慢病毒介导炭疽毒素受体1基因靶向沉默对γ珠蛋白基因表达的影响

The effect of the targeted silencing of anthrax toxin receptor 1 gene mediated by lentivirus on the expression of γ-globin gene

目的探讨沉默炭疽毒素受体1 (anthrax toxin receptor 1,ANTXR1)基因对γ珠蛋白基因表达的影响。方法构建ANTXR1基因特异性序列的shRNA重组质粒及阴性对照重组质粒,经酶切验证及测序鉴定后,将干扰载体和慢病毒质粒共转染入293T细胞后收集病毒上清液,再将其转染至K562细胞中,并经puromycin筛选稳转细胞株。通过实时荧光定量PCR技术检测各组ANTXR1基因和γ珠蛋白基因mRNA的表达情况;通过Western蛋白印迹技术检测各组ANTXR1蛋白和γ珠蛋白的表达情况。采用t检验进行统计分析。结果获得有效干扰ANTXR1基因的K562稳转细胞株,实时荧光定量PCR结果显示,ANTXR1-shRNA实验组中ANTXR1基因和γ珠蛋白基因mRNA相对表达水平分别为0.19±0.02和1.46±0.24;Western蛋白印迹技术结果显示,蛋白相对表达水平分别为0.20±0.01和1.45±0.14。ANTXR1-shRNA实验组与阴性对照组、空质粒载体组和空白对照组相比,ANTXR1基因相对低表达,γ珠蛋白基因相对高表达,差异均有统计学意义(P <0.05)。结论靶向沉默ANTXR1基因后,促进γ珠蛋白基因mRNA和蛋白水平的表达。

ObjectiveTo explore the effect of silent anthrax toxin receptor 1 （ANTXR1） gene on theexpression of γ-globin gene. MethodsA shRNA recombinant plasmid containing the ANTXR1 gene-specificsequence and a negative control recombinant plasmid were constructed. After restriction enzyme digestion andsequencing identification, the interfering vector and lentiviral plasmids were co-transfected into 293T cells andthe virus supernatant was harvested. The virus recombinant plasmid vector was transfected into K562 cells andthe stable cell line was screened by puromycin. The expression levels of ANTXR1 and γ globin gene of RNA ineach group were detected by real time-PCR （RT-PCR） and the expression levels of γ globin in each group were detected by Western blotting. Statistical analysis was performed by t-test. ResultsStably transfected K562 cellline with effectively interfered by ANTXR1 gene was obtained. RT-PCR results showed that in the ANTXR1-shRNA group, the relative expression levels of ANTXR1 gene and γ-globin gene were 0.19±0.02 and1.46±0.24 respectively at mRNA level. Western blotting results showed that the relative level of protein were0.20±0.01 and 1.45±0.14 respectively . There were significant differences in ANTXR1-shRNA group vs thenegative control group, blank plasmid group and the blank control group, the expression level of ANTXR1 genewith down-regulation and the γ-globin gene with up-regulation （P ﹤ 0.05）. Conclusion Targeted silencing ofthe ANTXR1 gene promotes the expression of γ-globin gene at mRNA and protein level.