人神经营养素-4的克隆及其在原核细胞的表达

Cloning and prokaryotic cell expressing of gene encoding human neurotrophin-4

目的：体外扩增人神经营养素－4(NT-4)基因，经克隆后在原核细胞中表达并鉴定人重组NT-4，为研究其生物学特性及临床应用奠定基础。方法：以人类基因组DNA为模板，扩增NT-4成熟活性蛋白的DNA编码序列，将此DNA片段克隆到载体PBV220中，对重组质粒进行PCR筛选和限制性酶切分析，确定后将该重组质粒转化到大肠杆菌DH5α进行诱导表达。用抗NT-4抗体进行Western blot鉴定重组蛋白。结果：体外成功扩增NT-4基因，转染的大肠杆菌经诱导后表达出能被抗NT-4抗体识别的重组蛋白，其表达量为15%。结论：在原核细胞能成功表达NT-4。

Objective:To amplify in vitro the gene for h uman neurotrophin－4(NT－4) gene from chromosomal DNA and induce the expression of recombinant NT －4 in prokaryotic PBV220 system,and to set up the base for further resear ch on its biological character and clinical application. Methods:By the means of PCR the human NT－4 gene was amplified from chromosomal DNA extracted from fetus brain. PCR products were then ligated to PB V220 and the recombinant was transformed to DH5α. Screened by PCR and res triction enzymes digestion,the cells were induced to express the desired NT－4 w hich was then collected by the way of time course and detected by Western－blot. Results:NT－4 gene was amplified and a recombinant plasmid expressi ng human NT－4 in Ecoli was constructed.The induced recombinant protein was rec ognized by anti－NT－4 serum. The yield of recombinant protein was 15% of total cell lysis. Conclusions:NT－4 was successfully expressed in prokaryotic PBV 220 system.