牛结节性皮肤病病毒ORF006蛋白的原核表达及其多克隆抗体的制备

Prokaryotic expression and polyclonal antibody preparation of lumpy skin disease virus ORF006 protein

为探究牛结节性皮肤病病毒(LSDV)ORF006蛋白的生物学功能,制备了ORF006基因的多克隆抗体,用PCR技术获得LSDV ORF006的基因片段克隆至pET-28a原核表达载体,并转化至大肠杆菌BL21(DE3)后进行IPTG诱导表达,将纯化后的蛋白免疫新西兰大白兔制备多克隆抗体。结果显示,LSDV ORF006基因全长约696 bp;pET-28a-ORF006重组质粒在大肠杆菌中于37℃经1 mmol/L的IPTG诱导12 h,成功表达约28.0 ku目的蛋白;ELISA结果显示,制备的多克隆抗体效价为1∶102400;Western-blot结果显示,制备的多克隆抗体能特异性识别ORF006蛋白;间接免疫荧光试验表明,制备的多克隆抗体能够特异性识别LSDV感染MDBK细胞表达的ORF006蛋白。本研究为进一步探究LSDV ORF006蛋白生物学功能奠定了基础。

To explore the biological function of the open reading frame 006(ORF006) protein of lumpy skin disease virus(LSDV),polyclonal antibody against the ORF006 gene was prepared.In this study,the gene fragment of LSDV ORF006 obtained by PCR was cloned into the prokaryotic expression vector p ET-28a and transformed to E.coli BL21(DE3) for IPTG induced expression,and the purified protein was immunized to New Zealand white rabbits to prepare polyclonal antibodies.The results showed that the full length of LSDV ORF006 gene was about 696 bp.The p ET-28a-ORF006 recombinant plasmid was successfully expressed about 28.0 ku of the target protein in E.coli after being induced by 1 mmol/L of IPTG at37 ℃ for 12 h.ELISA showed that the titer of the prepared polyclonal antibody was 1 ∶ 102 400.The Western-blot results showed that the prepared polyclonal antibody specifically recognized the ORF006 protein.Indirect immunofluorescence assays showed that the prepared polyclonal antibody could specifically recognize ORF006 protein expressed in MDBK cells infected by LSDV.This study laid a foundation for further study on the biological function of LSDV ORF006 protein.