RGD-重组葡激酶-人α微球蛋白融合蛋白原核表达、纯化及鉴定

Prokaryotic expression,purification and functional identification of RGD-recombinant SAK- α1M fusion protein

目的构建RGD-重组葡激酶-人α微球蛋白融合蛋白的原核表达质粒,在大肠杆菌中表达融合蛋白,纯化并初步鉴定其生物学活性。方法利用重叠延伸PCR获得目的融合基因片段RGD-r SAK-α1M,插入带有His-GST标签的原核高效可溶性表达载体PEGX-6P-1中。经酶切鉴定后,将重组表达质粒转化大肠杆菌BL21菌株,IPTG诱导目的蛋白表达,经Ni+亲和层析柱纯化后用3C蛋白酶切除重组蛋白的His-GST标签,再利用DEAE离子交换柱和分子筛纯化蛋白。最后应用纤维蛋白平板溶圈法和血小板聚集抑制试验分别测定评价重组融合蛋白的溶栓活性和抗血小板聚集作用。结果 1)成功构建了RGD-重组葡激酶-人α微球蛋白融合蛋白。2)实现了目的融合蛋白在大肠杆菌上清液中的高效表达,并纯化了融合蛋白。3)体外实验表明纯化后的融合蛋白纤溶活性同尿激酶标准品无明显差异。4)融合蛋白抗血小板聚集作用较单纯重组葡激酶明显提高。结论经文献检索确认为首次获得了纯化的RGD-重组葡激酶-人α微球蛋白融合蛋白,并验证了其纤溶活性和尿激酶标准品无明显差异,而抗血小板聚集作用较单纯重组葡激酶增强,为下一步进行融合蛋白免疫原性鉴定奠定了基础。

Objective To construct a recombinant prokaryotic expression plasmid of RGD-r SAK- α1M in E. col BL21. Furthermore,it aims to purify the fusion protein and determine its bioactivity. Methods The target fusion gene,RGD-r SAK-a1 M,was amplified by overlapping PCR and inserted into a prokaryotic expression vector PEGX-6P-1 with a His-GST tag to construct a recombinant expression plasmid PEGX-6P-1- RGD-r SAK- α1M,which had a high efficiency of prokaryotic fusion-expression. E. coli BL21 was used to transform the recombinant plasmid and the expression of fusion protein was induced by IPTG. The tag of His-GST was excised by a 3C protein after the purification through the nickel ion affinity chromatography column,and the fusion protein was purified by a DEAEion exchange column and molecular sieve. Soluble fibrin plate methods and inhibition test of platelet aggregation were applied to evaluate fibrinolytic activity and anti-platelet aggregation. Results 1） The fusion protein of RGDr SAK- α1M was construted. 2） The high express of target fusion protein in the supernatant of E. coli lysate was achieved,and purify the fusion protein. 3） The fibrinolytic activity of purified fusion protein was basically the same as that of urokinase standards. 4） The effect on anti-platelet aggregation was significantly enhanced by fusion protein rather than simple recombinant staphylokinase. Conclusions Literature retrieval has confirmed that the soluble fusion protein of RGD-r SAK- α1M is acquired,which has an equal fibrinolysis activity and a higher ability of antiplatelet aggregation compared to urokinase standards,and the effect on anti-platelet aggregation is significantly enhanced rather than simple recombinant staphylokinase,laying the foundation for following identification of fusion protein immunogenicity.