HGV NS5A区部分基因的克隆及表达  

Molecular cloning and expression of the NS5A genomic region of HGV

作　　者：马会慧[1] 李刚[1] 廖家杰 梁英杰 杨绍基[1] Berwyn Clarke 姚集鲁[1] 

机构地区：[1]中山大学附属三院传染科,广州510630 [2]香港玛丽医院胃肠肝病科 [3]粤港肝炎研究中心 [4]VirologyUnit.GlaxowellcomeMedicinesCentreEngland

出　　处：《热带医学杂志》2001年第2期123-125,共3页Journal of Tropical Medicine

基　　金：国家自然科学基金 (No .396 0 0 130 );广东省医药卫生科研基金部分资助项目

摘　　要：目的　构建含HGVNS5A区基因的原核表达载体。方法　采用PCR方法从重组了HGV 6 6 72 - 72 92区段基因的pUC19中扩增出HGV 6 86 4- 72 77nt片段 ,定向克隆入pGEMEX1质粒 ,再经SacⅠ、HindⅢ酶切亚克隆入高效表达载体pRSETA。结果　重组pRSETA 经序列测定证实插入基因为目的基因 ,符合表达框架 ,经IPTG诱导 ,在BL2 1(DE3)菌株中表达连接 6个组氨酸的HGVNS5A融合蛋白。Westernblotting显示在Marker约 2 5kDa处可见明显的蛋白带 ,与预期结果一致。结论　成功构建了重组HGVNS5A区基因表达载体 ,在BL2 1(DE3)中能有效表达。Objective To construct recombinant expression vector containing the NS5A genomic region of HGV. Methods The 6864-7277nt fragment of HGV NS5A region from the recombinant pUC19 containing 6672-7292nt region of HGV genome was amplified with PCR. The PCR product was directionally cloned into pGEM EXⅠ.The insert was excised win SacⅠ and Hind Ⅲ and subcloned into high expression vector pRSET A.Results The inserted fragments in pRSET A was confirmed by DNA sequeencing and was inframe with the expression vector.IPTG induced the exprssion of the 6 histidine-HCG NS5A queion protein in BL21(DE3) transformed cells.Western blotting showed a position band around 25 kDa in size.Conclusion The recombinant expression vector is constructed successfully, which could express the recombinant HGV.NS5A protein in BL21(DE3) cells.

关 键 词：庚型肝炎病毒 分子克隆 基因表达 

分 类 号：R373.21[医药卫生—病原生物学]