梅毒螺旋体金属蛋白酶水解纤维蛋白原的活性研究

目的　研究梅毒螺旋体金属蛋白酶Tp0751对细胞外基质纤维蛋白原的水解能力，为深入研究TP的致病机制提供实验依据。　方法　采用两点法构建H198A突变型及H202A突变型Tp0751基因，分别构建野生型和突变型Tp0751原核载体进行诱导表达；体外实验检测Tp0751野生型Tp0751蛋白、H198A　Tp0751突变型蛋白、H202ATp0751突变型蛋白对纤维蛋白原的降解活性。　结果　成功表达并纯化了野生型Tp0751蛋白、H198A　Tp0751突变体蛋白、H202A　Tp0751突变体蛋白，各蛋白相对分子量大小约为26kD；纤维蛋白原在与野生型Tp0751蛋白作用约24h后被完全水解；与H198A　Tp0751突变体蛋白作用约24　h后部分水解，尚有部分未被降解；与H202A　Tp0751突变体蛋白作用约24h后几乎不被水解。　结论　野生型Tp0751蛋白具有水解纤维蛋白原的作用；Tp0751蛋白水解纤维蛋白原的活性可能与金属蛋白酶HEXXH域相关,影响Tp0751蛋白对纤维蛋白原的降解的活性位点可能是202位的H而不是198位的H。

The Fibrinolytic Potential of Treponema Pallidum Metalloprotease

Objective To investigate the fibrinolytic potential of Treponema pallidum metalloprotease Tp0751,providing the experimental basis for further research on syphilis pathogenic mechanism. Methods Constructed the H198A mutant Tp0751 and H202A mutant Tp0751 with two-point technology.Wide-type and Mutant Tp0751 recombinant protein were expressed in E.coli BL21.And detected their degradation activity to fibrinogen. Results Three fusion protein with molecular weight about 26KDa were attained after expression and purification,The wild type Tp0751 can degradate plasminogen-free human fibrinogen,fibrinogen was absolutely degradated 24h post-incubation,The H198A mutant Tp0751 can also degradate plasminogen-free human fibrinogen,but which was partly degradated 24h post-incubationThe H202A mutant Tp0751 can not degradate plasminogen-free human fibrinogen,fibrinogen was scarcely degradated 24h post-incubation. Conclusion The wild type Tp0751 can degradate plasminogen-free human fibrinogen,The HEXXH motif may have a important role on Tp0751 to the degradation of plasminogen-free human fibrinogen,residue H202,but not H198,from the pallilysin HEXXH motif is required for fibrinogen degradation.