问号钩端螺旋体LA_2144基因产物PAF-AH和PLA2酶活性研究  

PAF-AH and PLA2 activity of Leptospira interrogans LA_2144 gene product

作　　者：张琴超 孙爱华[2] 张金良[3] 胡玮琳[1] 严杰[1] 林旭瑷[1] Zhang Qinchao;Sun Aihua;Zhang Jinliang;Hu Weilin;Yan Jie;Lin Xu′ai(Department of Pathogen Biology and Microbiology, Zhejiang University School of Medicine, Hangzhou 310058, China;Faculty of Basic Medicine, Hangzhou Medical College, Hangzhou 310058, China;Jinhua Polytechnic College School of Medicine, Jinhua 310058, China)

机构地区：[1]浙江大学医学院病原生物学和微生物学系,杭州310058 [2]杭州医学院基础医学部,31OO58 [3]金华职业技术学院医学院,310058

出　　处：《中华微生物学和免疫学杂志》2019年第7期519-526,共8页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(81671974);浙江省科技计划项目(2014C33143).

摘　　要：目的了解问号钩端螺旋体LA_2144基因产物血小板活化因子乙酰水解酶(PAF-AH)和磷脂酶A2(PLA2)的酶活性。方法生物信息学软件预测问号钩端螺旋体赖株LA_2144基因跨膜区、信号肽和结构域。构建无信号肽LA_2144基因原核表达系统,Ni-NTA亲和层析法提纯目的重组蛋白rLep2144后复性。采用分光光度法检测rLep2144水解PAF-AH底物2-thioPAF的活性及其Km和Kcat值以及水解PLA2底物花生四烯酸硫代卵磷脂的活性。采用实时荧光定量RT-PCR和Westernblot法检测问号钩端螺旋体感染人和小鼠血管内皮细胞(HUVEC和EOMA)后LA_2144基因转录、蛋白质表达和外分泌情况。结果LA_2144基因无跨膜区但有信号肽和SGNH水解酶超家族结构域。所构建的无信号肽LA_2144基因原核表达系统能高效表达rLep2144,提纯后的rLep2144在分离胶上为单一蛋白质条带并成功复性。rLep2144有较强PAF-AH活性,其Km和Kcat值分别为688.235μmol/L和0.976/s,但PLA2活性较弱。问号钩端螺旋体感染HUVEC和EOMA后LA_2144基因mRNA转录和蛋白质表达水平迅速升高(P<0.05)且外分泌。结论问号钩端螺旋体LA_2144基因产物具有较强PAF-AH活性和一定的PLA2活性,可在钩端螺旋体病出血性病变和炎症反应中发挥作用。Objective To analyze the enzymatic activity of Leptospira interrogans (L.interrogans) LA_2144 gene product to hydrolyze platelet activating factor acetylhydrolase (PAF-AH) and phosphatidase A2 (PLA2). Methods Bioinformatic softwares were used to predict transmembrane regions, signal peptides and domains of the LA_2144 gene of L. interrogans strain Lai. A prokaryotic expression system for signal peptide-free LA_2144 gene was established. The expressed target recombinant protein rLep2144 was extracted by Ni-NTA affinity chromatography and then renatured. Spectrometry was used to detect the activity of rLep2144 to hydrolyze PAF-AH substrate 2-thio PAF and the Km and Kcat values as well as the activity to hydrolyze PLA2 substrate arachidonoyl 2-thio PC. Real-time fluorescence quantitative RT-PCR and Western blot were performed to detect the transcription, protein expression and secretion of LA_2144 gene during infection of human and mouse vascular endothelial cells (HUVEC and EOMA) with L. interrogans. Results L. interrogans LA_2144 gene contained a signal peptide and a domain belonging to SGNH hydrolase superfamily, but no transmembrane regions. The established prokaryotic expression system for signal peptide-free LA_2144 gene could efficiently express rLep2144. The extracted rLep2144 was shown as a single protein fragment in separation gel and then successfully renatured. rLep2144 had a stronger PAF-AH activity with the Km and Kcat values of 688.235 μmol/L and 0.976/s, but its PLA2 activity was relatively weak. Expression of the LA_2144 gene at mRNA and protein levels in HUVEC and EOMA was rapidly increased after the cells were infected with L. interrogans (P<0.05) and the secretion of LA_2144 gene product could be detected. Conclusions L. interrogans LA_2144 gene product had a stronger PAF-AH and a certain PLA2 activity, which might involve in the hemorrhage and inflammatory response in leptospirosis.

关 键 词：问号钩端螺旋体 LA_2144基因 血小板活化因子乙酰水解酶 磷脂酶A2 酶活性 

分 类 号：R377[医药卫生—病原生物学]