猪干扰素α－1基因的原核表达与多克隆抗体制备

摘要

Objective] The porcine interferon alpha 1 gene (PoIFNα-1) was expressed by using Escherichia coli prokaryotic expression system, and polyclonal antibodies against the recombinant protein PoIFNα-1 were prepared. [Method] The codon-optimized PoIFNα-1 gene was synthesized according to the nucleotide sequence published by GenBank, and then the synthesized PoIFNα-1 gene was cloned into the vector pET-28a. After transformation of the recombinant plasmid into E. coli BL21 (DE3) host bacteria, the recombinant bacterium was induced by using IPTG. After purification of the recombinant protein PoIFNα-1 expressed in E. coli BL21 (DE3), the polyclonal antibodies against the recombinant protein PoIFNα-1 was prepared in Kunming mice, and then reactivity of the prepared polyclonal antibodies with native porcine interferon alpha 1 was determined by Western blot. [Result] The synthesized PoIFNα-1 gene was successfully expressed in E. coli BL21 (DE3), and the polyclonal antibody against recombinant protein PoIFNα-1 could recognize the native porcine interferon-α-1. [Conclusion] The synthesized PoIFNα-1 gene was successfully expressed in E. coli, which provided some basis for following further research of antivirus.%[目的]利用大肠杆菌原核表达系统对猪干扰素α-1（PoIFNα-1）基因进行表达，制备PoIFNα-1重组蛋白多克隆抗体。[方法]根据GenBank发表的PoIFNα-1核苷酸序列，合成密码子优化的PoIFNα-1基因，构建PoIFNα-1基因的pET-28a原核表达载体，转化大肠杆菌BL21（DE3）宿主菌后，利用IPTG进行诱导表达。表达的PoIFNα-1重组蛋白纯化后，免疫昆明系小鼠，制备多克隆抗体。采用Western blot技术检测PoIFNα-1重组蛋白的多克隆抗体与天然PoIFNα-1的反应性。[结果]合成的PoIFNα-1基因在大肠杆菌BL21（DE3）宿主菌中成功表达，针对PoIFNα-1重组蛋白的多克隆抗体能够识别天然的猪干扰素α-1。[结论]合成的PoIFNα-1基因在大肠杆菌中获得了成功表达，为后续研究奠定了基础。