利用PRNP^(-/-)羊制备朊蛋白多克隆抗体及其特性分析

Production and Characterization of Anti-PrP Polyclonal Antibody by PRNP Gene Knockout Goat

【目的】利用朊蛋白双基因敲除(PRNP-/-)羊制备朊蛋白多克隆抗体,并对其特性进行分析。【方法】构建山羊朊蛋白(PrP)原核表达载体,转入大肠杆菌并诱导表达,纯化获得羊PrP;将获得的PrP免疫PRNP-/-山羊,制备朊蛋白特异性的多克隆抗体;并对获得的朊蛋白多克隆抗体进行ELISA及Western-blot检测。【结果】获得了大量的朊蛋白特异性抗血清,间接ELASA检测抗血清中朊蛋白多克隆抗体的效价为25600;Western-blot检测显示所制备抗体不仅可以识别鼠、牛、羊脑组织内源性朊蛋白,而且能识别鼠脑组织内朊病毒。【结论】PRNP-/-转基因山羊可用于制备大量高亲和力朊蛋白多克隆抗体,获得的抗体可用于多种动物朊蛋白及朊病毒类疾病的检测。

【Objective】The study was conducted in order to produce and characterize anti-PrP polyclonal antibodies by prion protein knockout（PRNP-/-） goat.【Method】Using polymerase chain reaction（PCR） method,the DNA sequence encoding the goat mature PrP（Prion Protein） was amplified from goat DNA.It was then cloned into the vector pET-22b to express recombinant PrP protein.After purification,the recombinant PrP was used as antigen to immunize PRNP-/-goat for producing anti-PrP antibodies.ELISA and Western blot were conducted to characterize the titer and specificity of polyclonal antibodies.【Result】A large scale of anti-PrP polyclonal antibodies was successfully produced by PRNP-/-goat.ELISA assay revealed that the titer of the polyclonal antibodies against PrP was as high as 1﹕25 600.Western blot test showed that the antibody was not only able to react with the native prion proteins（PrPC） of mouse,cattle and goat brain,but also could combine with misfolded prion proteins（PrPSc） from mouse brain.【Conclusion】Ablation of the prion protein（PrP） gene in goat facilitates the production of a large scale anti-PrP polyclonal antibodies,the produced antibody can be widely used in detecting PrPC and PrPSc derived from a variety of animals.