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

作　　者：胡琼[1] 谢富安 洪笑迁 刘宽灿[1,2] 

机构地区：[1]厦门大学附属东方医院,福建福州350025 [2]南京军区福州总医院全军临床检验医学研究所,福建福州350025

出　　处：《细胞与分子免疫学杂志》2017年第3期342-346,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81302068)

摘　　要：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.

关 键 词：LLGL2 慢病毒载体 KYSE450细胞系 TE-1细胞系 

分 类 号：R392-33[医药卫生—免疫学] Q25[医药卫生—基础医学]