FMDV 2A介导的乳腺上皮细胞双基因表达载体构建及表达

Construction and Coexpression of Bicistronic Expression Vector Based on FMDV 2A in Mammary Gland Cells

构建FMDV 2A介导的人白细胞介素2基因(hIL-2)和增强型绿色荧光蛋白(EGFP)基因双顺反子乳腺特异性表达载体,并验证其在乳腺上皮细胞中的表达。克隆奶山羊β-酪蛋白启动子序列,将hIL-2基因序列置于启动子之后,然后利用口蹄疫病毒2A(FMDV 2A)自剪切序列连接EGFP基因,构建出乳腺特异性的双顺反子表达载体pFIENβ,并利用脂质体转染山羊乳腺上皮细胞,然后用RT-PCR技术和Western blot检测hIL-2基因与EG-FP基因的表达。重组质粒pFIENβ经酶切鉴定后表明构建成功,转染质粒PFIENβ和pEGFP-C1的细胞均观察到绿色荧光;从转染pFIENβ的乳腺上皮细胞中扩增出hIL-2基因和EGFP基因,而转染pEGFP-C1的细胞中只扩增出EGFP基因;经Western blot检测,转染pFIENβ的细胞中均表达了hIL-2和EGFP蛋白。结果表明山羊β-酪蛋白启动子能同时启动hIL-2基因和EGFP基因在山羊乳腺上皮细胞中的表达,并且利用FMDV 2A元件实现了hIL-2基因和EGFP基因的非融合型表达。

The aim of this study was to construct mammary gland specific bicistronic expression vector based on FMDV 2A,and verify the expression of sequences of EGFP and human IL-2 in mammary epithelial cells.We utilized self-cleaving peptide 2A from foot-and-mouth disease virus（FMDV 2A） connecting hIL-2 and EGFP sequence,and cloned into goat β-casein regulatory sequence,then we got mammary gland specific bicistronic expression vector pFIENβ,after transient transfection into goat mammary epithelial cells by LipofectamineTM 2000,we used RT-PCR and Western blot to detect the expression of hIL-2 and EGFP.Restriction analysis suggested that the recombinant plasmid pFIENβ is correct,then we found that the cells which were transfected with pFIENβ and pEGFP-C1 expressed EGFP by observing with fluorescent microscope,and there is no green flourescence in which were untransfected,and we amplificating hIL-2 and EGFP sequence in the cells transfected with pFIENβ,and amplificating EGFP sequence only in the cells transfected with pEGFP-C1;After detection by Western blot,the cells transfected with pFIENβ expressed both hIL-2 and EGFP.The results showed that goat β-casein regulatory sequence can trigger hIL-2 and EGFP expression in mammary epithelial cells,and FMDV 2A facilitate expresson of hIL-2 and EGFP independently.