Npu DnaE内含肽构建及其对293T细胞高效反式剪接活性分析  

Construction of Npu DnaE intein and its highly efficient protein trans-splicing activity in 293T cells

作　　者：张矫[1] 崔文静[1] 马祥敏[1] 王雯雯[1] 王欣[1] 

机构地区：[1]天津医科大学附属肿瘤医院,乳腺癌防治教育部重点实验室,天津市肿瘤防治重点实验室,天津300060

出　　处：《山东医药》2013年第44期10-13,I0002,共5页Shandong Medical Journal

基　　金：天津市高等学校科技发展基金计划项目(20090139)

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 pCDH-CMV-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.

关 键 词：NPU DnaE 内含肽 蛋白质反式剪接 

分 类 号：R34[医药卫生—基础医学]