人促血小板生成素(TPO)原核表达载体pQE30-TPO的构建

Construction of human thrombopoietin(TPO)prokaryotic expression vector pQE30-TPO

目的:获得人血小板生成素全长cDNA克隆,并构建重组表达载体pQE30-TPO。方法:利用RT-PCR技术从人胎肝细胞mRNA中扩增目的基因,将扩增产物克隆至pMD18-T载体,经菌落PCR鉴定后,DNA序列分析重组质粒pMD18-T-TPO。构建并鉴定原核表达载体pQE30-TPO。结果:构建了重组克隆载体pMD18-T-TPO和重组表达载体pQE30-TPO,序列分析表明,获得的TPOcDNA序列与国内外报道的人促血小板生成素核苷酸序列完全一致。结论:成功构建了重组表达载体pQE30-TPO,为TPO的进一步研究奠定了基础。

Objective:To clone human thrombopoietin cDNA and to construct human TPO prokary-otic expression vector.Method:RT-PCR was used to amplify the target gene with total RNA from human fetal liver cell.The gene was inserted into pMD18-T,identified by bacteria colonies PCR,then sequenced.After that,the prokaryotic expression vector pQE30-TPO was constructed and iden tified.Results:Clonal re combi nant of pMD18-T-TPO and prokaryotic expression vector pQE30-TPO were constructed.The ob-tained TPO cDNA sequence is the same as what had been reported previously.Conclusion:Prokaryotic expression vector pQE30-TPO was successfully constructed.