非洲猪瘟病毒pE248R蛋白的原核表达及其多克隆抗体制备

Prokaryotic Expression of African Swine Fever Virus pE248R Protein and Preparation of Polyclonal Antibodies

本研究利用PCR方法从非洲猪瘟病毒的灭活样品中扩增出747 bp的E248R基因全长序列,利用同源重组法构建原核表达质粒pCold I-pE248R,经1 mmol/L的IPTG诱导1 h后,利用SDS-PAGE对重组蛋白进行表达鉴定和反应原性分析,表达蛋白的分子量约为32 kDa,利用纯化后得到pE248R重组蛋白作为免疫原经过4次免疫后制备鼠源抗pE248R多克隆抗体。利用该多克隆抗体检测实验室构建并拯救的已证明能够稳定表达ASFV pE248R蛋白的重组病毒rPRRSV-E248R,结果显示制备的抗pE248R的多克隆抗体能够与rPRRSV-E248R发生特异性结合反应,证明利用本试验中原核表达的pE248R蛋白制备的多克隆抗体具有抗pE248R蛋白的特异性,为进一步针对ASFV E248R基因建立快速,特异性的血清学检测方法奠定了基础,也为针对pE248R蛋白的功能性研究提供了研究基础。

In this study,the 747 bp E248R gene was amplified by PCR from inactivated samples of African swine fever virus(ASFV),and the prokaryotic expression plasmid pCold I-pE248R was constructed by homologous recombination method.After induced with 1 mmol/L IPTG for 16 h,the recombinant protein was identified with a molecular mass of 32 kDa in SDS-PAGE.The purified pE248R recombinant protein was used to immunize mice 4 times to prepare anti-pE248R polyclonal antibodies.Then the polyclonal antibodies were used to detect the recombinant viruses rPRRSV-E248R,which was constructed and rescued by our laboratory and had been proved to stably express ASFV pE248R protein.The results showed that the E248R.The availability of the polyclonal antibodies against the prokaryotic pE248R protein laid a foundation for developing a rapid and specific serological detection method for ASFV E248R gene,and also provided a research foundation for the functional study of pE248R protein.