异源基因α1,3半乳糖转移酶与增强型绿色荧光蛋白融合对荧光蛋白表达的影响

Impact of fusion of exogenous gene α1,3 galactosyltransferase and enhance green fluorescent protein on fluorescent protein expression

目的研究异源(猪)基因α1,3半乳糖转移酶(α1,3GT)与增强型绿色荧光蛋白(EGFP)基因形成的融合蛋白对其荧光表达量的影响。方法 BamHI,EcoRI酶切pcDNA3.1-α1,3GT重组载体后,回收含α1,3GT的片段,与BamHI、EcoRI酶切回收的pEGFP-N1载体连接,并酶切、测序鉴定重组真核表达载体pEGFP/α1,3GT。将该表达载体转染人肺癌细胞A549、人胚肾细胞293FT,EGFP的表达用荧光显微镜观察和流式细胞仪进行荧光定量分析。结果 pEGFP-N1载体转染A549和293FT后48h的转染阳性率分别80.5﹪和86.5﹪;pEGFP/α1,3GT载体转染A549和293FT细胞后48h的转染效率则为75.8﹪和81.2﹪。A549细胞空白对照,转染pEGFP-N1和pEGFP/α1,3GT载体的A549细胞平均荧光强度分别为1.21、0.956;293FT细胞空白对照,pEGFP-N1和pEGFP/α1,3GT载体的293FT细胞平均荧光表达量分别为7.66、1.00。结论 pEGFP/α1,3GT融合蛋白的生成抑制了绿色荧光蛋白的表达强度。

Objective To investigate the effects of the fusion protein of the heterologous gene （pig-derived） α1,3 galactosyltransferase（α1,3GT） and the enhanced green fluorescent protein （EGFP） on the fluorescence expression level. Methods The α1,3GT gene fragment of pcDNA3.1 recombinant vector was digested with Barn HI and EcoRI, and ligated with pEGFP vector that was digested with two same enzymes to form a new recombinant vector which was subjected to sequencing identification. This eukaryotic expression vector was transfected into human pulmonary carcinoma cell A549 and HEKC 293FT, and the expression of EGFP was quantitatively analyzed by using the fluorescent microscope and the flow cytometer. Results The GFP positive rate ofA549 and 293 FT cells transfected with pEGFP-N1 after 48 hours was 80.5 % and 86.5 % respectively; while the GFP positive rate of that with pEGFP/α1,3GT was 75.8 % and 81.2 % respectively. The mean fluorescence intensities of the blank control of A549 cells and the A549 ceils that were transfected with pEGFP-N1 and pEGFP/α1,3GT vectorswere 1.21 and 0.956 respectively, while the mean fluorescence intensities of the blank control of 293FT cells and the 293FT cells that were transfected with pEGFP-N1 and pEGFP/α1,3GT vectors were 7.66 and 1.00. Conclusien The production of the fusion protein significantly inhibited the expression level of the green fluorescent protein.