纤维蛋白原γ链D结构域基因点突变导致遗传性纤维蛋白原缺陷症的分子机制研究

项目名称： 纤维蛋白原γ链D结构域基因点突变导致遗传性纤维蛋白原缺陷症的分子机制研究

项目编号： No.81501810

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 朱丽青

作者单位： 温州医科大学

项目金额： 18万元

中文摘要： 纤维蛋白原（Fibrinogen，Fg）γ链D区包含了Ca2+、凝血酶等多个配体的结合位点，对Fg正常功能的发挥至关重要。本研究组前期发现了三例位于γ链D结构域世界首报的氨基酸点突变：Lys232Thr、Trp208Leu及Thr277Arg，均引起先证者及部分家系成员Fg水平显著下降，导致遗传性纤维蛋白原缺陷症。蛋白模型分析发现：这三个突变氨基酸可能通过改变空间位阻、改变氨基酸疏水性、诱发糖基化位点等机制，导致所在蛋白错误折叠、组装异常，继而影响其稳定性或正常分泌，导致血浆Fg水平降低。本项目将通过构建突变体表达质粒，体外表达和纯化突变蛋白，进行一系列实验，深入研究突变后引起Fg含量降低的机制，阐明突变蛋白影响Fg发挥正常止凝血功能的机制。同时通过丙氨酸置换、同源氨基酸置换等突变体的研究，探讨该三个位点在纤维蛋白原γD结构域中的特异性功能，深入了解突变引起纤维蛋白原缺陷症的具体发病机制。

中文关键词： 遗传性纤维蛋白原缺陷症；纤维蛋白原；基因突变；体外表达；分子机制

英文摘要： γD domain of fibrinogen (Fg) contains many ligand binding sites, such as ca2+ and thrombin, which are vital to the normal functions of Fg. Our previous researches have been identified three novel mutations (Lys232Thr, Trp208Leu and Thr277Arg) in this domain, which cause congenital fibrinogen deficiency in three families. Protein model analysis speculated that these mutations probably made a change in the electrostatic potential of protein surface, or create a void and cause local refolding in order to close the gap, also may induced glycosylation site that increased instability of the protein. All of those are eventually result in the low concentration of Fg. We plan to construct these three mutant plasmids and express in vitro, purified the mutant protein, then do a series of experiments to find out the mechanism of mutations leading to congenital fibrinogen deficiency. Besides these, Alanine replaced mutants, homologous amino acid substituted mutants and polar/nonpolar exchanged mutants are also be planed to figure out the special function of these amino acids in the γD domain.

英文关键词： congenital fibrinogen deficiency; fibrinogen;gene mutation;expression in vitro;molecular mechanism