摘要
目的构建Npu DnaE内含肽,探讨Npu DnaE内含肽是否在293T细胞中具备高效反式剪接活性。方法制备融合基因Vn-NDn-myc和NDc-Vc,插入pCDH-CMV-MCS-EF1-Puro载体,构建质粒。转染293T细胞,荧光显微镜下观察目的蛋白Venus是否形成。48 h后收集细胞蛋白,应用Western blot技术进一步印证。结果构建的质粒经限制性内切酶鉴定及测序比对正确,转染293T后可见共转染细胞组出现明亮荧光,弥漫分布于细胞质中,Western blot证明高量目的蛋白Venus形成。结论成功构建Npu DnaE内含肽,其能在293T细胞中发挥反式剪接的生物学功能,并具有高效的剪接效率及功能目的蛋白形成率。
Objective To construct Npu DnaE intein and to prove that Npu DnaE have highly efficient protein transsplicing activity in 293T cells. Methods We inserted the prepared fusion gene Vn-NDn-myc and NDc-Vc into pCDHCMV-MCS-EF1-Puro vector to construct the plasmid,then the plasmid was transfected into 293T cells,after that we observed the target protein to find the product of Venus with fluorescence microscope. We collected the protein after 48 hours and further proved the Venus protein by using Western blot. Results The recombinant plasmid was verified by enzyme digestion and sequence analysis. Fluorescence microscope showed that the fluorescence appeared in the co-transfected group and distributed all over the cytoplasm after transfection and the protein was highly produced in 293T cells,which was confirmed by Western blot. Conclusion Npu DnaE intein can play the biological functions of trans-splicing in 293T cells and with highly efficient protein trans-splicing activity and formation rate of the function target protein.
引文
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