关键词: 重组葡激酶, 人α微球蛋白, RGD, 融合蛋白, 溶栓活性, 抗血小板聚集

Abstract: Objective Our study aims to construct a recombinant prokaryotic expression plasmid of RGD-rSAK- a1M in E.col BL21. Furthermore, it aims to purify the fusion protein and determine its bioactivity. Methods The target fusion gene, RGD-rSAK-a1M，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 -rSAK- a1M, 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 DEAE ion exchange column and molecular sieve. Soluble fibrin plate methods and inhibition test of platelet aggregation were applied to determine and evaluate its fibrinolytic activity and anti-platelet aggregation, respectively. Results 1) The fusion protein of RGD-rSAK- a1M was construted successfully. 2) The high express of target fusion protein in the supernatant of E.coli lysate was achieved, and purify the fusion protein. 3) Vitro experiments indicated that 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 confirm that the soluble fusion protein of RGD -rSAK- a1M is firstly acquired, which has an equal fibrinolysis activity and a higher ability of anti-platelet aggregation compared to urokinase standards, and the ?effect on anti-platelet aggregation was significantly enhanced rather than simple recombinant staphylokinase，laying the foundation for following identification of fusion protein immunogenicity.

Key words: recombinant staphylokinase, human α-microglobulin, RGD, fusion protein, expression and purification, anti-platelet aggregation