人神经营养素-3全基因在大肠杆菌内的表达

Expression of the intact human neurotrophin-3 gene in E.Coli

目的 应用基因工程方法制备人神经营养素 3(hNT 3)蛋白 ,为研究hNT 3的生物学作用和开展应用hNT 3进行中枢神经损伤和退行性疾病的治疗提供材料。方法 将经过序列分析确定的hNT 3全基因重组至原核表达质粒pBV2 2 0中 ,构建了重组表达载体pBV/hNT 3,在大肠杆菌中表达后 ,SDS PAGE法测定目的蛋白的相对分子质量 ,鸡胚背根节培养法检测其生物学活性。结果 pBV/hNT 3在大肠杆菌中表达出了三个分子质量分别为 1 5× 1 0 3、1 8× 1 0 3和 33× 1 0 3的蛋白质 ,恰与hNT 3蛋白、其前导肽及前体的分子量相符。表达蛋白主要以包涵体形式存在 ,经纯化复性后 ,可明显促进鸡胚背根节的存活和神经元突起的生长。结论 人神经营养素 3全基因可在大肠杆菌内正确地转录和翻译 。

Objective To prepare the human neurotrophin 3(hNT 3) by genetic engineering method, and provide materials for probing its biological roles and therapeutical effects on neurodegenerative disorders of central nervous system. Methods Identified by sequence analysis, the hNT 3 intact gene was recombined into the prokaryotic expression plasmid pBV220, thus the expression vector pBV/hNT 3 was constructed and expressed in E. coli. The relative molecular weight of the novel protein was estimated by SDS PAGE,and its bioactivity was measured by chicken embryo dorsal root ganglion (DRG) culture. Results The recombinant pBV/hNT 3 could produce 3 novel proteins of 15×10 3,18×10 3 and 33×10 3 in E. coli,which match the molecular weight of hNT 3,the prepro peptide and the precursor. The expressed protein mainly recovered as inclusion body. After purification and renaturation,it could promote survival and neurite outgrowth of DRG neurons. Conclusion The hNT 3 intact gene could be correctly transcribed,translated and partially processed in E. coli.