人睫状神经营养因子基因在大肠杆菌中的表达  

EXPRESSION OF HUMAN CILIARY NEUROTROPHIC FACTOR GENE IN ESCHERICHIA COLI

作　　者：黄晓明[1] 胡向阳[1] 高翼之[1] 

机构地区：[1]南京铁道医学院医学科研所

出　　处：《中华医学遗传学杂志》1998年第2期69-71,共3页Chinese Journal of Medical Genetics

摘　　要：目的克隆人睫状神经营养因子（ｈＣＮＴＦ）基因，并在大肠杆菌宿主系统中高效表达出具有生物活性的ｈＣＮＴＦ蛋白。方法从人外周神经组织总ＲＮＡ经逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）扩增得到编码人ＣＮＴＦ的全长ｃＤＮＡ片段，此片段经回收和纯化后克隆进ＰＧＥＭ－５Ｚｆ（＋）质粒载体，并进行了ＤＮＡ序列分析，然后进一步亚克隆进Ｔ７启动子表达载体，用ＩＰＴＧ在大肠杆菌宿主中诱导ｈＣＮＴＦ蛋白表达，并以体外培养的鸡胚背根神经节（ＤＲＧ）神经元测定了重组ｈＣＮＴＦ蛋白的神经营养活性。结果成功克隆了人ＣＮＴＦ基因，含重组ＣＮＴＦ质粒的大肠杆菌经０．４ｍｍｏｌ／ＬＩＰＴＧ诱导３小时后，靶蛋白占细菌总蛋白的２５％以上。Objective To express biologically active human ciliary neurotrophic factor (hCNTF) gene in Escherichia coli. Methods Total RNA was extracted from human peripheral nerves and cDNA was synthesized by superscript reverse transcriptase, a polymerase chain reaction(PCR) was conducted to obtain a full length cDNA fragment encode for hCNTF gene. After recovery from gel and purification, the PCR product was cloned into the pGEM 5Zf(+) vector and DNA sequence analysis was performed to verify hCNTF gene. The hCNTF gene was then placed under control of T7 promoter in the expression vector pET 11d and transformed into Escherichia coli strain BL21(DE3). Cultures of this transformat were induced by IPTG for the expression of recombinant protein. The bioactivity of recombinant protein was evaluated by its ability to support the survival of embryonic chick dorsal root ganglion neurons in culture. Results The human CNTF gene was cloned and biologically active hCNTF was expressed efficiently. Based on densitometry of stained gel, the recombinant hCNTF accounted for more than 25% of the total bacterial protein. Conclusion The cloning and expression at high level of hCNTF gene in E.coli provides a basis for understanding the structure activity relationship of CNTF and its potential clinical application.

关 键 词：神经营养因子 hCNTF 大肠杆菌 基因表达 克隆 

分 类 号：Q78[生物学—分子生物学]