人神经营养素-4基因的克隆及表达

Cloning and expression of the human neurotrophin-4

目的通过基因工程的方法扩增人神经营养素-4(hNT-4)cDNA,在大肠杆菌中表达hNT-4,为研究hNT-4的生物学作用提供材料。方法以人胎脑RNA为模板,采用RT-PCR技术扩增hNT-4 cDNA,将其插入的质粒pGEM-T中。将经过序列分析确定的hNT-4 cDNA插入原核表达载体pGEX-6p-1中,构建重组表达载体pGEX-6p-1-NT-4,用SDS-PAGE法测定融合蛋白在大肠杆菌中的表达。结果经序列测定分析,克隆的hNT-4 cDNA核苷酸序列与已发表序列(GenBank,M86528)完全一致,并获得了高效表达hNT-4的重组菌株。结论体外成功扩增hNT-4 cDNA,并在原核细胞中高效表达了hNT-4。

Objective Clone human neurotrophin-4 cDNA through the biotechnical method, express its protein in E. coli, and provide material for researching the biological activity of hNT-4. Methods With the RNA of human fetal brain as template, hNT-4 cDNA was amplified by RT-PCR and recombinated into phage pGEM-T. After being sequenced, the cloned hNT-4 cDNA was recombinated into expressed vector pGEX-6p-1, and the fusion protein was expressed in E. coli and identified by SDS-polyacarylamide gel electrophoresis. Results The sequence of the cloned hNT-4 cDNA was completely the same as the reported one （ GenBank, M86528）, and the bacterium line which could express the fusion protein efficiently was screened out. Conclusion The hNT-4 cDNA was amplified and hNT-4 was expressed in vitro successfully.