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目的构建人类中间丝聚合蛋白(filaggrin,FLG)基因慢病毒载体及观察慢病毒表达载体介导的RNA干扰RNAi对人HaCat细胞FLG表达的影响。方法应用基因工程技术筛选出3条针对FLG基因的RNAi靶序列,分别与GV115载体连接,构建3个重组慢病毒表达载体FLG-vshRNA 10154,FLG-vshRNA 10155,FLG-vshRNA 10156;将连接产物转化到DH5α感受态细胞,经PCR筛选阳性克隆、测序鉴定,将FLG-vshRNA,pHelper 1.0,pHelper 2.0共转染293T细胞,包装产生慢病毒颗粒并测定病毒滴度,将包装产生的3种重组慢病毒分别感染HaCat细胞,实时定量PCR检测HaCat细胞FLG mRNA表达。根据筛选的结果,选取最有效的载体进行病毒的大量包装。结果慢病毒载体PCR和测序结果表明3对碱基成功插入到预计位点,序列完全一致。慢病毒载体感染HaCat细胞后,FLG基因mRNA的表达量与未感染慢病毒的细胞组及空载体感染组相比均明显下降,下降程度达70%以上(P<0.05)。3个慢病毒载体经包装产生的病毒滴度分别为1.2×109,1.5×109,1×109TU/mL。结论成功构建针对FLG基因的3个慢病毒载体FLG-vshRNA,体外感染HaCat细胞后可有效抑制FLG基因的表达。
Objective To construct human lentiviral vector (FLG) gene lentiviral vector and observe the effect of lentiviral vector mediated RNA interference RNAi on FLG expression in human HaCat cells. Methods Three RNAi target sequences of FLG gene were screened by genetic engineering and ligated with GV115 vector respectively to construct three recombinant lentiviral vector FLG-vshRNA 10154, FLG-vshRNA 10155 and FLG-vshRNA 10156, To DH5α competent cells, positive clones were screened by PCR and identified by sequencing. 293T cells were cotransfected with FLG-vshRNA, pHelper 1.0 and pHelper 2.0. The lentiviral particles were packaged and assayed for virus titer. The viruses were respectively infected with HaCat cells, and the real-time quantitative PCR was used to detect the FLG mRNA expression in HaCat cells. According to the results of the screening, the most effective vector is selected for mass packaging of the virus. Results The lentiviral vector PCR and sequencing results showed that 3 pairs of bases were successfully inserted into the predicted sites and the sequences were identical. The lentiviral vector infected HaCat cells, FLG gene mRNA expression levels compared with non-infected lentiviral cells and empty vector infection group were significantly decreased, the degree of decline of more than 70% (P <0.05). The virus titers of the three lentiviral vectors packaged were 1.2 × 109, 1.5 × 109 and 1 × 109 TU / mL, respectively. Conclusion Three lentiviral vector FLG-vshRNAs targeting FLG gene were constructed successfully. Infection of HaCat cells in vitro could effectively inhibit FLG gene expression.