重组变异纤维蛋白原体外表达及对凝血功能的影响  

Expression in Vitro of Recombinant Mutant Fibrinogen and Its Effect on Coagulation Function

作　　者：毛用敏[1] 平立芳 程津新[1] 陈倩[1] 赵福梅[1] 赵炳让[1] 崔让庄[1] 

机构地区：[1]天津市心血管病研究所,300051

出　　处：《天津医药》2002年第7期420-423,共4页Tianjin Medical Journal

基　　金：天津市自然科学基金资助项目(编号:013610711)

摘　　要：目的:采用基因重组技术和细胞培养方法制备纤维蛋白原（Fbg）β链K 318E/D 320 S变异蛋白,观察此变异蛋白对凝血反应的影响。方法:用Site-directed mutagenesis方法构建变异重组质粒;磷酸钙法将此质粒转染CHO细胞;经细胞培养表达变异蛋白;硫酸铵沉淀法和免疫亲和层析法获取蛋白。进行纤维蛋白多肽A、B释放实验和在3种Ca2+浓度下的纤维蛋白聚合实验,并评价此变异蛋白的功能。结果:变异与正常Fbg纤维蛋白多肽A、B释放实验无差异。纤维蛋白聚合实验显示,在不同Ca2+条件下两者间Lag period,Vmax和最终光密度均有显著性差异（P<0.05）。结论:B　β链变异蛋白可能引起Fbg D区结构的变化,使纤维蛋白聚合位点和Ca2+结合位点不能发挥正常功能,致使纤维蛋白聚合反应速度减慢及纤维蛋白纤维粗细出现异常。Objective :To study the expression in vitro of recombinant mutant fibrinogen(Fbg)and its effect on coagulation function. Methods: The recombinant mutant plasmid was constructed by site-directed mutagenesis, and then the plasmid was transfected into a CHO cell line to express mutant protein. The Fbg was precipitated with (NH4)2SO4 from medium and purified by immunoaffinity chromatography. The function of variant Fbg was measured. Results: There was no difference between normal and mutant fibrinopeptide A, B release tests. Lag period of fibrin polymerization was delayed for B β K318 E D 320 S with both 1 mmol /L and 10 mmol /L Ca2+. Vmax and final absorbance of normal Fbg were elevated with the increase of Ca2+ concentration ,but no change was observed in β K 318 E /D 320 S. Conclusion: B β chain variant protein may cause the structure change in D domain of fibrinogen,so the function of Ca2+ binding and fibrin polymerization site will not be normal. It also may impair the polymerization speed of fibrinogen and cause the size alteration of fibrin.

关 键 词：重组变异纤维蛋白原 体外表达 凝血功能 影响 

分 类 号：R446.11[医药卫生—诊断学]