基于口蹄疫病毒2A片段的多基因共表达质粒的构建及表达  

Construction and expression of a multi-gene co-expression plasmid based on foot and mouth disease virus 2A segment

作　　者：刘丹清 廖勇[2] 刘敏慧[1] 刘俊英[1] 盛云建[1] 童师雯[1] 汤慧[2] 任红[1,2] 

机构地区：[1]重庆医科大学附属第二医院感染科,重庆400016 [2]重庆医科大学病毒性肝炎研究所,重庆400016

出　　处：《中国生物制品学杂志》2014年第3期342-347,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(63004601)

摘　　要：目的构建基于口蹄疫病毒(foot and mouth disease virus,FMDV)2A片段的多基因共表达质粒,并在293T细胞中共表达HBx、人Sox2及人c-Myc基因。方法以FMDV 2A自剪切片段连接HBx、Sox2和c-Myc基因编码序列,克隆至载体pEGFP-C1,构建多基因共表达质粒pEGFP-XSM;在脂质体LipofectamineTM2000的介导下,将重组质粒pEGFP-XSM转染293T细胞,同时设空载体组(转染空载体pEGFP-C1)和空白对照组(未转染),转染48 h后,荧光显微镜下观察细胞中绿色荧光蛋白(green fluorescent protein,GFP)的表达;Real-time PCR和Western blot法分别检测各组293T细胞中HBx、Sox2和c-Myc基因mRNA的转录水平和蛋白的表达水平。结果多基因共表达质粒pEGFP-XSM经酶切和测序鉴定构建正确;空载体组和pEGFP-XSM转染组细胞荧光镜下均可见GFP的表达;pEGFP-XSM转染组中HBx、Sox2和c-Myc基因mRNA的转录水平较空白对照组及空载体组均明显升高(P﹤0.001);pEGFP-XSM转染组可见相对分子质量约48 000的GFP-HBx-2A融合蛋白条带和约38 000的HA-Sox2-2A融合蛋白条带,各组均可见相对分子质量约49 000的c-Myc蛋白条带,pEGFP-XSM转染组c-Myc蛋白的表达水平明显高于空白对照组及空载体组(P﹤0.01)。结论成功构建了多基因共表达质粒pEGFP-XSM,并在293T细胞中实现了HBx、Sox2和c-Myc蛋白的共表达,为进一步深入研究该共表达蛋白在肝细胞肝癌(hepatocellular carcinoma,HCC)发生发展中的生物学机制奠定了基础。Objective To construct a muhi-gene co-expression plasmid based on foot and mouth disease virus （FMDV） 2A segment, and express HBx, human Sox2 and human c-Myc genes in 293T cells. Methods The encoding sequences of HBx, Sox2 and c-Myc genes were linked with a single FMDV self-cleaving 2A peptide sequence, and cloned into vector pCR4-TOPO. The constructed muhi-gene co-expression vector pEGFP-XSM was transfected to 293T cells in mediation of LipofectamineTM 2000, using that transfected with empty vector and that untransfected as controls, then observed for ex- pression of green fluorescent protein （GFP） by fluorescence microscopy 48 h later, and for those of HBx, Sox2 and c-Myc mRNAs and proteins by real-time fluorescent quantitative PCR and Western blot respectively. Results Restriction analy- sis and sequencing proved that muhi-gene co-expression vector pEGFP-XSM was constructed correctly. The expression of GFP was observed in both the cells transfected with empty vector and pEGFP-XSM under fluorescent microscope. The mRNA transcription expression levels of HBx, Sox2 and c-Myc in the cells transfected with pEGFP-XSM were significant- ly higher than those transfected with empty vector and those untransfected （P 〈 0. 001 ）. Fusion protein GFP-HBx-2A with a relative molecular mass of about 48 000 and HA-Sox2-2A with a relative molecular mass of 38 000 were observed in pEGFP-XSM transfection group, while c-Myc protein with a relative molecular mass of about 49 000 in various groups. However, the expression level of c-Myc in 293T cells transfected with pEGFP-XSM was significantly higher than thoseuntransfected and those transfected with empty vector pEGFP-C1 （P 〈 0. O1 ）. Conclusion Multi-gene non-fusion co- expression plasmid pEGFP-XSM was successfully constructed, and HBx, Sox2 and c-Myc proteins were co-expressed in 293T cells, which laid a foundation of further study on biological mechanism of the co-expressed proteins in pathogenesis and progress hepatocellular carcinoma （HCC）.

关 键 词：口蹄疫病毒2A片段 多基因 共表达 

分 类 号：S855.3[农业科学—临床兽医学] Q786[农业科学—兽医学]