Hu Qiong, Xie Fu'an, Hong Xiaoqian, Liu Kuancan
Dongfang Hospital, Xiamen University, Fuzhou 350025, China.
PLA Institute for Laboratory Medicine, Fuzhou General Hospital of Nanjin Military Area Command, Fuzhou 350025, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2017 Mar;33(3):342-346.
Objective To prepare a lentiviral vector expressing LLGL2 and establish KYSE450 and TE-1 cell lines for the stable expression of LLGL2. Methods The full-length LLGL2 sequence was amplified by high-fidelity PCR, and then it was inserted into pCDH-CMV-IRES-GFP-EF1-Puro vectors. The recombinant plasmid was confirmed by double enzyme digestion and sequencing. After co-infection of pCDH-CMV-LLGL2-IRES- GFP-EF1-Puro with vesicular stomatitis virus glycoprotein (VSVG) and PHR into HEK293T cells, the lentivirus was harvested and used for infecting esophageal squamous cell carcinoma cell lines including KYSE450 and TE-1 cells. These two cell lines infected with the lentivirus were screened with puromycin, and the stable cell lines were further confirmed with green fluoresence and Western blotting. Results Dual-enzyme digestion and sequencing confirmed that the pCDH-CMV-LLGL2-IRES-GFP-EF1-Puro vector, a lentiviral expression vector for the overexpression of LLGL2, was successfully constructed through high-fidelity PCR and ligation. Western blotting showed the increased expression level of LLGL2 protein in KYSE450 and TE-1 stable cell lines compared with the controls. Conclusion The experiment successfully established KYSE450 and TE-1 stable cell lines for the overexpression of LLGL2.
目的 制备表达LLGL2的慢病毒载体，并建立稳定表达LLGL2的KYSE450和TE-1细胞系。方法 通过高保真PCR扩增LLGL2全长序列，然后将其插入pCDH-CMV-IRES-GFP-EF1-Puro载体。经双酶切和测序鉴定重组质粒。将pCDH-CMV-LLGL2-IRES-GFP-EF1-Puro与水疱性口炎病毒糖蛋白（VSVG）和PHR共转染HEK293T细胞后，收获慢病毒并用于感染食管鳞状细胞癌细胞系，包括KYSE450和TE-1细胞。用嘌呤霉素筛选这两种感染慢病毒的细胞系，并用绿色荧光和蛋白质免疫印迹进一步鉴定稳定细胞系。结果 双酶切和测序证实，通过高保真PCR和连接成功构建了用于过表达LLGL2的慢病毒表达载体pCDH-CMV-LLGL2-IRES-GFP-EF1-Puro。蛋白质免疫印迹显示，与对照相比，KYSE450和TE-1稳定细胞系中LLGL2蛋白表达水平升高。结论 本实验成功建立了用于过表达LLGL2的KYSE450和TE-1稳定细胞系。
