E3泛素连接酶RNF165在A亚群禽白血病病毒复制中的作用

Role of E3 Ubiquitin Ligase RNF165 in the Replication ofAvian Leukosis Virus Subgroup A

【目的】探明鸡源环指蛋白165(ring finger protein 165,RNF165)在A亚群禽白血病病毒(ALV-A)复制中的作用,为进一步研究鸡源RNF165对ALV-A致病性的影响提供参考。【方法】将ALV-A接种于DF-1细胞和1日龄雏鸡,通过Western blotting和实时荧光定量PCR方法分别从体外和体内两个方面验证ALV-A对宿主RNF165表达的影响;参考已公布的RNF165基因序列设计过表达引物,通过PCR扩增RNF165基因,将其克隆至真核表达载体pcDNA3.1;将验证表达的重组质粒转染DF-1细胞,1 d后接种ALV-A,使用Western blotting检测过表达RNF165对病毒复制的影响;最后设计突变引物,构建RNF165功能结构域突变质粒,将过表达质粒和突变质粒分别转染DF-1细胞,1 d后接种ALV-A,Western blotting测定gp85蛋白表达差异。【结果】ALV-A感染DF-1细胞后未能检测到内源性的RNF165蛋白,但基因转录水平较对照组明显下调(P<0.01)。在体内试验中,与对照组相比,感染病毒雏鸡肝脏中RNF165基因和蛋白表达水平均有所下调(P<0.01);通过PCR成功扩增到大小为1068 bp的目的片段并成功克隆至pcDNA3.1,Western blotting结果显示,在39 ku处出现目的条带,RNF165过表达后促进了病毒复制;测序结果显示,成功构建出功能结构域突变质粒,与过表达质粒组相比,RNF165突变质粒组病毒的复制水平受到显著抑制。【结论】ALV-A感染抑制DF-1细胞和雏鸡肝脏RNF165的表达,在体外试验中,过表达RNF165会促进ALV-A的复制,且这种影响与其保守结构域有关。

【Objective】The aim of the experiment was to explore the role of chicken derived ring finger protein 165(RNF165)in Avian leukosis virus subgroup A(ALV-A)replication,and provide reference for further research on the impact of chicken derived RNF165 on ALV-A pathogenicity.【Method】ALV-A was inoculated into DF-1 cells and 1-day-old chicks.Western blotting and Real-time PCR were used to verify the effect of ALV-A on the expression of host RNF165 in vitro and in vivo.The expression primer was designed according to the published sequence of RNF 165 gene.The RNF 165 gene was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.1.The recombinant plasmid was transfected into DF-1 cells and inoculated with ALV-A one day later.The effect of RNF165 expression on virus replication was detected by Western blotting.Finally,the mutation primer was designed to construct the RNF165 functional domain mutation plasmid.The overexpression plasmid and the mutant plasmid were transfected into DF-1 cells respectively.One day later,they were inoculated with ALV-A.Western blotting was used to determine the difference of gp85 protein expression.【Result】The endogenous RNF165 protein could not be detected after ALV-A infected DF-1 cells,but the gene transcription level was significantly lower than that of control group(P<0.01).In vivo experiments,the gene and protein expression level of RNF165 in chicken liver was lower than that of control group(P<0.01).The 1068 bp target fragment was successfully amplified by PCR and cloned into pcDNA3.1.Western blotting results showed that the target band appeared at 39 ku,and the over-expression of RNF165 promoted virus replication.The sequencing results showed that the functional domain mutant plasmid was successfully constructed,and the replication level of the RNF165 mutant plasmid group was significantly inhibited compared with the overexpression plasmid group.【Conclusion】The infection of ALV-A inhibited the expression of RNF165 in DF-1 cells and chicken liver.In vitro,overexpression of RNF165 would promote the replication of ALV-A,and this effect was related to its conservative domain.