稳定过表达幼虫巨大致死基因2(LLGL2)的食管鳞癌细胞系的建立

Establishment of esophageal squamous carcinoma cell lines stably over-expressing LLGL2

目的构建人幼虫巨大致死基因2(LLGL2)慢病毒表达载体,并建立稳定表达的人食管鳞癌KYSE450细胞系和TE-1细胞系。方法 PCR扩增人LLGL2全长基因序列,连接插入至pCDH-CMV-IRES-GFP-EF1-Puro慢病毒载体中,并对重组质粒进行双酶切及测序验证。将重组质粒pCDH-CMV-LLGL2-IRES-GFP-EF1-Puro和PHR、水疱性口炎病毒G蛋白(VSVG)共转染HEK293T细胞进行病毒包装,利用该病毒液感染KYSE450细胞和TE-1细胞。经嘌呤霉素筛选建立LLGL2稳定过表达的KYSE450细胞系和TE-1细胞系,用Western blot法检测LLGL2的表达情况。结果双酶切和测序分析,成功构建pCDH-CMVLLGL2-IRES-GFP-EF1-Puro慢病毒表达载体质粒。Western blot法检测结果显示,与对照组相比,感染病毒液的细胞LLGL2蛋白水平明显增高。结论成功建立了稳定过表达LLGL2基因的KYSE450细胞系和TE-1细胞系。

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 HEK293 T 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.