| 王淑君 张德蒙 李娜 王贝贝 谢红国 郭昕 白凤武.TurboFect介导骨形态发生蛋白2 转染CHO 细胞的研究[J].现代生物医学进展英文版,2016,16(10):1801-1806. |
| TurboFect介导骨形态发生蛋白2 转染CHO 细胞的研究 |
| CHO Transfected by TurboFect-mediated Bone Morphogenetic Protein 2 |
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| DOI： |
| 中文关键词: 骨形态发生蛋白2 转染 阳离子聚合物 壳聚糖 TurboFect |
| 英文关键词: Bone morphogenetic protein-2 Transfection Cationic polymers Chitosan TurboFect |
| 基金项目:国家自然科学基金项目(51103157，81171555) |
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| 中文摘要: |
| 目的：构建真核表达载体pIRES-EGFP-BMP-2，通过TurboFect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cDNA，克隆入pMD18-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pIRES-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TurboFect 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pIRES-EGFP-BMP-2 质粒。与壳聚糖组相比，TurboFect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TurboFect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：TurboFect 成功的介导了pIRES-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。 |
| 英文摘要: |
| Objective:To construct the eukaryotic vector pIRES-EGFP-BMP-2 and get the BMP-2 expressed in CHO cells by
cationic polymers.Methods:Human BMP-2 cDNA was amplified by RT- PCR, and inserted into cloning pMD18-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 TurboFect 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 pIRES-EGFP-BMP-2 was verified by restriction enzyme
digestion. When the dosage of TurboFect 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 TurboFect is 1:2, which was higher than
(28.59± 2.38)%and (37.52± 2.14)%when the dosage of TurboFect 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× 103 cells /cm2. After screened by G418 BMP-2 protein was stabled expressed
in CHO cells.Conclusion:The pIRES-EGFP-BMP-2 has been successfully transfected into CHO cells by TurboFect. The study has
established CHO cell lines stable expressed BMP-2 and EGFP. |
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