凝血因子ⅩⅢ A亚基mRNA大片段缺失所致遗传性凝血因子ⅩⅢ缺乏症的分子机制研究  

Study on the molecular mechanisms of a novel large deletion of FⅩⅢ A mRNA in a new hereditary factor ⅩⅢ deficiency

作　　者：马秋玲 金洁[2] 蔡望伟[3] 

机构地区：[1]河南中医学院第二附属医院血液科,450002 [2]浙江大学医学院附属第一医院血液科 [3]海南医学院生物化学教研室

出　　处：《中华血液学杂志》2015年第2期131-134,共4页Chinese Journal of Hematology

基　　金：国家自然科学基金（30060037）;教育部科学技术研究重点项目（03147）

摘　　要：目的 研究凝血因子ⅩⅢ（FⅩⅢ）A亚基mRNA DelCD 11-279大片段缺失引起遗传性FⅩⅢ缺乏症的分子致病机制.方法 构建正常人、先证者母亲FⅩⅢA亚基mRNA表达质粒pET-22b（＋）/FⅩⅢA和先证者FⅩⅢA亚基mRNA DelCD 11-279大片段缺失的表达质粒pET-22b（＋）/FⅩⅢA-Del,转化大肠杆菌BL21,SDS-PAGE和Western blot法检测FⅩⅢA亚基蛋白的表达,Ni-NTA树脂结合柱纯化FⅩⅢA亚基蛋白质,EZ-LinkTM5-（Biotinamido） Pentylamine掺入法检测纯化FⅩⅢA亚基蛋白质活性.结果 pET-22b（＋）/FⅩⅢA和pET-22b（＋）/FⅩⅢA-Del经酶切及PCR鉴定构建成功,转化大肠杆菌BL21,经异丙基硫代-β-D-半乳糖苷诱导后获特异高效表达,SDS-PAGE显示重组蛋白质的相对分子质量分别为83 200和51 900,用Ni-NTA树脂结合柱分离得到纯化蛋白质,Western blot方法分析表明重组的蛋白质是人FⅩⅢA亚基蛋白,先证者及其母亲的纯化FⅩⅢA亚基蛋白活性分别为正常人的0、95.87％.结论 FⅩⅢA亚基mRNA DelCD 11-279大片段缺失导致编码截短的464个氨基酸的无活性FⅩⅢA亚基蛋白是先证者遗传性FⅩⅢ缺乏症的分子机制之一.Objective To investigate the mechanisms of DelCD11-279 of factor ⅩⅢ subunit A mRNA in the pathogenesis of hereditary factor ⅩⅢ deficiency.Methods The recombinant plasmids containing pET-22b （＋）/F ⅩⅢA of normal subject and proband＇ s mother and pET-22b （＋）/FⅩⅢA-Del of the proband were constructed and transformed into E.coli BL21.Expressing protein was analyzed by the SDSPAGE and purified by Ni-NTA resin.Purified proteins were detected by the Western-blot.The activity of purified protein was detected by the incorporation test with EZ-LinkTM5-（Biotinamido） Pentylamine.Result The recombinant plasmids containing pET-22b （＋）/F ⅩⅢ A and pET-22b （＋）/F ⅩⅢ A-Del which constructed and identificd successfully by enzyme digestion and PCR,were transformed into E.coli BL21 and efficiently expressed by IPTG induction.The molecular weights of expressing proteins are 83 200 and 51 900 by the SDS-PAGE.Expressing proteins were purified by Ni-NTA resin,and were proved to be human F ⅩⅢ A proteins by Western-blot.Purified protein activity of proband＇ s mother and proband was 95.87% and 0 of the purified FⅩⅢ A protein activity from the normal subject,respectively.Conclusions DelCD 11-279 of F ⅩⅢ A mRNA which encoding a 464 amino acids of inactive F ⅩⅢA protein is one of the molecular mechanisms resulting in FⅩⅢ deficiency in the patient.

关 键 词：因子ⅩⅢ 因子ⅩⅢ缺乏 质粒 突变 FACTOR ⅩⅢ 

分 类 号：R725.9[医药卫生—儿科]