鸡干扰素调节因子7的原核表达及多克隆抗体的制备

Prokaryotic Expression of chIRF7 Protein and Preparation of Polyclonal Antibody

为表达鸡干扰素调节因子7蛋白并制备其多克隆抗体,通过RT-PCR技术扩增chIRF7基因的编码序列,构建重组质粒PET30a-chIRF7。将重组质粒转化至大肠埃希菌(E.coil)BL21(DE3)后经IPTG诱导,SDS-PAGE结果显示,重组蛋白rchIRF7分子量约为60ku。将纯化的重组蛋白免疫接种小鼠制备多克隆抗体,间接ELISA测定血清抗体滴度在1∶51 200以上,IFA检测结果显示制备的鼠抗chIRF7蛋白多抗能够与AIV刺激的CEF细胞内的chIRF7蛋白发生特异性反应,表明通过原核表达系统表达的重组蛋白rchIRF7具有很好的免疫原性,以其制备的鼠抗chIRF7蛋白多克隆抗体滴度高、特异性好。

The chIRF7 gene was amplified by RT-PCR and inserted into pET30a vector to construct recombinant plasmid pET30a-chIRF7. Then, the pET30a-chIRF7 was transformed into BL21（DE3） and induced with IPTG, the SDS-PAGE results indicated that the recombinant protein rchIRF7 was about 60 ku. The purified recombinant protein was used to immunize mice to prepare polyclonal antibody, the titer of the polyclonal antibody was above 1 ： 51 200 detected with indirect ELISA, the IFA test results indicated that the polyclonal antibody could recognize chIRF7 protein expressed in CEF cells stimulated by AIV specifically,it revealed that the chIRF7 recombinant protein had high immunogenicity and the prepared polyclonal antibody had high titer and specificity.