血管活性肠肽与乙肝病毒核心抗原的融合表达、鉴定及其纯化  

Expression, Verification and Purification of Fusion Protein of Vasoactive Intestinal Polypeptide and Hepatitis B Virus Core Antigen

作　　者：黄运茂[1] 施振旦[1] 刘颖[1] 毕英佐[1] 刘丽[1] 

机构地区：[1]华南农业大学动物科学学院,广州510642

出　　处：《农业生物技术学报》2003年第5期506-510,共5页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金资助项目(39700102);广东省自然科学基金资助项目(970013[粤科基(1997)23])。

摘　　要：将血管活性肠肽(VIP)插入到乙肝病毒核心抗原(HBcAg)刺突区的融合基因VIP-HBc分别插入表达质粒pRSET-A的两对克隆位点BamHⅠ\HindⅢ与NheⅠ\HindⅢ,构建重组表达质粒pRSET-VHBH和pRSET-VHNH,经IPTG诱导,均能在大肠杆菌(Escherichiacoli)BL21(DE3)中高水平表达融合蛋白,分别占菌体蛋白的20%和15%,但都以包涵体形式存在。将菌体破碎后上清液在50%Ni-NTA树脂上过柱纯化或经硅胶浓缩后蔗糖浓度梯度超速离心都没有得到可溶的融合蛋白。将包涵体用8mol/L尿素变性后在50%Ni-NTA树脂上过柱纯化能够得到高纯度的融合蛋白。A fusion gene VIP-HBc with vasoactive intestinal polypeptide (VIP) gene inserted into domain coding for loop area of hepatitis B virus core antigen (HBcAg) was cloned into Bam HⅠ\HindⅢ or NheⅠ\HindⅢ cloning sites of expression plasmid pRSET-A to generate reconstructed plasmids pRSET-VHBH and pRSET-VHNH. Induced with IPTG, the two reconstructed plasmids were capable of expressing the target fusion protein in Escherichia coli. strain BL21(DE3). The fusion protein produced by pRSET-VHBH accounted for 20% of the total protein content of the bacteria, whereas that by pRSET-VHNH only 15%, but the 2 fusion proteins were expressed in the form of inclusion body. The soluble fraction of the bacteria cells was purified through 50%Ni-NTA agarose column and centrifuged under sucrose concentration gradient after following concentration by silica gel, but no soluble fusion protein was detected. The inclusion body was denatured with 8 mol/L urea and the soluble fraction was purified through 50% Ni-NTA agarose column, a large amount of the fusion protein was obtained.

关 键 词：血管活性肠肽 乙肝病毒核心抗原 融合蛋白 表达 纯化 

分 类 号：R512.62[医药卫生—内科学] R373.21[医药卫生—临床医学]