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目的:探讨RNAi过程中TRBP传递双链RNA的机制,构建RNA结合结构域突变的人TRBP基因真核表达载体。方法:设计突变引物,通过拼接PCR将突变型TRBP基因克隆入pFLAG-CMV4真核表达载体,经双酶切和测序鉴定正确后,命名为pFLAG-CMV4-TRBPm。瞬时转染HEK-293细胞,western-blot检测目的蛋白表达。结果:成功扩增了突变序列,构建了突变型TRBP基因的真核表达载体,转染HEK-293细胞后检测到带标签的目的蛋白表达。结论:突变型TRBP基因真核表达载体的成功构建,为进一步研究TRBP的生物学效应奠定了基础。
OBJECTIVE: To investigate the mechanism of TRBP transmitting double-stranded RNA in RNAi and to construct eukaryotic expression vector of human TRBP gene with RNA binding domain mutation. Methods: The mutant TRBP gene was cloned into the eukaryotic expression vector pFLAG-CMV4 by splicing PCR. The double-digested and sequenced TRBP gene was named as pFLAG-CMV4-TRBPm. HEK-293 cells were transiently transfected, and the expression of the target protein was detected by western-blot. Results: The mutant sequence was successfully amplified and the eukaryotic expression vector of mutant TRBP gene was constructed. The expression of the target protein was detected after HEK-293 cells were transfected. Conclusion: The successful construction of mutant TRBP gene eukaryotic expression vector lays the foundation for further study on the biological effects of TRBP.