重组人转化生长因子β1原核表达及多克隆抗体制备

摘要

Purpose To clone human TGF-pl gene and prokaryotic expression of TGF-pl protein through the expression of this gene, and then to get the rabbit-anti-human TGF-pi polyclonal antibody from the immune rabbit with this protein. Methods Using RT-PCR technique to amplified human TGF-pl gene sequence. Then the target gene was cloned into a prokaryotic expression vector pET-28a by using the recombinant DNA technique. After induced by IPTG,the protein TGF-pl was expressed in E. coli BL21 ( DE3) . The protein testified its antigenicity by Western-blot. Next, we immunized New Zealand white rabbits with the obtained recombinant protein as antigen. We collected the immune sera of rabbit. Then purified antibody by ammonium sulfate fractionationthen, the indirect Enzyme-linked immunosorbent assay was used to detect the polyclonal antibodies titer. Western-blot techniques were used to identify antibody specificity. Results The encoding sequence and expression vector of TGF-pl was obtained while the interest protein was mainly expressed in the inclusion body. Western-blot detection of the target protein displays antigenicity. The purified rabbit anti-human polyclonal antibodies titer is l:10 000. Conclusion The rabbit anti-human TGF-pi polyclonal antibodies titer is higher and has a better specificity.%目的 克隆人转化生长因子β1( TGF-β1)基因,原核表达TGF-β1蛋白,制备兔抗人TGF-β1多克隆抗体.方法 应用RT-PCR技术扩增人TGF-β1基因序列,构建pET-28a-TGF-β1重组质粒.转化至E.coli BL21( DE3)中,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达.Western-blot检测目的蛋白的抗原性.免疫新西兰白兔,获得兔抗人TGF-β1多克隆抗血清.饱和硫酸铵盐析法纯化多克隆抗体,间接ELISA法检测多克隆抗体效价,Westernblot技术进行抗体特异性检测.结果 获得了TGF-β1编码序列和表达载体,目的蛋白主要存在于超声破碎后的包涵体中;经Western-blot检测目的蛋白存在抗原性;获得纯化的兔抗人多克隆抗体效价达1:10 000.结论 获得的兔抗人TGF-β1多克隆抗体效价较高,具有良好的特异性.