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本研究应用分子生物学技术 ,构建小鼠 β2 m反义核酸重组荧光蛋白表达载体pEGFP β2 mAN ,经过酶切、PCR鉴定 ,以及序列分析证实克隆的正确性。然后用脂质体转染NIH3T3细胞 ,经过RT PCR及Westernblot检测 ,观察重组质粒对细胞β2 mmRNA和蛋白表达的影响 ,同时在荧光显微镜下直接观察细胞转染的结果。结果证明pEGFP β2 mAN已成功导入NIH3T3细胞中 ,且有效表达 ,在荧光显微镜下可见梭形绿色荧光细胞 ;与同步转染的小鼠 β2 m正、反义核酸表达载体pcDNA3 β2 mSN、pcDNA3 β2 mAN的转染结果一起进行分析 ,转染pcDNA3 β2 mSN细胞的 β2 mmRNA和蛋白表达水平升高 ,而转染pcD NA3 β2 mAN和pEGFP β2 mAN的细胞 β2 mmRNA和蛋白表达水平降低。小鼠 β2 m反义核酸重组荧光蛋白表达载体pEGFP β2 mAN的转染结果显示 :它不但可以降低NIH3T3细胞 β2 m基因的表达 ,而且为观察脂质体转染效果提供了直观、即时的方法
In this study, the molecular biology technique was used to construct pEGFP β2 mAN, a recombinant fluorescent protein expression vector of mouse β2 m antisense oligonucleotides. The correctness of cloning was confirmed by restriction enzyme digestion, PCR and sequence analysis. Then, the NIH3T3 cells were transfected with liposomes. The expression of β2 mmRNA and protein was observed by RT PCR and Western blotting. The transfection results were observed under fluorescence microscope. The results showed that pEGFP β2 mAN was successfully introduced into NIH3T3 cells and expressed efficiently. Fluorescent green fluorescent cells were observed under the fluorescence microscope. The positive and negative antisense RNAi expression vectors pcDNA3 β2 mSN, pcDNA3 β2 mAN The results showed that the expression of β2 mmRNA and protein in pcDNA3 β2 mSN cells was increased, while the expression of β2 mmRNA and protein in pcDNA3 β2 mAN and pEGFP β2 mAN transfected pcDNA3 β2 mAN cells was decreased. The transfection results of the mouse β2 m antisense nucleic acid recombinant fluorescent protein expression vector pEGFP β2 mAN showed that it not only can reduce the β2 m gene expression in NIH3T3 cells but also provide an intuitive and immediate method for observing the transfection efficiency of the liposome