人睫状神经营养因子基因克隆、表达、纯化及其生物活性

［摘 要］目的 克隆人睫状神经营养因子（hCNTF）基因，在大肠杆菌宿主系统中高效表达并纯化，获得具有生物活性的hCNTF蛋白。方法 以人染色体DNA为模板，经PCR扩增到编码CNTF成熟蛋白的cDNA序列，并克隆进原核表达载体 pBV220，在大肠杆菌 BL21（Gold）中进行表达，产物经 CM-Sepharose FF柱纯化后，以体外培养的鸡胚背根神经节（DRG）测定生物活性。结果 重组表达质粒pBV22Z- CNTF在大肠杆菌BL21（Gold）中获得了非融合的稳定、高效表达，目的蛋白占细胞总蛋白的45％左右，以可溶性和包涵体两种形式存在，表达上清及包涵体复性后经CM柱一步纯化纯度达90％以上；表达的重组hCNTF能促进培养的鸡胚背根神经节神经突起的生长。结论 基因工程rhCNTF的获得为进一步研究其结构功能关系和临床应用奠定基础。

Cloning and Expression of Human Ciliary Neurotrophic Factor Gene and Purification and Bioactivity of Expressed Product

Abstract] Objective To clone and highly express human ciliary neurotrophic factor(hCNTF)gene in E. coli and obtain purified hCNTF protein with bioactivity. Methods The cDNA sequence encoding hCNTF mature peptide was amplified from human chromosome DNA by PCR, cloned into expression vector pBV220 and transformed to E. coli BL21(Gold) .The expressed product was purified by CM-Sepharose FF column chroma-tography,and the bioactivity of it was detected by the dorsal root ganglia(DRG)of chick embryo cultured in vitro.Results The recombinant plasmid pBV220-QNTF was stably and highly expressed in E. coli BL21 ( Gold) . The expressed product existing in both the froms of soluble protein and inclusion body contained about 45% of total somatic protein and reached a purity of above 90% after being purified. It promoted the growth of tubercles of DRG. Conclusion The development of rhCNTF lays a foundation of further study on the relationship between structure and function as well as clinical application of it.