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目的构建人TPX2基因过表达慢病毒质粒,并筛选该基因的人宫颈癌HeLa细胞稳定表达株。方法经PCR法扩增人TPX2基因序列,克隆至线性化的慢病毒载体LV11中,构建重组慢病毒质粒,进行酶切及测序鉴定。将鉴定正确的重组慢病毒及空载体LV11(LV11-NC)分别与辅助质粒共转染293T细胞进行病毒包装,并检测病毒滴度。用重组慢病毒感染HeLa细胞,经不同浓度G418(0、0.2、0.4、0.6、0.8、1.0、1.2、1.5、2.0 mg/ml)筛选TPX2基因过表达稳转细胞株,同时设阴性对照(感染LV11-NC慢病毒)及空白对照(未感染病毒)。采用实时荧光定量PCR和Western blot法分别检测各组HeLa细胞中TPX2基因m RNA转录及蛋白的表达水平。结果经酶切及测序鉴定证明重组慢病毒质粒构建成功。经293T细胞包装后,获得重组慢病毒的滴度为3×10~8 TU/ml。采用0.6 mg/ml G418成功筛选出TPX2过表达细胞株。与阴性对照及空白对照比较,感染重组慢病毒的HeLa细胞中,TPX2基因m RNA转录及蛋白表达水平均明显升高(P<0.05)。结论成功构建了人TPX2过表达慢病毒质粒,并筛选出TPX2稳定表达的HeLa细胞株,为进一步研究TPX2基因在宫颈癌中的作用奠定了基础。
Objective To construct a lentiviral plasmid overexpressing human TPX2 gene and screen the stable expression of human cervical cancer HeLa cells. Methods The human TPX2 gene was amplified by PCR and cloned into linearized lentiviral vector LV11. The recombinant lentiviral plasmid was constructed and digested with restriction endonuclease and sequenced. The recombinant lentivirus and empty vector LV11 (LV11-NC) which were identified correctly were cotransfected into 293T cells with the helper plasmids respectively for virus packaging and the virus titers were detected. HeLa cells were infected with recombinant lentivirus and the TPX2 overexpression stable cell line was screened by different concentrations of G418 (0,0.2,0.4,0.6,0.8,1.0,1.2,1.5,2.0 mg / ml), and negative control (infection LV11-NC lentivirus) and blank control (uninfected virus). Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression levels of TPX2 gene in HeLa cells. Results confirmed by restriction enzyme digestion and sequencing of recombinant lentiviral plasmid was successfully constructed. After 293T cells were packaged, the titer of recombinant lentivirus was 3 × 10 ~ 8 TU / ml. The TPX2 overexpression cell line was successfully screened with 0.6 mg / ml G418. Compared with the negative control and blank control, the transcription and protein expression of TPX2 mRNA and protein in HeLa cells infected with recombinant lentivirus were significantly increased (P <0.05). Conclusion The human TPX2 overexpression lentiviral plasmid was successfully constructed and the HeLa cell line stably expressing TPX2 was screened out for further study of the role of TPX2 in cervical cancer.