永生化绵羊肺成纤维细胞系的构建及鉴定

ESTABLISHMENT AND IDENTIFICATION OF IMMORTALIZED SHEEP LUNG FIBROBLAST CELL LINE

为了利用携带人源端粒酶逆转录酶(human telomerase reverse transcriptase,h TERT)基因的慢病毒表达系统将原代绵羊肺成纤维细胞进行永生化。本研究利用PCR技术从p Babe-neo-h TERT中扩增h TERT基因,并利用infusion技术构建重组慢病毒表达载体p LOV-puro-h TERT。将重组慢病毒三质粒系统共转染293T细胞,拯救出慢病毒颗粒并感染原代绵羊肺成纤维细胞,经嘌呤霉素抗性筛选获得阳性克隆,分别应用RT-PCR和Western blot方法检测传代细胞系中h TERT基因的转录和表达水平。筛选获得的阳性克隆,命名为SLT细胞系。SLT细胞系连续传代后RT-PCR及Western blot检测均显示h TERT稳定表达。本研究表明,我们成功构建了永生化绵羊肺成纤维细胞系。

The lentivirus system carrying hTERT gene was inserted to sheep lung fi broblasts(SLT)in order to establish an immortal cell line.Firstly,the recombinant lentiviral expression vector(pLOV-puro-hTERT)carrying hTERT gene was transfected into 293T cells.Subsequently,the primary SLT cells were infected with the rescued lentiviruses.The positive SLT cells expressing hTERT were selected with puromycin and examined with RT-PCR and Western blot.The results showed that hTERT gene was stably expressed in SLT cells,indicated that an immortalized SLT cell line had been established.