分离型痘苗病毒表达载体的构建及HIV-1 gag p17-24的表达  

Construction of Vaccinia Virus Vectors and Expression of HIV-1 gag p17-24 Protein

作　　者：金宁一[1] 王宏伟[1] 殷震[1] 李体远 王玉红 罗坤 安汝国 

机构地区：[1]解放军农牧大学研究所病毒室,长春130062 [2]深圳市人民医院中心实验室,深圳518020

出　　处：《中国肿瘤生物治疗杂志》1999年第1期8-11,共4页Chinese Journal of Cancer Biotherapy

基　　金：总后卫生部杰出中青年基金和国家自然科学基金(39770661)资助

摘　　要：目的:探索荧光蛋白作为报告基因以及His·Tag作为纯化标签在重组痘苗病毒表达系统中的应用.方法:将N-端融合His·Tag基因序列,插入痘苗病毒表达载体pSFJ2-16,并在其多克隆位点下游装入带有巨细胞病毒(CMV)启动子的突变型荧光蛋白基因(GFP),构建成N-端融合His·Tag并带有荧光蛋白报告基因的分离型痘苗病毒表达载体.再从pET-28中切出C-端融合His·Tag及多克隆位点序列,并与CMV-FP基因一同装入pSFJ2-16,构建成C-端融合His·Tag的分离型痘苗病毒表达载体.将人免疫缺陷病毒-1型(HIV-1)核心蛋白(gag)p17-24基因分别插入上述载体,并筛选出重组病毒.结果:实验表明,重组病毒表达了gag蛋白.结论:将His-Tag的纯化标签加入到通用型痘苗病毒表达载体中用于纯化表达的目的蛋白,该方法是可行的.荧光蛋白基因作为一个筛选标记基因应用于痘苗病毒载体系统还有待进行进一步研究.Objective: To research the possibility of using fluorescence protein as the selecting reporter gene and the His Tag as the purification marker in recombinant vaccinia virus expression system. Methods: Two recombinant plasmids, p16HbF/p17-24 and p16HlF/p24, were constructed by inserting the HIV-1 p24^(gag) and p17-24gag gene into the vaccinia virus vectors, p16HlFP and p16H2FP, in which the mutant fluorescence protein (FP) marked as report gene, respectively . These two recombinants were designed to express the 6 × His-tagged N- or C-terminus fusion protein. Results: The results of indirect immunoflorescence assay, Western blot and Dot-ELISA showed that all the recombinant vaccinia virus could express the p24gag, p17-24gag fusion protein in infected BHK21 cells. Conclusion: The way for the purification of foreign protein by inserting His Tag gene into vaccinia virus vectors was reasonable and using fluorescence protein as a selecting marker should be researched further.

关 键 词：痘苗病毒 荧光蛋白 HIV-1 p17-24 载体构建 

分 类 号：Q782[生物学—分子生物学] R512.91[医药卫生—内科学]