| 金 丹,李 风,高 玥,姬广全,高殿帅.CEP55慢病毒表达载体构建和U251-CEP55细胞株的建立[J].,2017,17(33):6422-6426 |
| CEP55慢病毒表达载体构建和U251-CEP55细胞株的建立 |
| Constructing a Lentiviral Vector of CEP55 and Establishing Stably transfected U251 Cell Line |
| 投稿时间：2017-05-06 修订日期：2017-05-28 |
| DOI：10.13241/j.cnki.pmb.2017.33.005 |
| 中文关键词: CEP55基因 慢病毒载体 U251细胞 |
| 英文关键词: CEP55 gene Lentiviral vector U251 cell |
| 基金项目:国家自然科学基金项目（81402073）；江苏省自然科学基金项目（BK20130218） |
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| 中文摘要: |
| 摘要 目的：构建CEP55慢病毒表达载体，建立稳定表达CEP55的人脑胶质瘤U251细胞株。方法：使用PCR扩增方法将CEP55基因序列进行扩增，转入质粒中，并整合到载体上，构建重组质粒GV358-CEP55载体。与pHelper 1.0和pHelper 2.0质粒共转染293T细胞使其产生慢病毒，以GV358空载体包装的慢病毒作为对照。显微镜观察绿色荧光蛋白GFP表达强度，确定慢病毒的感染效率。采用病毒梯度稀释法测定病毒滴度。慢病毒感染人胶质瘤细胞株U251后，经嘌呤霉素筛选出稳定表达CEP55基因的细胞株U251-CEP55。qRT-PCR和Western blot 两种方法分别检测CEP55 mRNA及蛋白的表达。结果：测序证实慢病毒表达载体GV358-CEP55构建成功；转染293T细胞获得高滴度的病毒。病毒感染U251细胞后，使用嘌呤霉素筛选得到稳定转染细胞株U251-CEP55；qRT-PCR和Western blot方法分别检测后发现，U251-CEP55细胞株中CEP55 mRNA和蛋白水平的表达明显高于对照组。结论：成功构建CEP55慢病毒表达载体，获得稳定表达CEP55的人胶质瘤U251细胞株，为CEP55基因功能的探究给予了一定的实验基础。 |
| 英文摘要: |
| ABSTRACT Objective: To construct lentivirus expression vector containing CEP55 gene and establish a stable overexpression hu- man neuroglioma U251 cell line of CEP55. Methods: The full-length CEP55 gene sequence amplified by PCR was inserted into a GV358 lentiviral vector to construct GV358-CEP55 vector. GV-358 was then transfected into 293T cells with pHelper1.0 and pHelper2.0 vectors for lentivirus package, with GV358 vector lentivirus as control. The green fluorescent protein GFP expression intensity was observed by microscope and the infection efficiency of lentivirus was determined. Supernatant were harvested and filtered and the viral titer was de- termined by gradient dilution method. Subsequently, the lentivirus was transduced into the U251 cells and a stably overexpressed cell line named U251-CEP55 was established. U251 cells with CEP55 overexpression were screened with puromycin, and the mRNA and protein expression levels of CEP55 were detected by qRT-PCR and Western blot. Results: Lentiviral vector GV358-CEP55 was successfully con- structed, identified by sequencing, and transduced into 291T cells to obtain high titer lentivirus. Transfected cells were selected by puromycin and then obtained the stable cell line overexpress of CEP55. The expression of CEP55and CEP55 mRNA were successfully detected in U251cells. Conclusion: CEP55 expression lentiviral vector and its stable CEP55 overexpression human neuroglioma U251 cell line were successfully constructed, which lay the foundation for the study of the gene function. |
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