新型重组纤维蛋白原受体拮抗剂的表达、纯化及活性研究

目的:构建两个预期能够成为纤维蛋白原受体拮抗剂的重组融合蛋白.方法:人工合成北美水蛭素Decorsin的基因序列,利用基因工程的方法构建两个重组蛋白,分别是AnnV-D39:Annexin V加上Decorsin全序列和AnnV-D27:Annexin V加上Decorsin的羧基端27个氨基酸序列.在AnnV-D39的Annexin V和Decorsin序列之间加入了由10个氨基酸组成的连接肽GGGGSGGGGS.利用质粒pET-28(a )作为载体,将上述融合蛋白在大肠杆菌E.coli BL21(DE3)中以包涵体的形式进行高效表达,纯化后,进行抗血小板聚集活性测试.结果:基因表达的产物在变性条件下用DEAE-Cellulose52和SepharoseCL-4B层析纯化.随后进行的血小板聚集分析表明,AnnV-D39具有良好的抗血小板凝集活性,而AnnV-D27没有显示相似活性.结论:AnnV-D39作为一种新的纤维蛋白原受体抑制剂显示了良好的活性,而Annv-D27需要进行进一步的修饰.

Expression, Purification,and Activity Assay of Two New Recombinant Fibrinogen Receptor Antagonists

AIM:To construct and characterize two recombinant p roteins, which are expected to be antagonists of fibrinogen receptors. METHOD:The gene sequence of decorsin which is extracted from a kind of North American leeches was synthesized.Two recombinant proteins,Annexin V plus decorsin (AnnV-D39) and Annexin V plus the carboxyl terminal 27 amino acids variant of decorsin (AnnV-D27), were constructed. And a 10 amino acids linke r peptide of GGGGSGGGGS was inserted between annexin V and decorsin in AnnV-D39 . Using pET-28 (a+) as an expressing vector, both two recombinant proteins we re expressed in E. coli BL21(DE3) with high efficiency as inclusion bodies .RESULT:The expression products were purified by DEAE-Cellulos e 52 and Sepharose CL-4B chromatography under denaturing condition.The results of platelet aggregation assay (PAA) show that AnnV-D39 shows a good anti -platelet aggregation activity. However, AnnV-D27 shows no such activities i n any PAA test .CONCLUSION:AnnV-D39 showed a good anti-platelet aggregation a c tivity as a promising antagonist of fibrinogen receptor, while Annv-D27 needs further modification.