目的:克隆肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因片段(114~281氨基酸残基)并构建原核表达载体。方法:取健康人外周血提取总RNA,设计合成引物并引入EcoR I和Xho I酶切位点,RT-PCR扩增TRAIL基因的胞外区片段,克隆入原核表达载体pGEX-6P-1中,经双酶切、PCR及测序鉴定阳性克隆。结果:从外周血cDNA中扩增出501 bp的目的片段,测序结果证实成功构建重组质粒pGEX-6P-1/TRAIL。结论:成功构建TRAIL基因的原核表达载体pGEX-6P-1/TRAIL,为肿瘤细胞的凋亡研究提供理论依据。
Objective:To clone and construct the prokaryotic expression vector of the TNF-related apoptosis inducing ligand gene(amino acids 114~281).Methods:RNA was extracted from the peripheral blood of health adult.A pair of primers with restriction site EcoR I/Xho I was designed.The extracellular domain region of TRAIL gene was amplified by RT-PCR and then cloned into the prokary-otic expression vector pGEX-6P-1.The recombinants were confirmed by EcoR I/Xho I digestion,PCR,and DNA sequencing.Result:The extracellular domain region with a molecular size of 501 bp of TRAIL gene was amplified successfully.DNA sequencing showed that the recombinant plasmid was successfully constructed.Conclusion:A prokaryotic expression vector pGEX-6P-1/TRAIL was con-structed successfully,which provides theory for tumor apoptosis research.