睫状神经营养因子突变体的克隆、表达、纯化及生物学活性

目的设计具有更高生物学活性的睫状神经营养因子突变体,并对其进行表达、纯化及生物学活性检测。方法以计算机分子模拟系统设计突变体,重叠延伸PCR方法获得突变体的编码区DNA序列,克隆入表达载体pThioHisA,转化E.coliBL21,以IPTG诱导表达。复性纯化后,用鸡胚背根神经节无血清培养法和小鼠减重法进行生物学活性测定。结果目的蛋白以包涵体形式存在,表达量在35%以上,纯化后的纯度达95%以上,能有效地促进鸡胚背根神经节的生长,并能使正常小鼠的体重降低,脂肪指数下降。结论所设计的突变体经表达及纯化后,具有良好的生物学活性,为进一步研究突变体蛋白的促神经生长和减肥作用奠定了基础。

Gene Cloning,Expression,Purification and Biological Activity of Ciliary Neurotrophic Factor Mutant

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.