TurboFect介导骨形态发生蛋白2转染CHO细胞的研究

CHO Transfected by Turbo Fect-mediated Bone Morphogenetic Protein 2

目的:构建真核表达载体p IRES-EGFP-BMP-2,通过Turbo Fect转染得到表达BMP-2蛋白的CHO细胞系。方法:利用逆转录PCR方法扩增获得人的BMP-2基因c DNA,克隆入p MD18-T载体,经PCR、酶切和基因测序分析等方法鉴定重组质粒;将BMP-2连入p IRES-EGFP真核表达载体中,经限制性酶切和PCR扩增鉴定重组质粒。以壳聚糖和Turbo Fect分别作为基因载体转染CHO细胞,荧光显微镜检测分析转染结果;G418筛选富集转染阳性细胞。结果:成功的克隆得到了BMP-2基因,酶切鉴定成功构建了p IRES-EGFP-BMP-2质粒。与壳聚糖组相比,Turbo Fect用量为1:1时,细胞阳性率为(31.92±1.31)%,高于壳聚糖的细胞阳性率(6.33±1.53)%。目的基因与Turbo Fect比例为1:2时转染效率为(42.90±1.10)%高于1:1的(28.59±2.38)%和1:3的(37.52±2.14)%。细胞密度调节到5×103 cells/cm2阳性细胞率可达到(44.43±3.23)%。荧光检测可见荧光阳性细胞得到稳定传代。Western Blot检测可见BMP-2蛋白表达。结论:Turbo Fect成功的介导了p IRES-EGFP-BMP-2载体转染CHO细胞,建立了稳定表达BMP-2和EGFP的CHO细胞株。

Objective： To construct the eukaryotic vector p IRES-EGFP-BMP-2 and get the BMP-2 expressed in CHO cells by cationic polymers. Methods： Human BMP-2 c DNA was amplified by RT- PCR, and inserted into cloning p MD18-T vector. The recombinant plasmid was identified by processes of PCR amplification, restriction expression vector, the vector was identified by enzyme digestion and PCR amplification. Chitosan and Turbo Fect were taken as carriers to transfect CHO cells, respectively. The transfection efficiency was analyzed with fluorescence microscope analysis results of transfection. Finally, the positive cells screened by G418.Results： The BMP-2 gene was been successfully cloned and the construction of p IRES-EGFP-BMP-2 was verified by restriction enzyme digestion. When the dosage of Turbo Fect is 1：1, fluorescent cell positive rate was（31.92±1.31） %, which was higher than chitosan group[（6.33±1.53）%]. The fluorescent cell positive rate was（42.90±1.10） %, when the dosage of Turbo Fect is 1：2, which was higher than（28.59±2.38） % and（37.52±2.14） % when the dosage of Turbo Fect is 1：1 and 1：3. The fluorescent cell positive rate could reach up to（44.43±3.23） % by adjusting cell inoculum density to 5×10^3cells /cm^2. After screened by G418 BMP-2 protein was stabled expressed in CHO cells. Conclusion： The p IRES-EGFP-BMP-2 has been successfully transfected into CHO cells by Turbo Fect. The study has established CHO cell lines stable expressed BMP-2 and EGFP.