重组人睫状神经营养因子突变体的构建、表达及生物活性分析  

Expression and Biologic Activity Analysis of Recombinant Human Ciliary Neurotrophic Factor(CNTF) Mutant

作　　者：应莲芳[1] 雷清[1] 梁凌宇[1] 高雪峰[1] 蒋琳[1] 

机构地区：[1]兰州生物制品研究所,甘肃兰州730046

出　　处：《生物技术》2007年第4期16-18,共3页Biotechnology

摘　　要：目的:通过对原始CNTF的突变改造,降低表达蛋白的免疫原性,同时增加其稳定性,以期能用于临床治疗肥胖症。方法:以原始CNTF为模板,去掉N端的12个氨基酸、C端的14个氨基酸,并将C端末2个氨基酸点突变为极性较强的赖氨酸,在大肠杆菌中表达,获得CNTF-T突变体。结果:基因序列分析与原设计吻合,目的蛋白表达量占全菌体的35%左右,表达蛋白以包涵体形式存在,经变性、复性、DEAE-FF纯化,纯度可达95%以上,小鼠体内生物活性测定,能明显抑制小鼠体重增长,且CNTF-T的生物活性比原始序列CNTF的活性高。结论:突变的CNTF-T有望成为新一代的生物减肥制剂应用于临床。Objective：The CNTF- T gene was gencrated by site-specific mutagencsis based on the natural human CNTF. It was wished to decrease the immunogenicity and increase the stability of CNTF - T expression protein. Methods： Based on the natural human CNTF genc, both of 12 amino acids of N - terminal and 14 amino acids of C - terminal were removed, and 2 amino acids of C - terminal were mutated to Lysine by site- specific mutagenesis method so as to acquire CNTF- T mutant. Results： The mutated gene sequence accorded with the original design and was highly expressed in E. coli. The expressed product was up to approximately 35 % in total protein and existed in a form of inclusion body. After deraturing, refolding and purifying by DEAE- FF, the purity of final product was more than 95%. The biological activity of CNTF- T was measured by mouse in vivo test and the result showed that mutant protein could depress obviously the weight increasing of tested mouse, the activity of CNTF - T was higher than that of nature CNTF. Conclusion： The mutated CNTF - T protein was hoped to be a new gencrafion of biological diet drug and apply in clinic in the future.

关 键 词：睫状神经营养因子(CNTF) 突变体 基因表达 生物活性 减肥 

分 类 号：Q786[生物学—分子生物学] R392.11[医药卫生—免疫学]