摘要
目的:构建丙型肝炎病毒核心抗原结合蛋白12(HCVCore binding protein,HCBP12)原核表达载体,在大肠埃希菌中进行表达、纯化HCBP12融合蛋白并制备兔抗HCBP12多克隆抗体。方法:应用逆转录聚合酶链反应(RT-PCR),以提取的HepG2细胞mRNA为模板,扩增获得HCBP12基因片段,插入至原核表达载体pET-32a(+)中,构建原核表达载体pET-32a(+)-HCBP12,转化大肠埃希菌BL21,以异丙基硫代β-D-半乳糖苷(IPTG)诱导,获得HCBP12融合蛋白的可诱导性表达,并通过SDS-PAGE电泳、Western Blot免疫印迹分析和证实融合蛋白表达的特异性。利用Ni+亲和柱对表达蛋白进行纯化及柱上复性。纯化蛋白免疫新西兰兔,获得抗HCBP12多克隆抗体。以纯化HCBP12为抗原,利用Western blot和ELISA法对多克隆抗体进行特异性和效价检测。结果:HCBP12融合蛋白表达成功。SDS-PAGE分析表明其为包涵体表达。成功获得了融合蛋白纯品及兔抗HCBP12多克隆抗体。ELISA法表明多克隆抗体效价>1512000,Western blot检测证明多克隆抗体的特异性良好。结论:成功表达、纯化HCBP12融合蛋白,并获得高特异性、高效价兔抗HCBP12多克隆抗体,为研究HCBP12蛋白的生物学功能及丙肝的临床治疗提供了重要的实验工具。
AIM： To express and purify HCV core binding protein （HCBP12） with prokaryotic cell ex- pressive vector and to prepare HCBP12 specific rabbit polyclonal antibody. METHODS： The DNA segment of HCBP12 was amplified by RT-PCR with mRNA template from HepG2 cells and inserted into inducible prokaryotic expressive vector pET-32a（ ＋ ）. The recombinant plasmid, pET-32a （ ＋ ）-HCBP12, was trans- formed into the competent E. coli BL21. The inducible HCBP12 fusion protein was analyzed by SDS-PAGE and conformed by Western blot. Then the inducible HCBP12 protein was purified and renatured by Ni＋ affinity column chromatography. The purified HCBP12 protein was used to immunize New Zealand rabbits for preparing polyclonal antibody. The specificity and po-tency of polyclonal antibody was evaluated by Western blot and ELISA. RESULTS： The HCBP12 fusion protein was highly expressed and purified. The polyclonal antibody against HCBP12 was obtained successfully with the titer 〉 1： 512000 and confirmed specifically with Western blot. CONCLUSION：The successful expression and purification of HCBP12 fusion protein and the preparation of specific anti-HCBP12 polyclonal antibody will be valuable for the study on the biological function of HCBP12 and clinical therapy of Hepatitis C.
出处
《中国临床药理学与治疗学》
CAS
CSCD
2008年第4期425-430,共6页
Chinese Journal of Clinical Pharmacology and Therapeutics
