Xin Chuan, Wang Jiong-Ke, Li Jing, Zeng Xin
State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Dept. of Oral Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2020 Feb 1;38(1):6-10. doi: 10.7518/hxkq.2020.01.002.
To construct a PA28γ overexpression cell line and determine its effects after infecting an oral squa-mous cell carcinoma (OSCC) cell line.
The PA28γ gene was cloned into the pLOV.CMV.cherry.2A.EF1a.PuroR lentiviral vector by polymerase chain reaction (PCR), and PCR and DNA sequencing alignment analysis were used for identification. Then, 293T cells were used to package viral diseases. Infected OSCC cells were used to construct a cell line with stable PA28γ overexpression. Finally, the level of PA28γ expression in the OSCC cell line was detected through Western blot.
The successful construction of PA28γ recombinant lentiviral vectors was confirmed by DNA sequencing. The results of immunofluorescence showed that the PA28γ overexpression lentivirus successfully infected the OSCC cells and showed cherry red fluorescence. The results of Western blot demonstrated that the constructed cells with stable PA28γ overexpression significantly increased the expression of PA28γ.
The PA28γ overexpression lentiviral vector can significantly increase its protein expression in OSCC cells. We provide a stable OSCC cell line for further study on the effect of PA28γ in OSCC.
构建PA28γ过表达细胞系，并确定其感染口腔鳞状细胞癌（OSCC）细胞系后的作用。
通过聚合酶链反应（PCR）将PA28γ基因克隆到pLOV.CMV.cherry.2A.EF1a.PuroR慢病毒载体中，并采用PCR和DNA测序比对分析进行鉴定。然后，利用293T细胞包装病毒。用感染后的OSCC细胞构建PA28γ稳定过表达细胞系。最后，通过蛋白质免疫印迹法检测OSCC细胞系中PA28γ的表达水平。
DNA测序证实成功构建了PA28γ重组慢病毒载体。免疫荧光结果显示，PA28γ过表达慢病毒成功感染了OSCC细胞，并呈现樱桃红色荧光。蛋白质免疫印迹法结果表明，构建的PA28γ稳定过表达细胞显著提高了PA28γ的表达。
PA28γ过表达慢病毒载体可显著提高其在OSCC细胞中的蛋白表达。我们提供了一个稳定的OSCC细胞系，用于进一步研究PA28γ在OSCC中的作用。
