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

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。

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

Objective:The CNTF-T gene was generated by site-specific mutagenesis 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 gene,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 generation of biological diet drug and apply in clinic in the future.