重组ABHD17A的表达及功能验证  

Expression and functional verification of recombinant ABHD17A

作　　者：史雪盟 王靖仪 陈帅武 张鑫彤 温鑫 毛琳 许君[1] SHI Xuemeng;WANG Jingyi;CHEN Shuaiwu;ZHANG Xintong;WEN Xin;MAO Lin;XU Jun(College of Life Sciences,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学生命科学学院,河南郑州450046

出　　处：《河南农业大学学报》2024年第5期775-782,共8页Journal of Henan Agricultural University

基　　金：国家自然科学基金项目(32300140,U1804108);河南省自然科学基金项目(232300421157);河南省科技攻关项目(232102310305);河南农业大学大学生创新创业训练项目(2023CX122)。

摘　　要：【目的】通过表达重组α/β水解酶超家族17A(α/β-hydrolase domain-containing 17A,ABHD17A),验证其在蛋白质棕榈酰化修饰及病毒感染过程中的调控作用,为动物病毒性疾病的治疗提供理论依据及新思路。【方法】原核诱导重组ABHD17A表达,通过酰基-聚乙二醇交换法(Acyl-PEGyl exchange gel-shift,APEGS)及实时荧光定量PCR、免疫印迹等技术检测重组ABHD17A对N-Ras棕榈酰化修饰以及乙脑病毒(Japanese encephalitis virus,JEV)感染的影响。【结果】成功将abhd17a编码序列克隆至pGEX-4T-1原核表达载体,将其转化入BL21(DE3)大肠杆菌诱导表达,发现在22℃、1 mmol·L^(-1)异丙基硫代半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)诱导可得到高浓度的可溶性重组ABHD17A。利用亲和层析纯化出重组ABHD17A,经考马斯亮蓝染色及免疫印迹确认了该蛋白的特异性表达。将重组ABHD17A与表达有Flag-N-Ras的HEK293细胞裂解液在4℃孵育12 h,借助APEGS法检测N-Ras的棕榈酰化,结果显示,重组ABHD17A显著下调N-Ras棕榈酰化修饰水平,表明重组ABHD17A在体外可发挥去棕榈酰化酶的作用。将重组ABHD17A孵育HEK293细胞24 h后感染JEV,24 h后采用实时荧光定量PCR及免疫印迹法分别检测JEV mRNA及蛋白水平,发现孵育重组ABHD17A能显著抑制JEV感染,参与免疫调控。【结论】证实了纯化的重组ABHD17A可特异性去棕榈酰化N-Ras,并抑制JEV感染。【Objective】The recombinantα/β-hydrolase domain-containing 17A(ABHD17A)was expressed to verify its regulation role in the process of protein palmitoylation modification and virus infection,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 abhd 17 a 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) isopropylβ-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 inhibited JEV infection,and involved in immune regulation.【Conclusion】The purified recombinant ABHD17A 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 

分 类 号：Q786[生物学—分子生物学] S852.65[农业科学—基础兽医学]