目标:壁虎,由中医命名的“天龙”,在自然生态中截尾后经历快速凝固和无疤痕再生,为开发有效和安全的血液凝固药物提供了一个绝佳的机会。这里,重组制备了壁虎凝血酶(gtaxin),并对其促凝活性进行了比较研究。
方法:使用I-TASSER的同源建模方法构建了g凝血酶的3D结构。通过在293T细胞中表达壁虎前凝血酶-2制备活性凝血酶,然后用Ni2+螯合柱层析纯化,然后用蛇毒衍生的Ecarin活化。通过合成底物S-2238的水解和纤维蛋白原凝固来测定g凝血酶的酶活性。使用易损神经细胞在分子和细胞水平上评估gtaxin的毒性。
结果:在不同温度和pH条件下,活性重组gtaxin表现出比人高的催化和纤溶效率。此外,gtaxin对包括神经元在内的中枢神经细胞产生无毒作用,与哺乳动物的对应物相反,导致神经元损伤,星形胶质增生,和脱髓鞘.
结论:从爬行动物中鉴定出一种超高活性但安全的促凝血候选药物,这为快速凝血的临床应用提供了很有希望的前景。
Gecko, the \"sky dragon\" named by Traditional Chinese Medicine, undergoes rapid coagulation and scarless regeneration following tail amputation in the natural ecology, providing a perfect opportunity to develop the efficient and safe drug for blood clotting. Here, gecko thrombin (gthrombin) was recombinantly prepared and comparatively studied on its procoagulant activity.
The 3D structure of gthrombin was constructed using the homology modeling method of I-TASSER. The active gthrombin was prepared by the expression of gecko prethrombin-2 in 293 T cells, followed by purification with Ni2+ -chelating column chromatography prior to activation by snake venom-derived Ecarin. The enzymatic activities of gthrombin were assayed by hydrolysis of synthetic substrate S-2238 and the fibrinogen clotting. The vulnerable nerve cells were used to evaluate the toxicity of gthrombin at molecular and cellular levels.
The active recombinant gthrombin showed super-high catalytic and fibrinogenolytic efficiency than those of human under different temperatures and pH conditions. In addition, gthrombin made nontoxic effects on the central nerve cells including neurons, contrary to those of mammalian counterparts, which contribute to neuronal damage, astrogliosis, and demyelination.
A super-high activity but safe procoagulant candidate drug was identified from reptiles, which provided a promising perspective for clinical application in rapid blood clotting.