抗炎、抗凝双效融合蛋白TAP-SSL5表达载体的构建及其功能研究

Construction of a novel fusion protein TAP-SSL5 and evaluation of its function as anti-inflammatory and anticoagulant protein in vitro

目的构建一种具有抗炎和抗凝双效功能的融合蛋白,并对其功能进行初步鉴定。方法将重组金黄色葡萄球菌超抗原样蛋白-5(staphylococcal superantigen-like protein-5,SSL5)与蜱抗凝血肽(tick anticoagulant peptide,TAP)融合,从金黄色葡萄球NCTC8325菌株中克隆SSL5基因;经DNA重组技术,将编码pelB前导序列、SSL5和TAP的基因重组并克隆于原核表达载体pHOG21中,再转染于大肠杆菌BL21中扩增表达,制备融合蛋白SSL5-TAP或TAP-SSL5,采用流式细胞仪检测融合蛋白是否和SSL5一样,具有与粒细胞表面P-选择素糖蛋白配体-1(P-selectin glycoprotein ligand-1,PSGL-1)结合的特性;采用反应底物法在体外检测融合蛋白对活化的凝血因子X(factor Xa,FXa)活性的抑制作用。结果TAP-SSL5保留了SSL5与PSGL-1结合的特性,并且融合蛋白TAP-SSL5可显著抑制FXa的活性(P<0.01),抑制率达39.5%。结论融合蛋白TAP-SSL5是一种以PSGL-1为靶向的新型抗炎、抗凝双效分子。

Objective To construct a double-effect fusion protein functioning as an anti-inflammatory and anticoagulant, which can inhibit the inflammatory response and thrombosis simultaneously in atherosclerosis lesions. Methods Firstly, staphylococcal superantigen-like protein-5 （SSL5） gene sequence was cloned by PCR from Staphylococcus aureus （strain NCTC8325）. Secondly, N-terminal pelB leading sequence, SSL5 gene sequence, tick anticoagulant peptide （TAP） gene sequence, with C-terminal both c-Myc and His6 tags were cloned into the pHOG21 expression vector using recombined DNA technique, and the obtained recombinant were further transformed into E. coli BL21 （DE3） cells to express the fusion protein TAP-SSL5. Flow eytometry was used to investigate whether the fusion protein could bind with P-selectin glyeoprotein ligand-1 （ PSGL-1 ） on monoeytes as SSL5. Finally, the inhibitory effect of TAP-SSL5 on factor Xa （FXa） activity were detected by chromogenic substrate assay in vitro. Results TAP-SSL5 fusion protein binded to PSGL-1 on HL60 monocytes, and inhibited the FXa activity in vitro as well, with an inhibitory rate of 39.5% （P 〈0.01 ）. Conclusion The fusion protein TAP-SSL5 is a novel molecule targeting PSGL-1, which has an double-effects of antiinflammation and anticoagulation.