马疱疹病毒1型ORF2蛋白的原核表达及多克隆抗体的制备

Prokaryotic expression of equine herpesvirus type 1 ORF2 protein and preparation of its polyclonal antibodies

研究旨在克隆马疱疹病毒1型(EHV-1)ORF2基因,通过大肠杆菌原核表达系统获得ORF2蛋白,制备多克隆抗体。根据EHV-1 YM2019株全基因组序列(GenBank:MT063054)设计1对引物,将ORF2基因克隆至pET-28a-ORF2原核表达载体中,通过Transetta(DE3)感受态细胞,异丙基硫代半乳糖苷(IPTG)诱导表达ORF2蛋白,使用Ni-NTA琼脂糖纯化树脂纯化,免疫BALB/c小鼠,检测多克隆抗体的效价和特异性。结果显示:ORF2基因成功克隆至原核表达载体pET-28a中,并通过大肠杆菌表达系统得到ORF2蛋白,大小约为38 ku,以包涵体的形式存在。通过Ni-NTA纯化树脂获得纯度较高的重组蛋白,能够与EHV-1阳性血清特异性结合;间接ELISA方法检测多克隆抗体的效价为1∶64000,RK-13表达的ORF2蛋白可被多克隆抗体能特异性识别。研究表明,大肠杆菌表达的ORF2重组蛋白具有良好反应原性,ORF2蛋白制备的多克隆抗体效价高、特异性强,可为ORF2蛋白的生物学功能和基因缺失疫苗的研究奠定基础。

The purpose of this study was to clone the ORF2 gene of equine herpesviruses type 1(EHV-1),obtain the ORF2 protein by Escherichia coli prokaryotic expression system,and prepare polyclonal antibodies.According to the whole genome sequence of EHV-1 YM2019 strain(GenBank:MT063054),a pair of primers were designed,and ORF2 gene was cloned into pET-28a-ORF2 prokaryotic expression vector.ORF2 protein was induced by Transetta(DE3)competent cells and IPTG,purified by Ni-NTA agarose purification resin,and immunized with balb/c mice to detect the titer and specificity of polyclonal antibody.The results showed that the ORF2 gene was successfully cloned into the prokaryotic expression vector pET-28a,and the ORF2 protein was obtained by Escherichia coli expression system.The size of ORF2 protein was about 38 ku,and it existed in the form of inclusion body.The recombinant protein with high purity obtained by Ni-NTA resin can specifically bind to EHV-1 positive serum.The titer of polyclonal antibody detected by indirect ELISA was 1∶64000,and ORF2 protein expressed by RK-13 could be specifically recognized by polyclonal antibody.The study indicated that the recombinant ORF2 protein expressed by Escherichia coli had good reactivity,and the polyclonal antibody prepared by ORF2 protein had high titer and specificity,which could lay a foundation for the study of biological function of ORF2 protein and gene deletion vaccine.