福建农业学报2025，Vol.40Issue(5)：467-475,9.DOI:10.19303/j.issn.1008-0384.2025.05.005
副结核分枝杆菌85A、85B蛋白生物信息学特征及其原核表达
Bioinformatics and Prokaryotic Expressions of 85A and 85B in Mycobacterium paratuberculosis
摘要
Abstract
[Objective]Bioinformatics of 85A and 85B of Mycobacterium avium subsp.paratuberculosis(MAP)were studied,and the proteins obtained by prokaryotic expression technology to determine the reactogenicity for the development of an ELISA detection kit and vaccine.[Methods]The amino acid sequences of 85A and 85B were secured using an online bioinformatics platform.The transmembrane region and signal peptide structure were removed,and the codons optimized.pET-30a-MAP-85A and pET-30a-MAP-85B recombinant plasmids were successfully constructed.Using BL21(DE3),the induction time,temperature,and IPTG concentration for the prokaryotic expression were optimized.The solubility of the proteins with the induced expression was analyzed,the purification conducted on nickel columns,and the immunoreactivity evaluated by western blotting.[Results]The recombinant 85A was successfully obtained showing a molecular weight of approximately 31.3 kDa.An optimal expression was achieved after 4 h of induction at 37℃with a final IPTG concentration of 1.0 mmol·L-1.The recombinant 85B had a molecular weight of 31.8 kDa exhibiting an optimal expression after 6 h of induction at 37℃with 0.8 mmol·L-1 of IPTG.Both recombinant proteins were expressed in the form of inclusion bodies and high-purified on nickel ion columns.Using the MAP-positive bovine serum as primary antibody,the immunoreactivity of the purified proteins was verified by western blot.[Conclusion]The recombinant 85A and 85B were successfully expressed with confirmed immunoreactivity for the development of immunological detection on M.avium subsp.paratuberculosis.关键词
副结核分枝杆菌/原核表达/85A/85BKey words
Mycobacterium aviumsubsp.paratuberculosis/prokaryotic expression/85A/85B分类
农业科技引用本文复制引用
平宇明,常华,张宏莉,王佳,李明鑫,李超,赵萍萍,王娅荣,张立华,李劼..副结核分枝杆菌85A、85B蛋白生物信息学特征及其原核表达[J].福建农业学报,2025,40(5):467-475,9.基金项目
云南省重大科技专项计划项目(202302AA310020) （202302AA310020）
呼和浩特市科技创新领域人才项目(2022RC-联合体-1) （2022RC-联合体-1）
云南省重点研发计划——建设面向南亚东南亚科技创新中心专项(202403AP140033) （202403AP140033）
