重组人睫状神经营养因子突变体的制备及PEG修饰

Preparation and PEGylation of Recombinant Human Ciliary Neurotrophic Factor Mutant

通过大肠杆菌重组表达人睫状神经营养因子突变体(CNTFm),并进行PEG修饰,旨在降低免疫原性。该突变体将天然CNTF的C端15个氨基酸删除,大肠杆菌表达的CNTFm以包含体形式存在,经复性、纯化获得纯度达到95%的目的蛋白。体内生物学活性测定结果显示,给药10d,小鼠最大体重减少率达31%,产生的最高中和抗体滴度达到1∶6400;经PEG修饰,CNTF突变体的生物学活性降低了34%,但最高中和抗体滴度降低到1∶800。该PEG修饰后的CNTFm制备工艺有望为CNTF的临床应用开辟道路。

Human ciliary neurotrophic factor（CNTF） is a neurotrophic factor（NTF） not included in NTF family. Recently body weight loss effect of CNTF was discovered in many studies, but clinical application has been limited as immunogenicity. Prepared ciliary neurotrophic factor mutant （CNTFm） with prokaryotic expression and analyzed effect on weight reduction. Chemical synthesis DNA of CNTFm, in which polypeptide is truncated 15aa at C-terminus. The DNA is subcloned in temperature and induced vetor pBV220, then the recombinant plasmid is transformed in E. coli BL21 and recombination protein expression is induced by culture at 42 ℃. The recombination protein is purified with ion exchange chromatography. The normal mice＇ weight boss tests are used for activity analysis. The expressed level of the CNTFm can reach over 40% of the total bacteria protein and exist in a form of inclusion body. After denaturing, refolding and purifying by Q Sepharose-FF, the purity of final product is more than 95%. Activity analysis indicates that the mutant protein obviously reduces body weight of tested mouse, maximal slip of body weight to reach 31.23% with administration 10 days, but the antibody levels induced by the CNTFm are significantly higher. CNTFm is modified by methyl polyethylene glycol succinimidyl propionate（mPEG-SPA）. Compared with unmodified CNTFm, the pEG-CNTFm has 66% biologic activity of unmodified CNTFm in vivo, but it has significantly decreased the immunogenicity.