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目的:构建并鉴定Arnt基因特异性RNAi慢病毒载体,将其导入HCCLM6肝癌细胞中,筛选有效抑制ARNT表达的稳定细胞株。方法:针对已经筛选确定的Arnt基因RNAi有效靶序列,合成靶序列的OligoDNA,退火形成双链DNA,与经HpaI和XhoI酶切后的pLVTHM载体连接[含U6启动子和绿色荧光蛋白(GFP)]产生LV-shArnt慢病毒载体,PCR筛选阳性克隆,测序鉴定。用LV-shArnt载体,pCMV-dR8.74载体,pMD2G载体三质粒共转染包装细胞293T细胞,包装产生慢病毒,以293T细胞GFP蛋白表达水平测定病毒滴度,挑选细胞克隆,收获病毒上清,将病毒上清转染HCCLM6中,经过GFP筛选得到稳定抑制Arnt表达的HCCLM6细胞株,通过RT-PCR鉴定抑制Arnt表达的效果。结果:双酶切与测序鉴定证实成功构建了ArntshRNA的慢病毒载体,转染包装病毒细胞株获得的病毒上清成功转染了HCCLM6,并且抑制了Arnt的表达。结论:运用pLVTHM载体构建的LV-shArnt慢病毒载体可有效抑制Arnt在肝癌细胞株HCCLM6中的表达,为观察转染shRNA慢病毒表达载体的肿瘤细胞的恶性生物学行为变化奠定了实验基础。
OBJECTIVE: To construct and identify Arnt gene-specific RNAi lentiviral vector and introduce it into HCCLM6 hepatocarcinoma cells and screen for stable cell lines that can effectively inhibit ARNT expression. Methods: According to the target RNAi sequences of Arnt gene that have been selected and screened, OligoDNA of the target sequence was synthesized and annealed to form double-stranded DNA. The oligonucleotide was ligated with the pLVTHM vector [containing U6 promoter and green fluorescent protein (GFP) ] Produce LV-shArnt lentiviral vector, PCR screening of positive clones, sequencing identification. 293T cells were co-transfected with the LV-shArnt vector, the pCMV-dR8.74 vector and the pMD2G vector and packaged to produce lentivirus. The virus titer was determined by the expression level of GFP protein in 293T cells. The cell clones were selected and the virus supernatant was harvested . The virus supernatant was transfected into HCCLM6. After GFP screening, HCCLM6 cell line stably inhibiting Arnt expression was obtained and the effect of inhibiting Arnt expression was identified by RT-PCR. Results: Double enzyme digestion and DNA sequencing confirmed that ArnthRNA was successfully constructed. The virus supernatant was transfected into HCCLM6 successfully and the expression of Arnt was inhibited. CONCLUSION: LV-shArnt lentiviral vector constructed with pLVTHM vector can effectively inhibit the expression of Arnt in HCCLM6 cell line, which lays the foundation for the observation of malignant biological behavior of tumor cells transfected with shRNA lentivirus vector.