重组人转化生长因子β1原核表达及多克隆抗体制备

Recombinant human TGF-β1 prokaryotic expression and polyclonal antibody preparation

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,Western-blot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。

Purpose To clone human TGF-β1 gene and prokaryotic expression of TGF-β1 protein through the expression of this gene,and then to get the rabbit-anti-human TGF-β1 polyclonal antibody from the immune rabbit with this protein.Methods Using RT-PCR technique to amplified human TGF-β1 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-β1 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-β1 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 1∶ 10 000.Conclusion The rabbit anti-human TGF-β1 polyclonal antibodies titer is higher and has a better specificity.