摘要
目的 了解并确定问号钩端螺旋体黄疸出血型赖株LA2582 和LA2901 基因产物金属内肽酶活性及其致病相关性.方法 采用生物信息学软件分析LA2582 和LA2901 基因结构与功能.构建LA2582 和LA2901 基因胞外区原核表达系统,Ni-NTA 亲和层析法提纯目的重组表达产物rLA2582 和rLA2901.采用分光光度法检测rLA2582 和rLA2901 水解偶氮酪蛋白底物的活性.采用荧光分光光度法检测rLA2582 和rLA2901 水解Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans 荧光标记五肽底物的活性并测定其Km 和Kcat 值.采用SDS-PAGE 和分光光度法分别检测rLA2582 和rLA2901 水解细胞外基质（ECM）分子Ⅰ型胶原蛋白（COL1）、纤维连接蛋白（FN）和刚果红标记弹性蛋白（ELN）的活性.采用实时荧光定量RT-PCR 和Western blot 法分别检测问号钩端螺旋体赖株感染人脐静脉内皮细胞（HUVEC）后LA2582 和LA2901 基因mRNA 和蛋白质表达水平.结果 LA2582 和LA2901 基因产物均为含信号肽和HXH 基质金属蛋白酶基序的锌离子依赖M23 家族Gly-Gly 金属内肽酶.rLA2582 和rLA2901 不能水解偶氮酪蛋白,但能水解荧光五肽底物,其Km 和Kcat 值分别为126. 54μmol/ L 和4. 67/ s 、190. 25 μmol/ L 和4. 86/ s .rLA2582 和rLA2901 具有水解COL1、FN 和ELN 的活性.问号钩体赖株感染HUVEC 后,LA2582 和LA2901 基因mRNA、蛋白质表达水平均显著升高（P〈0. 05）.结论 问号钩体赖株LA2582 和LA2901 基因产物是具有水解ECM 分子活性的锌离子依赖M23 金属内肽酶,与问号钩体侵袭力密切相关.
Objective To understand and determine the biological activity and pathogenicity of metalloendopeptidases encoded by LA2582 and LA2901 genes of Leptospira interrogans（L.interrogans） sero-group Icterohaemorrhagiaeserovar Lai strain Lai. Methods Structures and functions of LA2582 and LA2901 genes were analyzed by using bioinformatic software. Prokaryotic expression systems for expressing the extra-cellular regions of LA2582 and LA2901 genes were generated. The target recombinant expression products, rLA2582 and rLA2901,were extracted by Ni-NTA affinity chromatography. The Azo-casein-hydrolyzingactiv-ity of rLA2582 and rLA2901 was detected by spectrophotometry. Activities of rLA2582 and rLA2901 in the hydrolysis of Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans, a fluorescence-labeling pentapeptide substrate, were de-tected by fluorospectrophotometry,and then the Km and Kcat values were determined. SDS-PAGE and spec-trophotometry were performed to detect the activities of rLA2582 and rLA2901 in hydrolyzing extracellular matrix molecules such as collagen type-Ⅰ （COL1）, fibronectin （FN） and Congo red-labeling elastin （ELN）. Real-time fluorescent quantitative RT-PCR（qRT-PCR） and Western blot were respectively used to measure the expression of LA2582 and LA2901 genes at mRNA and protein levels after infecting human um-bilical vein endothelial cells（HUVEC） with L. interrogans strain Lai. Results The gene products of LA2582 and LA2901 genes were identified as the signal peptide and matrix metalloproteinase motif HXH-containing Zn2＋-dependent Gly-Gly metalloendopeptidases belonging to the M23 superfamily. rLA2582 and rLA2901 did not hydrolyze Azo-casein （Km=126.54 μmol/L, Kcat=4.67/s）, but could hydrolyze the pentapeptide substrate （Km=190. 25 μmol/L, Kcat 4. 86/s）. rLA2582 and rLA2901 could hydrolyze COL1, FN and ELN. Expression of LA2582 and LA2901 genes at both mRNA and protein levels was signifi-cantly increased after infection of HUVEC with L.interrogans strain Lai（P〈0.05）. Conclusion The
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2017年第10期753-760,共8页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金（81671974,81471907）
