牛结节性皮肤病病毒ORF141蛋白的原核表达及其多克隆抗体的制备

Prokaryotic expression of lumpy skin disease virus ORF141 protein and preparation of polyclonal antibody

为获得牛结节性皮肤病病毒(lumpy skin diseasevirus,LSDV)ORF141蛋白及其多克隆抗体,本研究参照Gen Bank收录的LSDV ORF141基因序列,利用Snap Gene软件设计引物,以LSDV基因组为模板,利用PCR扩增ORF141基因,构建原核表达重组质粒p ET-30a(+)-ORF141,转化至Rosseta大肠杆菌表达细胞中,用异丙基硫代半乳糖苷(IPTG)诱导表达,然后利用SDS-PAGE和Western-blot对目标蛋白进行鉴定,用该蛋白免疫新西兰大白兔,利用免疫荧光试验确定多克隆抗体的特性。结果表明,本研究成功构建了p ET-30a(+)-ORF141原核表达载体,在1 mmol/L IPTG、37℃条件下诱导表达目的蛋白,大小约25 ku,主要以包涵体的形式存在;制备的抗LSDV ORF141多克隆抗体的效价可达1∶409600,免疫荧光试验表明其能够识别LSDV病毒中ORF141编码的天然蛋白,这为后续明确LSDV ORF141基因编码蛋白的生物学功能研究奠定了基础。

In order to obtain the ORF141 protein of lumpy skin disease virus(LSDV)and its polyclonal antibody,primers were designed using Snap Gene software referring to the LSDV ORF141 gene sequence from Gen Bank,and ORF141 gene was amplified by PCR using the LSDV genome as template,and the prokaryotic expression recombinant plasmid p ET-30a(+)-ORF141 was constructed.Transformed into Rosetta E.coliexpressing cells,expression was induced with isopropylthiogalactoside(IPTG),and the interest protein was identified using SDS-PAGE and Western-blot.To prepare polyclonal antibodies,New Zealand white rabbits were immunized with expressed protein and the properties of polyclonal antibodies was determined by immunofluorescence assays.The results showed that the p ET-30a(+)-ORF141 prokaryotic expression vector was successfully constructed,and the target protein was induced to express at 1 mmol/L IPTG at37℃,with the size of about 25 ku,which mainly existed in the form of inclusion bodies.The titer of the prepared anti-LSDV ORF141 polyclonal antibody could reach 1∶409600,and the immunofluorescence test showed that it could recognize the natural protein encoded by ORF141 in LSDV virus,which lays a foundation for further research on the biological function of the protein encoded by LSDV ORF141 gene.