中国兽医科学2024，Vol.54Issue(8)：1072-1078,7.DOI:10.16656/j.issn.1673-4696.2024.0129
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
摘要
Abstract
In order to obtain the ORF141 protein of lumpy skin disease virus(LSDV)and its polyclonal antibody,primers were designed using SnapGene software referring to the LSDV ORF141 gene sequence from GenBank,and ORF141 gene was amplified by PCR using the LSDV genome as template,and the prokaryotic expression recombinant plasmid pET-30a(+)-ORF141 was constructed.Transformed into Rosetta E.coli-expressing 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 deter-mined by immunofluorescence assays.The results showed that the pET-30a(+)-ORF141 prokaryotic expression vector was successfully constructed,and the target protein was induced to express at 1 mmol/L IPTG at 37 ℃,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∶409 600,and the immunofluorescence test showed that it could recognize the natural protein encoded by ORF141 in LSDV virus,which lays a founda-tion for further research on the biological function of the protein encoded by LSDV ORF141 gene.关键词
牛结节性皮肤病病毒/ORF141基因/原核表达/多克隆抗体Key words
lumpy skin disease virus/orf 141 gene/prokaryotic expression/polyclonal antibody分类
农业科技引用本文复制引用
张慧,景志忠,何小兵,房永祥,谭金龙,余翰维,高真贞,景伟,苏洋,陈国华..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[J].中国兽医科学,2024,54(8):1072-1078,7.基金项目
兰州市科技计划项目(2023-1-38) （2023-1-38）
牛皮肤结节病抗原纯化工艺研究项目(22JR5RA780) （22JR5RA780）
2023动物疫情监测与防治经费项目(125161031000200002) （125161031000200002）
