构建重组表达质粒pBV220/mhNT-4及mhNT-4在大肠杆菌中的表达  

The strategy for constructing the recombinant pBV220/the matured human neurotrophin-4 gene molecular cloning and its expression in escherichia coli

作　　者：张华[1] 赵家良[1] 胡海涛[2] 

机构地区：[1]中国医学科学院北京协和医院眼科,北京100730 [2]西安交通大学医学院神经生物学中心,西安710061

出　　处：《中国科技论文》2006年第4期263-268,共6页China Sciencepaper

基　　金：首都医学发展科研基金(2002-1014);美国中华医学基金(No.82413)

摘　　要：利用基因重组技术将 mhNT-4亚克隆到表达载体 pBV220,构建重组表达质粒 pBV220/mhNT-4,诱导表达 mhNT-4成熟蛋白,鉴定 mhNT-4的生物活性。将经鉴定的 pGEM-TEasy/mhNT-4克隆用限制性内切酶EcoRI,BamHI 消化,琼脂糖凝胶电泳回收片断。酶切原核高表达载体 pBV220,用限制性内切酶 EcoRI,BamHI 消化,琼脂糖凝胶电泳回收线性化载体。用 T4DNA 连接酶连结两个回收片断,构建重组质粒 pBV220/mhNT-4。限制性内切酶消化,琼脂糖凝胶电泳鉴定重组质粒。经鉴定的重组质粒 pBV220/mhNT-4转染大肠杆菌 DH5α,温度诱导表达 mhNT-4蛋白。SDS-聚丙烯酰胺(SDS-PAGE)凝胶电泳技术分离表达的 mhNT-4蛋白。制备8～9d鸡胚,分离并培养背根神经节(DRG)细胞。分别加入回收的表达蛋白和牛血清白蛋白,培养鸡胚背根神经节 DRG细胞,检测 mhNT-4生物活性。重组表达质粒 pBV220/mhNT-4构建正确,表达框架未发生改变。成功诱导表达、分离了 mhNT-4成熟蛋白。表达蛋白组可见大量神经突起自神经节组织快周缘向四周呈放射状长出,对照组未见神经突起长出。mhNT-4成熟蛋白具有良好的生物活性。本研究为进一步开展 mhNT-4基因治疗青光眼提供了资料。Through genetic recombination technique,the matured human neurotrophin-4 gene(mhNT-4)is inserted into polylinker site of expression vector pBV220,to generate a recombination plasmid pBV220/ mhNT-4 as expression vector.Construction,recombinant plasmind pBV220/mhNT-4 and evaluation the bioactivity of mhNT-4.The splice donor sequence BamH I and pretect base pair is obtained and cloned into fusion vector pBV220, after restrict enzymes digesting.(The results showed that mhNT-4 is cloned correctly into expression vector pBV220, recombinant expression vector pBV220/mhNT-4 is constructed.).Recombinant plasmind pBV220/mhNT-4 is correctly constructed and open reading frame is not exchange.This study successfully detachment,puraficated and indificated the gene of mhNT-4.The bioactivity of the secreted proteins is detected.The mhNT-4 protein is a satisfactory bioactivity.In conclusion,the results of this study suggest that genomic productions of the mature hNT-4 genes can be used for the neuroprotective therapy.It is concluded that the prokaryocyte cell expression vector is constructed successfully for gene therapy of glaucoma and other central nervous system diseases.

关 键 词：人神经营养素-4成熟蛋白基因(mhNT-4) 基因重组 表达载体 生物活性 

分 类 号：Q78[生物学—分子生物学]