CAJ | 학술논문

目的研究异源(猪)基因α1,3半乳糖转移酶(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48 h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48 h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.
목적 연구이원(저)기인α1,3반유당전이매(3GT)여증강형록색형광단백(EGFP)기인형성적융합단백대기형광표체량적영향.방법 BamHI,EcoRI매절pcDNA3.1-α1,3GT중조재체후,회수함α1,3GT적편단,여BamHI、EcoRI매절회수적pEGFP-N1재체련접,병매절、측서감정중조진핵표체재체pEGFP/α1,3GT.장해표체재체전염인폐암세포A549、인배신세포293FT,EGFP적표체용형광현미경관찰화류식세포의진행형광정량분석.결과 pEGFP-N1재체전염A549화293FT후48 h적전염양성솔분별80.5％화86.5％；pEGFP/α1,3GT재체전염A549화293FT세포후48 h적전염효솔칙위75.8％화81.2％.A549세포공백대조,전염pEGFP-N1화pEGFP/α1,3GT재체적A549세포평균형광강도분별위1.21、0.956;293FT세포공백대조,pEGFP-N1화pEGFP/α1,3GT재체적293FT세포평균형광표체량분별위7.66、1.00.결론 pEGFP/α1,3GT융합단백적생성억제료록색형광단백적표체강도.
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 Bam 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 cells that were transfected with pEGFP-N1 and pEGFP/α1,3GT vectors were 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.Conclusion The production of the fusion protein significantly inhibited the expression level of the green fluorescent protein.