摘要目的 构建致病性问号钩端螺旋体(简称钩体)黄疸出血群赖型赖株vwA1和vwA2基因原核表达系统,初步了解目的重组表达产物rVwA1和rVwA2与血小板性出血相关性.方法 采用高保真PCR扩增问号钩体赖株vwA1和vwA2基因并测序,常规方法构建vwA1和vwA2基因原核表达系统.采用SDS-PAGE联合Bio-Rad凝胶图像分析系统检查rVwA1和rVwA2表达情况及其可溶性,Ni-NTA亲和层析柱提纯rVwA1和rVwA2.采用实时荧光定量RT-PCR检测问号钩体赖株感染人脐静脉内皮细胞株HUVEC前后vwAl-mRNA和vwA2-mRNA水平变化.采用流式细胞术检测rVwA1与人血小板膜糖蛋白结合能力.采用酶切试验及SDS-PAGE观察人血管性血友病因子裂解酶ADAMTS13水解rVwA2的情况.结果 所克隆的钩体vwA1和vwA2基因与报道的相应基因比较,其核苷酸和氨基酸序列相似性均为100％.所构建的vwA1和vwA2基因原核表达系统能分别表达可溶性rVwA1和rVwA2.问号钩体赖株感染HUVEC 8 h后,钩体vwAl-mRNA和vwA2-mRNA水平均显著上调(P＜0.05).流式细胞术检测结果显示,钩体rVwA1与人血小板结合率为60.8％.人ADAMTS13可水解重组人vWF-A2( rhvWF-A2),但不能水解钩体rVwA2.结论 vwA1和vwA2基因可能在钩体感染中发挥作用,其中vwA1基因产物功能与钩体病出血密切相关.

abstractsObjective To generate the prokaryotic expression systems of vwA1 and vwA2 genes of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai,and to determine the correlation among the target recombinant expressed products rVwA1 and rVwA2 and platelet-associated hemorrhage.Methods The vwA1 and vwA2 genes of L.interrogans strain Lai were amplified using high fertility PCRs and then the amplification products were sequenced.The prokaryotic expression systems of vwA1 and vwA2 genes were generated by using routine genetic techniques.The expression and dissolubility of rVwA1 and rVwA2 proteins were examined by SDS-PAGE plus Bio-Rad Gel Image Analyzer.Ni-NTA affinity chromatography was used to extract the expressed rVwA1 and rVwA2.Real-time fluorescence quantitative RT-PCRs were performed to determine the changes of vwA1-mRNA and vwA2-mRNA levels in the leptospires of L.interrogans strain Lai before and after infection of human umbilical vein endothelial cell line (HUVEC).The ability of leptospiral rVwA1 binding to human platelets was detected by flow cytometry.A hydrolytic test plus SDS-PAGE were applied to examine the cleavage of leptospiral rVwA2 by a human von Willebrand factor lysase (ADAMTS13).Results The nucleotide and putative amino acid sequences of the cloned leptospiral vwA1 and vwA2 genes were 100％ identities compared to the reported corresponding genes.The generated prokaryotic expression systems of vwA1 and vwA2 genes could express soluble rVwA1 and rVwA2,respectively.When L.interrogans strain Lai infected HUVEC for 8 h,both the vwA1-mRNA and vwA2-mRNA levels were significantly up-regulated (P＜0.05).The result of flow cytometry showed that the leptospiral rVwA1 was able to combine with human platelets with a 60.8％ binding rate. Human recombinant vWF-A2 ( rhvWF - A 2 ),but not the leptospiral rVwA 2,could be hydrolyzed by human ADAMTS 13.Conclusion The vwA1 and vwA2 genes may play roles during infection of L.interrogans species,and the function of vwA1 gene product is referring to the hemorrhage in leptospirosis.