鸡β-肌动蛋白第一内含子对EIAV载体质粒外源蛋白表达能力的影响

Influence of the Chicken β-actin Intron 1 on the EGFP Expression of Gene Transfer Vector Derived from Equine Infectious Anemia Virus

以马传染性贫血病毒(equine infectious anemia virus,EIAV)基因转移载体pcPPTW PRE为基础,用含不同长度片段的鸡β-肌动蛋白启动子替换原有的人巨细胞病毒立即早期启动子(CMVIEp)启动外源基因的表达,分别构建了2个基因转移载体,含部分第一内含子的pWCAGP0.8和含全长内含子的pWCAGP1.6。连同已构建的不含内含子的质粒pcPPTWCAG,采用磷酸钙法分别转染HEK293细胞和DF-1细胞,利用荧光显微镜观察报告基因eGFP蛋白的表达。并利用流式细胞仪定量。统计学分析结果表明,在HEK293细胞中,pcPPTWCAG、pWCAGP0.8和pWCAGP1.6表达阳性率分别为47.9%、46.1%和23.8%,转染后48h,收获细胞,利用流式细胞仪比较转染细胞中EGFP表达阳性细胞的百分率。结果表明,在HEK293细胞中,pcPPTWCAG、pWCAGP0.8和pWCAGP1.6的阳性细胞分别占计数细胞的47.9%、46.1%和23.8%;在DF-1细胞中,依次分别为12.4%、9.5%和4.2%,pcPPTWCAG与pWCAGP1.6差异显著。表明不含内含子的EIAV载体质粒表达EGFP的能力高于含部分和全长内含子的载体。

Based on the EIAV vector pcPPTWPRE, the plasmids pWCAGP0.8 containing part of the chicken β-actin intron 1 and pWCAGP1.6 containing the full intron were constructed by restriction enzyme digestion. Then the EIAV gene transfer vectors pWCAGP0. 8, pWCAGP1. 6 and pcPPTWCAG which were previously constructed without the chicken β-actin intron 1 were transfected into HEK293 cells and DF-1 cells using Calcium Phosphate Transfection Kit, respectively. The cells were investigated under fluorescence microscope at 12h,24h,36h and 48h after transfection. Flow-cytometric analysis was carried out at 48h. Results showed that the pcPPTWCAG had the equal EGFP expression level with pWCAGP0.8 ,while both of them could express EGFP much more efficiently than pWCAGP1.6 in HEK293 cells. In DF-1 cells, the pcPPTWCAG could express the EGFP a little more efficiently than pWCAGP0.8, while both of them could express EGFP much more efficiently than pWCAGP1.6. Results indicated that the chicken β-actin intron 1 could decrease, rather than increase the EGFP expression of EIAV vectors in both HEK293 cells and DF-1 cells.