中国动物检疫2026，Vol.43Issue(7)：103-112,10.DOI:10.3969/j.issn.1005-944X.2026.07.015
中华鳖黄病毒包膜蛋白的原核表达及多克隆抗体制备
Prokaryotic Expression of the Envelope Protein of Pelodiscus sinensis Flavivirus and Preparation of Its Polyclonal Antibodies
摘要
Abstract
In order to identify the evolutionary characteristics of the envelope(E)protein of Pelodiscus sinensis flavivirus(PSFV),and to enable its expression,purification,and the preparation of specific polyclonal antibodies,the PSFV E protein sequence was analyzed using bioinformatics approaches.With PSFV cDNA as a template,the gene encoding the ectodomain of the E protein was amplified by PCR,cloned into the prokaryotic expression vector pET-28a,and,after sequencing verification,transformed into Escherichia coli BL21(DE3)competent cells.The protein expression was then induced with IPTG,and purified by size-exclusion chromatography.Polyclonal antibodies were prepared by immunizing rabbits with the purified protein as the antigen,and subsequently characterized by SDS-PAGE,indirect ELISA,immunofluorescence and Western blot.The results showed that the PSFV E protein formed a monophyletic clade in the phylogenetic tree,was genetically distant from the E proteins of mosquito-borne and tick-borne flaviviruses,and shared amino acid sequence identities ranging from 38.89％to 46.26％with other members of the genus Orthoflavivirus.The recombinant plasmid pET28a-PSFV-E was successfully constructed,and a recombinant E protein with high purity and an apparent molecular mass of approximately 43 kDa was obtained through induced expression and purification.The prepared polyclonal antibodies presented high purity,and their titer,as determined by indirect ELISA,reached 1:162 000.Furthermore,these antibodies were capable of recognizing both the native and recombinant PSFV E proteins.In this study,the prokaryotic expression and purification of the PSFV E protein were successfully achieved,and the polyclonal antibodies were prepared with high titer and good immunoreactivity,thereby providing important experimental materials and technical support for molecular detection,epitope mapping and relevant researches on the functions of PSFV.关键词
中华鳖黄病毒(PSFV)/包膜蛋白/原核表达/多克隆抗体Key words
PSFV/envelope protein/prokaryotic expression/polyclonal antibody分类
农业科技引用本文复制引用
简思杰,傅晓晴,刘莉,聂品,宋晶伟..中华鳖黄病毒包膜蛋白的原核表达及多克隆抗体制备[J].中国动物检疫,2026,43(7):103-112,10.基金项目
国家重点研发计划项目(2025YFD2400304) （2025YFD2400304）
山东省自然科学基金项目(ZR2025QC208) （ZR2025QC208）
青岛市自然科学基金项目(25-1-1-31-zyyd-jch) （25-1-1-31-zyyd-jch）
