论文部分内容阅读
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 construct a lentivirus-mediated lentivirus-2 (LLGL2) lentivirus vector and establish a stable human esophageal squamous cell carcinoma cell line KYSE450 and TE-1 cell line. Methods The full-length human LLGL2 gene was amplified by PCR and ligated into pCDH-CMV-IRES-GFP-EF1-Puro lentiviral vector. The recombinant plasmids were double-digested and sequenced. The HEK293T cells were cotransfected with the recombinant plasmids pCDH-CMV-LLGL2-IRES-GFP-EF1-Puro and PHR and vesicular stomatitis virus G protein (VSVG), and infected with KYSE450 cells and TE-1 cells . The LLGL2 stably overexpressed KYSE450 cell line and TE-1 cell line were established by puromycin screening, and the LLGL2 expression was detected by Western blot. Results Double digestion and sequencing analysis, the successful construction of pCDH-CMVLLGL2-IRES-GFP-EF1-Puro lentiviral vector expression plasmid. Western blot results showed that compared with the control group, the virus liquid LLLL2 protein levels were significantly higher. Conclusion KYSE450 cell line and TE-1 cell line stably overexpressing LLGL2 gene were successfully established.