羊传染性脓疱病毒122蛋白的原核表达纯化及多克隆抗体的制备

Prokaryotic expression and purification of goat infectious pustular virus 122protein and preparation of polyclonal antibody

以提取的羊传染性脓疱病毒(ORFV)基因组为模版,PCR扩增ORFV122基因,并将该基因连接至原核表达载体pET-28a上,构建出pET-28a-122重组表达质粒。然后将该重组质粒转化至大肠杆菌BL21(DE3)感受态,经IPTG诱导后成功表达出ORFV122蛋白。利用His标签可与镍离子结合的特性对ORFV122蛋白进行分离纯化。将纯化后的ORFV122蛋白免疫BALB/c小鼠,制备相应的多克隆抗体,并利用Western blot技术检测该抗体的特异性。免疫小鼠获得的抗ORFV122蛋白多克隆抗体经ELISA检测,其效价约为1∶80000。此外,使用该抗体对ORFV122蛋白的表达规律进行了研究,发现ORFV122蛋白为早期表达蛋白。本试验获得的ORFV122蛋白多克隆抗体为以后ORFV122基因功能的后续研究奠定基础。

This experiment aimed to study the expression of ORFV122protein in the process of ORFV infection by preparing polyclonal antibody against ORFV122protein.The genome of ORFV was extracted,and the ORFV122gene of 972bp in length was amplified by PCR.The target gene was cloned into the prokaryotic expression vector PET-28ato construct a recombinant plasmid of PET-28a-122.The recombinant plasmid was then transformed into E.coli BL21(DE3),and the corresponding protein of interest was successfully expressed after induction by IPTG.The Histag is used to separate and purify the target protein by the characteristics of binding to the nickel column.The purified protein was immunized to BALB/c mice to prepare a polyclonal antibody against the protein of interest,and the specificity of the antibody was detected by western blot.The polyclonal antibody obtained after immunization of mice was tested by ELISA to obtain a titer of about 1∶80000.The polyclonal antibody of ORFV122protein obtained in this study laid the foundation for the subsequent study of ORFV122gene.