蛇毒金属蛋白酶Alfimeprase在大肠杆菌中的表达、纯化及活性检测

Expression,Purification and Activity Assay of Alfimeprase,a Snake Venom Metalloproteinase in Escherichia coli

目的:应用大肠杆菌表达系统表达纤溶性蛇毒金属蛋白酶Alfimeprase。方法:运用PCR技术扩增人工合成的目的基因,引入Nde Ⅰ、EcoR Ⅰ双酶切位点;目的基因经Nde Ⅰ、EcoR Ⅰ双酶切后,克隆到pET22b载体上,然后将重组质粒转化大肠杆菌BL21(DE3)进行胞内表达,对获得的包涵体进行体外透析复性;应用纤维蛋白原水解法、纤维蛋白平板法和纤维蛋白层叠法检测复性后蛋白的降纤活性。结果:目的蛋白以包涵体形式在大肠杆菌中获得高表达,复性后的蛋白具有降解纤维蛋白(原)的活性。结论:Alfimeprase包涵体可以通过复性获得活性产物。

Objective： To express snake venom metalloproteinase alfimeprase with fihrinolytic（fibrinogenolytic） activity in Escherichia coli expression system. Methods： The gene of interest was amplified by PCR and subcloned into the pET22b vector. This recombinant plasmid was transformed into E.coli BL21（DE3） cells. Transformants were cultured at 37℃ and an insoluble and inactive protein was expressed. The protein was isolated from the E.coli BL21（DE3） cells after sonication and refolded in vitro by dialysis. The refolding production was purified, and its activity analysis was carried out by fibrinogen degradation, fibrin plate method and fibrin overlay. Results： The active alfimeprase was obtained by refolding the inclusion body in vitro. Conclusion： The inclusion body of alfimeprase can be turned into the active form by refolding in vitro.