丙型肝炎病毒NS5A反式激活基因1的原核表达及多克隆抗体制备  

Prokaryotic expression of HCV NS5ATP1 gene and preparation of polyclonal antibody

作　　者：郑铁龙[1] 成军[2] 洪源[2] 李晓光[3] 张延峰[1] 

机构地区：[1]北京军事医学科学院研究生大队,100850 [2]北京地坛医院传染病研究所 [3]军医进修学院研究生大队

出　　处：《解放军医学杂志》2008年第1期59-61,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(C030114)

摘　　要：目的构建丙型肝炎病毒非结构蛋白NS5A的反式激活蛋白1(NS5ATP1)基因的原核表达载体并诱导其表达,纯化表达的融合蛋白并制备多克隆抗体。方法从pGBKT7-NS5ATP1质粒上切取NS5ATP1基因,克隆入pET32a(+)质粒,构建pET32a(+)-NS5ATP1表达载体,IPTG诱导融合蛋白表达。亲和镍柱层析纯化该融合蛋白后,免疫新西兰兔,获得多克隆抗体,ELISA法检测多抗效价。结果以pET32a(+)-NS5ATP1表达载体分别转化DH5α、BL21菌后,经IPTG诱导,成功获得分子量为56kD左右的重组蛋白。SDS-PAGE分析显示,IPTG诱导4.5h时重组蛋白的表达量最高。Western blot证实其具有良好的抗原性。用该蛋白成功免疫新西兰白兔,获得了该蛋白的多克隆抗体,ELISA检测其效价>1∶512000。结论成功构建原核表达载体pET32a(+)-NS5ATP1,表达纯化NS5ATP1融合蛋白并制备了该蛋白的多克隆抗体,为进一步的研究奠定了基础。Objective To construct prokaryofic expression vector of hepatitis C virus NSSATP1 gene, and to induce its expression in 17- coli. To purify the fusion protein and obtain its polyclonal antibody from immunized New Zealand rabbits. Methods The NSSATP 1 gene, which was cut from the vector pGBKT7-NS5ATP1, which was self constructed by the authors, was cloned into plasmid pET32a （＋） to construct the pET32a（＋）-NSSATP1 prokaryotic expression vector. It was proved that the recombinant plasmid was constructed correctly by sequencing. And then the expression vector was transformed into the competent E. coli DHSa and BL21. After being induced with IPTG, the NS5ATP1 fusion protein was expressed and analyzed with SDS-PAGE and Western blot. The transformed bacteria were fragmented by ultrasonic and then separated by SDS-PAGE. The fusion protein formed inclusion body. They were then purified and re-natured through Ni affinity column chromatography. The purified pET32a（＋）-N5SATP1 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and immunizing potence of polyclonal antibody were evaluated by Western blot and ELISA. Results After transferring pET32a（＋）-NSSATP1 plasmid into DH5a and BL21 and induced with TPTG, the NSSATP1 fusion protein of about 56kD was highly expressed. SDS-PAGE analysis showed that the fusion protein products were mainly in inclusion body and expressed in the highest level at 4. 5h of inductior. The purified protein and polyclonal antibody were obtained successfully. ELISA manifested the titer of polyclonal antibody was over 1：512 000. The high specificity was testified by Western blot. Conclusions The suc cessful expression and purification of NSSATP 1 fusion protein and the preparation of NSSATP 1 specific polyclonal antibody will be valuable for the study on the biological function of NSSATP1.

关 键 词：肝炎病毒 丙型 NS5ATP1基因 原核表达 

分 类 号：R512.63[医药卫生—内科学] R34-33[医药卫生—临床医学]