ARG2基因在ALV-J感染DF-1细胞中的作用研究

Effect of ARG2 gene on DF-1 cells infected by ALV-J

为了研究线粒体相关基因精氨酸酶2(arginase-2,ARG2)在J亚群禽白血病病毒(J subgroup leukosis virus,ALV-J)感染中的作用,试验采用qRT-PCR方法分别检测感染ALV-J后试验组鸡不同器官和DF-1细胞中ARG2基因的表达情况,分析ARG2基因和ALV-J感染之间是否有联系;设计ARG2基因的过表达载体(过表达ARG2组)和干扰片段(干扰ARG2组)转染DF-1细胞,并以pcDNA3.1或Si-NC为对照,采用qRT-PCR方法检测ARG2基因的过表达和干扰效率;采用qRT-PCR、Western-blot和间接免疫荧光试验检测过表达或干扰ARG2基因后感染ALV-J的DF-1细胞中gp85基因及Env蛋白表达情况,从而综合分析ARG2基因对ALV-J复制的影响。结果表明:与对照组相比,试验组鸡的脾脏、法氏囊中ARG2基因相对表达量极显著降低(P<0.01),而肾脏中ARG2基因相对表达量极显著升高(P<0.01)。同时在体外试验中,ARG2基因在感染后第6,12小时时相对表达量降低(P>0.05或P<0.05),而在第24,48,74,108小时相对表达量显著或极显著升高(P<0.05或P<0.01)。与对照组相比,过表达ARG2组ARG2基因相对表达量极显著升高(P<0.01),干扰ARG2组ARG2基因相对表达量降低(P>0.05或P<0.05),并选择干扰片段SI-ARG2-001进行后续试验。过表达ARG2基因后,与对照组相比,过表达ARG2组第12,24小时的gp85基因相对表达量显著或极显著升高(P<0.05或P<0.01),Env蛋白表达量显著上调(P<0.05),Env免疫荧光信号强度增强;干扰ARG2基因后,与对照组相比,干扰ARG2组第6,12,48小时的gp85基因相对表达量显著或极显著降低(P<0.05或P<0.01),Env蛋白表达量显著下调(P<0.05),Env免疫荧光信号强度减弱。说明ARG2基因可以促进ALV-J在DF-1细胞中的复制。

In order to investigate the role of mitochondrial-related gene arginase-2(ARG2)in the infection of J subgroup leukosis virus(ALV-J),qRT-PCR was used to detect the expression of ARG2 in different organs and DF-1 cells of chickens infected with ALV-J in the experimental group,and to analyze whether there was any association between ARG2 and ALV-J infection.The over expression vector of ARG2 gene(over expression ARG2 group)and interference fragment(interference ARG2 group)were designed to transfect DF-1 cells,and pcDNA3.1 or Si-NC as control group were used to detect the overexpression or interference efficiency of ARG2 gene by qRT-PCR method.The expression of gp85 gene and Env protein in ALV-J DF-1 cells infected with ARG2 gene overexpression or interference was detected by qRT-PCR,Western-blot and indirect immunofluorescence methods,so as to comprehensively analyze the effects of ARG2 gene on ALV-J replication.The results showed that compared with the control group,the relative expression of ARG2 gene in the spleen and bursa of chickens in the test group decreased extremely significantly(P<0.01),while the relative expression of ARG2 gene in the kidney increased extremely significantly(P<0.01).At the same time,in vitro showed a decrease in the relative expression of the ARG2 gene at 6 and 12 hours of infection(P>0.05 or P<0.05)and a significant or extremely significant increase in the relative expression at 24,48,74,and 108 hours(P<0.05 or P<0.01).Compared with the control group,the relative expression of ARG2 gene in the overexpression ARG2 group was highly significantly higher(P<0.01),and the relative expression of ARG2 gene in the interfering ARG2 group was lower(P>0.05 or P<0.05),and the interfering fragment SI-ARG2-001 was selected for the subsequent experiments.After overexpression of ARG2 gene,the relative expression of gp85 gene at 12 and 24 hours in the overexpression of ARG2 group increased significantly or extremely significantly(P<0.05 or P<0.01);the expression of Env protein was significantly up-regulated(P<0.05),and the intensity of Env immunofluorescence signals was enhanced,as compared with that in the control group;after interfering with ARG2 gene,the relative expression of gp85 gene was significantly or extremely significantly decreased(P<0.05 or P<0.01);Env protein expression was significantly down-regulated(P<0.05),and the intensity of Env immunofluorescence signal was weakened in the interfering with ARG2 group at 6,12,and 48 hours,compared with the control group.It indicated that ARG2 gene could promote the replication of ALV-J virus in DF-1 cells.