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［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。
[Abstract] Objective To clone human ciliary neurotrophic factor (hCNTF) gene and express it in Escherichia coli host system and purify it to obtain biologically active hCNTF protein. Methods The cDNA sequence encoding the mature CNTF protein was amplified by PCR using human chromosomal DNA as a template and cloned into prokaryotic expression vector pBV220. The recombinant plasmid was expressed in E. coli BL21 (Gold). After purification by CM-Sepharose FF column, Biological activity of chicken embryo dorsal root ganglion (DRG) cultured in vitro was measured. Results The recombinant plasmid pBV22Z-CNTF was stable and highly expressed in E. coli BL21 (Gold). The target protein accounted for about 45% of the total cellular protein. The recombinant protein was expressed in both soluble and inclusion bodies. The purity of inclusion bodies was 90% or more purified by CM column after refolding. The expressed recombinant hCNTF could promote the growth of cultured chick embryo dorsal root ganglion neurites. Conclusion The obtainment of genetic engineering rhCNTF lays the foundation for further study of its functional relationship and clinical application.