河南农业大学学报2024，Vol.58Issue(5)：775-782,8.DOI:10.16445/j.cnki.1000-2340.20240227.002
重组ABHD17A的表达及功能验证
Expression and functional verification of recombinant ABHD17A
摘要
Abstract
[Objective]The recombinant α/β-hydrolase domain-containing 17A(ABHD17A)was ex-pressed to verify its regulation role in the process of protein palmitoylation modification and virus infec-tion,which providing theoretical basis and new ideas for the treatment of animal viral diseases.[Method]Prokaryotic induction of recombinant ABHD17A expression was performed,and the effects of recombinant ABHD17A on N-Ras palmitoylation modification and Japanese encephalitis virus(JEV)infection were detected using Acyl-PEGyl exchange gel-shift(APEGS),quantitative real-time PCR,and immunoblotting,respectively.[Result]The coding sequence of abhd17a was successfully cloned into pGEX-4T-1 prokaryotic expression vector and transformed into BL21(DE3)E.coli for inducing expression.It was found that high concentration of soluble recombinant ABHD17A could be obtained at 22℃and 1 mmol·L-1 isopropyl β-D-thiogalactoside(IPTG)induction.The recombinant ABHD17A protein was purified by affinity chromatography,and its specific expression was confirmed by coomassie brilliant blue staining and immunoblotting.The recombinant ABHD17A was incubated with lysate of HEK293 cells expressing Flag-N-Ras at 4℃for 12 h,and the palmitoylation of N-Ras was detected by APEGS.The results showed that the recombinant ABHD17A significantly down-regulated the palmitoyl modification level of N-Ras,indicating recombinant ABHD17A could play the role of depalmitoylase in vitro.HEK293 cells were incubated with recombinant ABHD17A for 24 h,and then infected with JEV.After 24 h,the mRNA and protein levels of JEV were detected by quantitative real-time PCR and immunoblotting,respectively.It was found that recombinant ABHD17A could significantly inhibi-ted JEV infection,and involved in immune regulation.[Conclusion]The purified recombinant AB-HD17A can specifically deacetylate N-Ras and inhibit JEV infection.关键词
ABHD17A/蛋白表达纯化/酰基-聚乙二醇交换法/棕榈酰化修饰/病毒感染Key words
ABHD17A/protein expression and purification/Acyl-PEGyl exchange gel-shift/palmi-toylation modification/virus infection分类
生物科学引用本文复制引用
史雪盟,王靖仪,陈帅武,张鑫彤,温鑫,毛琳,许君..重组ABHD17A的表达及功能验证[J].河南农业大学学报,2024,58(5):775-782,8.基金项目
国家自然科学基金项目(32300140,U1804108) （32300140,U1804108）
河南省自然科学基金项目(232300421157) （232300421157）
河南省科技攻关项目(232102310305) （232102310305）
河南农业大学大学生创新创业训练项目(2023CX122) （2023CX122）
