论文部分内容阅读
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。结论ｈＣＮＴＦ基因的克隆和高效表达为进一步研究其结构功能关系和临床应用打下了基础
Objective To clone human ciliary neurotrophic factor (hCNTF) gene and express the biologically active hCNTF protein efficiently in E. coli host system. Methods The full-length cDNA encoding human CNTF was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA of human peripheral nerve tissue. The fragment was recovered and purified and cloned into PGEM-5Zf (+) plasmid vector The expression of hCNTF protein in E.coli host was induced by IPTG. The expression of hCNTF protein in the chick embryo dorsal root ganglion (DRG) neurons cultured in vitro was determined by recombinant DNA sequencing Neurotrophic activity of hCNTF protein. Results The human CNTF gene was cloned successfully. The target protein accounted for more than 25% of total bacterial protein after induced with 0.4 mmol / L IPTG for E. coli containing recombinant CNTF plasmid. Conclusion The cloning and high expression of hCNTF gene lay the foundation for further study of its structural and functional relationship and clinical application