PCV2 Rep蛋白N-糖基化位点突变对病毒复制的影响

The effect of N-glycosylation site mutation of PCV2 Rep protein on Viral replication

N-糖基化对病毒的感染与增殖均具有重要作用,与PCV2复制相关的Rep蛋白含有三个N-糖基化位点。为了分析PCV2Rep蛋白N-糖基化位点突变对病毒复制的影响,本试验构建了三个双拷贝突变体感染性克隆2M23、2M256、2M286,并成功拯救病毒。通过间接免疫荧光检测病毒的拯救效果,TCID50测定病毒的感染力,荧光定量PCR检测细胞病毒的载量。结果显示,PCV2Rep蛋白的23~25aa、256~258aa N-糖基化位点突变后降低病毒的复制能力,而286~288aa突变后增强病毒的复制能力,为进一步阐明PCV2的复制及致病机制提供参考。

N-glycosylation site is critical for the infection and proliferation of most virus. The Rep protein of PCV2 involved in virus replication contams three N-glycosylauon sites IN-glycosylation site mutation effect on virus replication, three double copy mutant infectious clone 2M23, 2M256, 2M286 were constructed and successfully rescued virus. Immune flurescent assay （IFA）,TCID30 and fluorescent quantitative RT-PCR to detect the virus rescue, the infectivity of virus and the virus load of cell. The results showed that mutation of the N-glycosylation site in the PCV2 Rep protein in 23~25 aa, 256~258 aa reduced virus replication and in 286~288 aa enhanced virus replication. This study arises a reference for the further elucidation of the mechanism of PCV2 replication and pathogenesis.