线虫CPL-1的原核表达和多克隆抗体的制备

Prokaryotic expression of C.elegans CPL-1 and production of polyclonal antibody

文章构建了线虫Cathepsin L相似蛋白酶(Cathepsin L-like proteases-1,CPL-1)原核表达体系成功获得原核表达蛋白,并制备兔源CPL-1多克隆抗体。首先提取线虫RNA运用逆转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)技术获得目的基因cpl-1,构建重组原核表达载pET28a-cpl-1,将其转化到大肠杆菌BL21中诱导表达。CPL-1融合蛋白经Ni亲和层析法纯化后,作为抗原免疫兔子获得CPL-1多克隆抗体。采用ELISA测定抗体效价发现制备的抗血清效价高达1∶256000,用western blot检测发现抗体特异性良好。CPL-1蛋白的表达和抗体的成功制备为研究CPL-1在线虫中的生物学功能奠定了基础。

The prokaryotic expression system of Cathepsin L-like proteases-l（CPL-l） in C. elegans was constructed, the prokaryotic expression protein was obtained, and the polyclonal antibody against CPL i for immunizing rabbit was produced. The cpl-1 gene was amplified by reverse transcription polymerase chain reaction（RT PCR）, and the recombinant prokaryotic expression plasmid（pET28a cpl- 1） for CPL-1 was constructed. The recombinant protein was successfully expressed in BL21 E. coli expression system. Subsequently, the recombinant protein was purified by Ni NTA Superflow Agar ose. After the confirmation of the purity, the protein was injected to rabbit with Freund＇s Adjuvant. Anti serum was collected to detect the titer and specificity by ELISA and western blot, respectively. The final titer of antibody is about 1 ： 256 000. The good specificity of the antibody was also identified by western blot. The expression of the CPL-1 protein and the successful production of its polyclonal antibody will be valuable for the study of CPL-1 in C. elegans.