摘要
目的构建丙型肝炎病毒NS5A蛋白反式激活蛋白6基因的原核表达载体并进行表达、鉴定。方法从已构建的pGBKT7-NS5ATP6质粒上切取NS5ATP6基因,再克隆入pET32a(+)质粒,构建pET32a(+)-NS5ATP6表达重组体。结果以pET32a(+)-NS5ATP6重组体分别转化DH5α和Rosseta大肠埃希菌后,经IPTG诱导,pET32a(+)-NS5ATP6表达出分子量为41KD左右的重组蛋白。免疫动物并经Westernblot检测证实其具有良好的抗原性。结论成功地构建了原核表达载体pET32a(+)-NS5ATP6,诱导表达和纯化了NS5ATP6融合蛋白,并制备了该蛋白的多克隆抗体,为下一步该基因功能研究奠定了基础。
Objective To construct prokaryotic expression vector carrying NS5ATP6 gene of hepatitis C virus and express it in E. coli. Rosseta. Methods The NS5ATP6 gene was come from the vector pGBKT7-NS5ATP6 constructed by ourselves. After sequencing, the gene was cloned into plasmid pET32a（ ＋ ） to construct recombinant prokaryotic expression vector pET32a（＋）-NS5ATP6 and transformed into the competent E. coli BL21. After the NS5ATP6 protein was induced with ]PTG,it was analyzed with SDS-PAGE and confirmed with Western blot. Expressed bacteria were quassationed by ultrasound and analyzed with sodium dodecylsulfate/polyacrylamide gel electrophoresis. The expressed product was purified and renatured by Nff affinity column chromatography. Then,the purified pET32a（ ＋ ）-NS5ATP6 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and potency of polyclonal anti- body were evaluated by Western blot and ELISA. Results The NS5ATP6 fusion protein was highly expressed. The pro- tein production was mainly in inclusion body through SDS-PAGE analysis. The purified protein and polyclonal antibody were obtained successfully. The titer of polyclonal antibody was 〉 1：512,000 by ELISA. The high specificity was testified with Western blot. Conclusions The successful expression and purification of NS5ATP1 fusion protein and the preparation of NS5ATP6 specific polyclonal antibody will be valuable for the further study on the biological function of NSSATP6.
出处
《实用肝脏病杂志》
CAS
2008年第1期2-5,49,共5页
Journal of Practical Hepatology
基金
国家自然科学基金资助项目(C030114)
