人CNTF突变体(CNTFM)基因的克隆、表达和产物纯化及活性鉴定

采用PCR的方法对睫状神经营养因子(CNTF)基因进行改造,获得CNTF突变体基因(CNTFM) ,将CNTFM基因克隆入表达载体pBV2 2 0 ,在大肠杆菌BL 2 1(Gold)中进行了表达.目的蛋白占细胞总蛋白5 5 %左右,以包涵体形式存在,经Superdex 75凝胶过滤柱一步纯化和复性,获得纯度达90 %目的蛋白.纯化的重组CNTFM蛋白能促进培养的鸡胚背根神经节长出神经突起,能明显减轻实验小鼠的体重,表明CNTFM具有良好的体内、体外生物学活性,为开发新型高效的减肥药奠定了基础.

Cloning, Expression and Purification of Human CNTF Mutant (CNTFM) and Characterization of Product

Ciliary neurotrophic factor(CNTF) is a trophic protein that promotes survival and/or differentiation of a variety of neuronal cell types including sensory,sympathetic,and motor neurons.Here we report construction of a human CNTF mutant (CNTFM) and analysis of its function.The mutant at position 17C/S,63G/A of rhCNTF expression plasmid was constructed by oligonucleotide-directed mutagenesis and cloned into E.coli BL21(Gold) to induce expression of the target protein.The recombinant protein account for about 55% of the total bacterial proteins,about 90% purity of rhCNTFM was obtained following denaturing and purifying via Superdex-75 chromatography.Its survival promoting activities were determined using cultures of embryonic chick dorsal root ganglion (DRG) neurons.The results showed that compared with control group,treatment of DRG neuron with 100 ng/ml rhCNTFM induces significantly growth of radial nerve fibers and has the same effect as the native CNTF. Furthermore,this mutant protein increase mouse weight loses after 2—6 weeks administration(p<0.05).These data provided experimental confirmation for development of new type and high efficient weight reducing drug.