1:贵阳医学院免疫学教研室
• 2:华中理工大学医学院
• 3:贵州省中心血站

摘要(Abstract)：

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 recombinant vector,pET28a(+)-Eb,was successfully constructed. SDS-PAGE assay showed that the gene E partial sequence could be highly expressed in BL21,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 10~4.88/0.1ml. 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(KeyWords)： 登革热病毒;基因,E;基因顺序;基因表达
dengue virus; genes,E; gene order; gene expression

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 刘世国,左丽,王娇
LIU Shiguo,ZUO Li,WANG Jiao(Department of Immunology

DOI: 10.19367/j.cnki.1000-2707.2008.06.006

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