摘要目的:确定问号钩端螺旋体vWF-A基因产物结合人胶原蛋白的作用与机制。方法:采用生物信息学软件分析问号钩端螺旋体黄疸出血群赖型赖株vWF-A基因(LA_0012、LA_0697和LA_4207)产物结构与功能。构建上述基因vWF-A功能结构域片段原核表达系统，采用SDS-PAGE和Ni-NTA亲和层析法检查目的重组蛋白rLep0012、rLep0697和rLep4207表达情况和提纯效果。采用ELISA和表面等离子共振法(SPR)检测目的重组蛋白与人Ⅰ、Ⅲ、Ⅳ和Ⅵ型胶原蛋白(hCOL1/3/4/6)的结合能力。采用实时荧光定量RT-PCR和Western blot检测问号钩端螺旋体赖株感染人和小鼠血管内皮细胞(HUVEC和EOMA)时vWF-A基因转录和表达水平变化。结果:vWF-A基因产物均为含vWF-A超家族结构域表面或跨膜蛋白，但LA_0697和LA_4207基因还含有金属离子依赖性黏附位点(MIDAS)。所构建的原核表达系统能有效表达目的重组蛋白，Ni-NTA亲和层析法提纯后SDS-PAGE检测均显示为单一蛋白条带。ELISA结果显示rLep0697与hCOL3/6、rLep4207与hCOL1/4呈强结合。SPR结果显示rLep0697快速结合hCOL3/6但快速解离( KD值＝5.71×10 -8和5.89×10 -8 mol/L)，rLep4207快速并稳定结合hCOL1/4( KD值＝6.4×10 -9和3.2×10 -9 mol/L)。感染HUVEC和EOMA细胞时，上述vWF-A基因转录和表达水平均显著升高( P<0.05)。 结论:LA_0697和LA_4207基因产物可作为问号钩端螺旋体感染过程中的黏附因子。

abstractsObjective:To investigate the role and mechanism of Leptospira interrogans ( L. interrogans) vWF-A gene products binding to human collagen proteins. Methods:Bioinformatic software was used to analyze the structure and function of the vWF-A genes (LA_0012, LA_0697 and LA_4207) of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai. Prokaryotic expression systems for the vWF-A domain segments in the vWF-A genes were generated. The target recombinant proteins, rLep0012, rLep0697 and rLep4207, were purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE. ELISA and surface plasmon resonance (SPR) were performed to detect the binding ability of the target recombinant proteins to humanⅠ, Ⅲ, Ⅳ and Ⅵ types of collagen proteins (hCOL1/3/4/6). Expression of the vWF-A genes at mRNA and protein levels were detected by real-time fluorescent quantitative RT-PCT and Western blot during infection of human umbilical vein endothelial cells (HUVEC) and mouse hemangioendothelioma endothelial cells (EOMA). Results:The products of vWF-A genes were vWF-A superfamily domain-containing surface or transmembrane proteins, but LA_0697 and LA_4207 genes also contained metal ion-dependent adhesion sites (MIDAS). The established prokaryotic expression systems efficiently expressed the target recombinant proteins and each of the proteins extracted by Ni-NTA affinity chromatography showed a single band in SDS-PAGE. ELISA results showed the strong binding of rLep0697 to hCOL3/6 and rLep4207 to hCOL1/4. SPR results showed the rapid binding and dissociation of rLep0697 with hCOL3/6 ( KD values=5.71×10 -8 and 5.89×10 -8 mol/L) and the rapid and stable biding of rLep4207 with hCOL1/4 ( KD values=6.4×10 -9 and 3.2×10 -9 mol/L). Expression of the vWF-A genes at both mRNA and protein levels were significantly elevated ( P<0.05) during infection of HUVEC and EOMA cells. Conclusions:The products of LA_0697 and LA_4207 genes could act as the adherence factors of L. interrogans during infection.