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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 fragment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene (114-281 amino acid residues) and construct prokaryotic expression vector. Methods: Total RNA was extracted from peripheral blood of healthy volunteers. Primers of EcoR I and Xho I were designed and synthesized. The extracellular region of TRAIL gene was amplified by RT-PCR and cloned into prokaryotic expression vector pGEX-6P-1 The positive clones were identified by double enzyme digestion, PCR and sequencing. Results: The 501 bp target fragment was amplified from the peripheral blood cDNA. Sequencing results confirmed the successful construction of the recombinant plasmid pGEX-6P-1 / TRAIL. CONCLUSION: The prokaryotic expression vector pGEX-6P-1 / TRAIL of TRAIL gene is successfully constructed and provides a theoretical basis for the study of tumor cell apoptosis.