人睫状神经营养因子基因的克隆、表达、纯化和生物活性鉴定  

Cloning,Expression,Purification and Characterization of Recombinant Human Ciliary Neurotrophic Factor Genes

作　　者：窦金民[1] 郭莉莉[1] 刘宏磊[1] 陈接根[1] 黄海燕[1] 宋后燕[1] 汤其群[1] 

机构地区：[1]复旦大学上海医学院生物化学与分子生物学系-教育部分子医学重点实验室,上海200032

出　　处：《复旦学报（医学版）》2005年第4期419-422,426,共5页Fudan University Journal of Medical Sciences

摘　　要：目的表达和纯化人睫状神经营养因子(CNTF)并观察其生物学活性。方法用PCR方法构建人CNTF的结构基因,并克隆于pLy4原核表达载体中。将重组表达质粒pLy4-CNTF转化大肠埃希菌JF1125,30℃培养至A600(nm)达到0.6时,迅速升温至42℃诱导目的蛋白的表达。细菌经超声破胞,包涵体经变性、复性、透析和阴离子交换层析,获得纯化的CNTF重组蛋白。然后,用鸡胚背根神经节神经元细胞存活实验观察其生物活性。结果42℃诱导后可以获得Mr≈22.9×103的目的蛋白,Western印迹结果进一步表明,其具有人CNTF的抗原性。表达蛋白主要以不溶形式存在,占菌体蛋白的35%以上,表达产物经变性、复性,纯化后可获得纯度90%以上的目的蛋白。该重组蛋白能够促进鸡胚背根神经节神经元细胞的存活。结论在大肠埃希菌JF1125中成功表达了人CNTF分子,经变性、复性,纯化后得到具有生物活性的蛋白。Purpose To obtain biologically active human ciliary neurotrophic factor. Methods Gene encoding for human ciliary neurotrophic factor(hCNTF) was amplified by PCR,and cloned in the expression vector pLy4 in which the target gene was under the control of a temperature-regulated promoter.The recombinant expression plasmid pLy4-CNTF was transformed into E.coli JF1125.When A 600(nm) reached 0.6 at 30?℃,this transformant was induced for expression of recombinant protein by temperature increasing to 42?℃.Recombinant hCNTF (rhCNTF) was in form of inclusion bodies and obtained after denaturation,renaturation,dialysis and ion-exchange chromatography.The bioactivity of recombinant protein was evaluated by its ability to support the survival of embryonic chick dorsal root ganglion (DRG) neurons in culture. Results After induction for 3 hours at 42?℃,rhCNTF was over 35% of total bacterial protein.SDS-PAGE and Western blot analysis indicated that its molecular weight was about 22.9×103 (Mr) and it could react with anti-CNTF.After purification by ion-exchange chromatography,the purity was up to 90%.The recombinant protein could support the survival of embryonic DRG neurons. Conclusions We successfully expressed human ciliary neurotrophic factor in E.coli JF1125 and obtained biologically active protein.

关 键 词：人睫状神经营养因子 生物活性鉴定 纯化 克隆 WESTERN印迹 背根神经节神经元 大肠埃希菌 CNTF 原核表达载体 重组表达质粒 离子交换层析 重组蛋白 生物学活性 PCR方法 神经元细胞 结构基因 实验观察 细胞存活 菌体蛋白 

分 类 号：R394.8[医药卫生—医学遗传学] R927[医药卫生—基础医学]