福建农业学报2025，Vol.40Issue(7)：656-661,6.DOI:10.19303/j.issn.1008-0384.2025.07.002
新型鸭呼肠孤病毒σB蛋白的可溶性原核表达与纯化
Prokaryotic Expression and Purification of σB of Novel Duck Reovirus
摘要
Abstract
[Objective]The soluble σB of novel duck reovirus(NDRV)was obtained with optimized prokaryotic expression for effective disease detection and control.[Method]The recombinant plasmid pET-32a-σB identified by double digestion was transformed into Escherichia coli BL21(DE3)competent cells.IPTG was used to induce the expression of recombinant σB(rσB).After ultrasonic crushing,the supernatant and precipitate were analyzed by SDS-PAGE.The rσB in the supernatant was then purified by a His-tag protein purification kit to establish an indirect ELISA for the viral detection.[Result]Under the conditions of 0.5 mmol·L-1 IPTG and induction at 16℃for 12 h,the rσB was detected in the supernatant with significantly improved expression.The SDS-PAGE analysis showed the purity of rσB to be higher than 90%,and the molecular weight approximately 60 kDa.The purified protein specifically reacted with His-tag monoclonal antibody as shown by western blot.The OD450 of the NDRV positive serum tested by the indirect ELISA was 1.75,which was much higher than that of positive sera from Muscovy duck reovirus and Muscovy duck goose parvovirus.The OD450 of MDRV and MDGPV positive sera were both less than 0.3.A strong specific reactivity of the purified rσB was confirmed by both western blot and ELISA.[Conclusion]The optimized prokaryotic expression conditions of NDRV σB protein established in this study applied low temperature and IPTG concentration to successfully induce the soluble NDRV σB protein that overcame the previously existing bottleneck for the functional and application studies.The secured soluble rσB would lead to the future development of NDRV serum antibody ELISA kit.关键词
新型鸭呼肠孤病毒/σB基因/原核表达/蛋白纯化Key words
novel duck reovirus/σ Bgene/prokaryotic expression/protein purification分类
农业科技引用本文复制引用
董慧,王劭,朱小丽,郑欣,陈少莺,陈仕龙..新型鸭呼肠孤病毒σB蛋白的可溶性原核表达与纯化[J].福建农业学报,2025,40(7):656-661,6.基金项目
福建省科技计划公益类专项(2021R1026009、2024R1025003) （2021R1026009、2024R1025003）
福建省农业科学院自由探索科技创新项目(ZYTS202225) （ZYTS202225）
福建省农业高质量发展超越"5511"协同创新工程项目(XTCXGC2021012、XTCXGC2021018) （XTCXGC2021012、XTCXGC2021018）
福建省农业科学院科技创新团队建设项目(CXTD2021034) （CXTD2021034）
