【作者】 李东升；

【导师】 李大伟；

【作者基本信息】 上海交通大学 ， 微生物与生化药学（专业学位）， 2016， 硕士

【摘要】 凝血Ⅷ因子是凝血级联反应中极为重要的一种凝血因子,凝血Ⅷ因子的缺乏将导致患者凝血功能障碍,引起血友病。目前血友病的治疗主要通过补充体内不足的凝血Ⅷ因子,以维持患者体内正常Ⅷ因子的水平。基因工程为人类治疗血友病提供了绝佳途径,使用基因工程方式能够获得充足的人凝血Ⅷ因子。人凝血Ⅷ因子全长2332个氨基酸,由于其肽链全长较长,且人凝血Ⅷ因子mRNA不稳定、翻译后的蛋白非正常折叠、转运过程中与内质网中的免疫球蛋白结合蛋白(immunoglobulin-binding protein,BiP)的结合、内部含有沉默子等因素的影响,使得其在宿主细胞内的表达量较低。研究表明,Ⅷ因子内某些位点氨基酸的突变、BiP结合区域内某些氨基酸的突变(如F309S或F309A)、以及BiP结合区域外糖基化位点的优化等方式可提高Ⅷ因子的分泌表达。本实验室前期结果也发现在BiP结合区域附近的某些位点可以影响Ⅷ因子的分泌表达。本实验在实验室原有研究的基础上,选择在FⅧ A1结构域中BiP结合区域外的E287位点,P290位点及H311位点进行突变。通过将突变质粒瞬时转染到293T细胞中,72 h后裂解细胞,使用蛋白免疫印迹检测胞内FⅧ表达量,使用Coamatic试剂盒检测分泌到胞外的FⅧ活性,以及对表达EGFP的细胞量的统计计数,结果表明,P290T,E287D,E287H这三种突变的胞内FⅧ表达效率没有明显提高,但是胞外Ⅷ因子活性相对WT-BDD-FⅧ提高了近2倍。提示这些突变可能提高了分泌到细胞外的FⅧ的比活,也可能通过改变FⅧ的结构,使得分泌到细胞外的FⅧ的量更多。更多还原

【Abstract】 FⅧ is a crucial factor which plays an important role in coagulation cascade reaction.Deficiency of FⅧ can cause severe coagulation disorders and thus lead to Hemophilia A.At present the treatment of Hemophilia A is to supplement the FⅧ,in order to maintain the normal FⅧ levels.Genetic engineering provides a good channel to derive enough FⅧ to cure the patients.Since the molecular of FⅧ is too large(2332 amino acids),the instability of FⅧ mRNA,informal misfolding of post-translational protein,binding to the BiP sites and internal silencer,the production of FⅧ is quite low.Former research demonstrates mutant of some amino acids in FⅧ,mutant of specific amino acid within BiP(for example,F309 S and F309A),optimization of glycosylation sites out of BiP sites can dramatically improve the expression and secretion of FⅧ.Research from our lab showed mutagenesis near the BiP sites can enhance the expression of FⅧ,based on the former results of our lab,we did site specific mutagenesis on the sites of E287,P290,H311.After transfecting the mutagenesis plasmid into 293 T cells for 72 h,used Western Blot to test the intracellular FⅧ expression and used the Coamatic assay to test the extracellular FⅧ activity in the supernatant,counted the number of cells which expressed EGFP,the results showed mutagenesis in P290 T,E287D,E287 H could not improve the intracellular FⅧ expression efficiency but could improve the activity of FⅧ to two times higher than the control(wild-type FⅧ).These suggests the above three mutagenesis can either improve the FⅧ specific activity or enhance the quantity of secreted FⅧ protein through the change of protein structure.更多还原