睫状神经营养因子突变体的克隆、表达、纯化及生物学活性

Objective To design and express the gene mutant encoding ciliary neurotrophic factor (CNTF) with higher bioactivity. Methods Design the gene mutant by computer molecular modeling system. Amplify the DNA sequence at encoding region of the gene mutant by overlap extension PCR and clone into expression vector pThioHisA, then transform to E. coli BL21 for expression under induction of IPTG. Re-naturalize and purify the expressed product for determination of bioactivity by serum-free culture of chick embryo dorsal root ganglion and mouse bodyweight decrease test. Results The expressed product in a form of inclusion body contained more than 35% of total somatic protein and reached a purity of more than 95% after purification. It promoted the growth of chick embryo dorsal root ganglion effectively and decreased the bodyweight and fat index of normal mice. Conclusion The expressed mutant protein showed good bioactivity, which laid a foundation of further study on the role of CNTF in promoting the growth of nerve and decreasing the bodyweight.