血管性血友病因子A1分子在大肠杆菌中的可溶性表达及功能鉴定  

Soluble expression and function of von Willebrand factor-A1 in Escherichia coli

作　　者：王依璐[1] 刘晓玲[1] 丁孝茹 方颖[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东省广州市510006

出　　处：《中国组织工程研究》2014年第38期6153-6159,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(10972081;11072080;11272125)~~

摘　　要：背景:血管性血友病因子A1通过介导随流血小板在受损血管部位的黏附在初始止血中发挥至关重要的作用,但重组A1在原核中多为包涵体表达,不利于纯化及体外的功能研究。目的:在大肠杆菌中稳定高效表达可溶性野生型血管性血友病因子A1以及突变型血管性血友病因子G1324S,并研究其生物学功能。方法:将血管性血友病因子A1(野生型)、血管性血友病因子G1324S(功能失去型突变体)重组质粒分别转化到感受态细胞DH5α中,筛选提取质粒。将提取的质粒分别转化到大肠杆菌(E.coli)BL21、E.coli M15中,筛选挑单克隆菌于LB培养基,扩大培养。比较溶菌酶破碎法和超声波破碎法两种不同的裂菌方法,使目的蛋白血管性血友病因子A1的可溶性表达,经过Ni柱亲和层析纯化蛋白,用SDS-PAGE和Western-blot鉴定纯化的血管性血友病因子A1、血管性血友病因子G1324S纯度和免疫学活性;采用流动腔实验系统分析在不同流体剪应力条件下,血小板在两种目的蛋白上的黏附能力。结果与结论:每100 mL上清液可分别纯化得到5.77 mg血管性血友病因子A1,6.83 mg血管性血友病因子G1324S纯品,并具有较高的纯度和免疫学活性。流动腔结果显示,对每个剪切应力下铺设不同A1分子的底板进行两两比较,随着流体剪应力从0.1 Pa提高1 Pa,G1324S突变组所黏附细胞数下降的幅度(46.8%)要远高于野生型组(20.5%),说明突变后的A1分子的功能有所减弱。结果表明纯化的蛋白介导血小板黏附的能力与临床特征一致,野生型血管性血友病因子的结合活性明显高于突变型。BACKGROUND： von Willebrand factoroA1 （VWF-A1） plays a crucial role in primary hemostasis by mediating blood platelet adhesion to sites of vascular damage under conditions of high shear stress. However, mostly expression of recombinant VWF-A1 in form of inclusion bodied within Escherichia coil （E. colt） that is not conducive to purify and functional study in vitro. OBJECTIVE： To acquire soluble recombinant VWF-A1 including wild type and its mutation G1324S which can be stable efficiently expressed in E. coil and to verify their biological function. METHODS： Wild-type VWF-A1 and its mutation G1324S recombinant plasmids were transformed into competent cells DH5a, to screen and extract the plasmids. Then the extracted plasmids were respectively transformed into E. coil BL21 and E. coil M15, to screen monoclonal bacterium. The bacterium was cultured in LB culture medium. The bacteria breaking methods with lysozyme and with ultrasonic were compared. Then, the solubilized proteins were passed over Ni-NTA agarose column to purify the VWF-A1 protein, and the purity and immune activity of purified products were identified with SDS-PAGE and western blot analysis. Finally, the biological function of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion by parallel-plate flowchamber system. RESULTS AND CONCLUSION： We obtained about 5.77 mg VWF-A1, 6.83 mg VWF-G1324S in culture supernatants per 100 milliliter, respectively, which all showed high purity and immune activity. The data of flow chamber experiment demonstrated that both VWF-A1 and VWF-G 1324S could mediate platelet adhesion. As the shear stress increased from 0.1 Pa to 1 Pa, the decline （46.8%） of the number of adhesive calls in G1324S mutation group was higher than that of wild type group （20.5%）. This evidence indicated that, the A1 molecule functions were attenuated after mutation. Our research shows that, wild type of VWFoA1 showed a stronger interaction with platelet in shear stress, which is con

关 键 词：血管性血友病疾病 血友病A 血小板 组织构建 组织工程 血管性血友病因子A1 血管性血友病因子G1324S 原核表达 可溶性 亲和层析 流动腔 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]