番鸭Viperin基因的克隆表达及其抗MDRV的作用研究

Cloning andexpression of Viperin gene in Muscovy duck and its antiviral effect against MDRV

为了研究番鸭Viperin蛋白在感染番鸭呼肠孤病毒(MDRV)细胞和组织中的表达情况,以及其是否具有抗MDRV作用,采用RT-PCR方法检测番鸭Viperin基因在感染MDRV不同时间点293T细胞、番鸭鸭胚成纤维细胞(MDEF)和雏番鸭脾脏中的表达情况;并采用RT-PCR方法从番鸭脾脏中扩增出Viperin基因片段,将其克隆到真核表达载体p Flag-CMV-5a中,测序验证后转染入293T细胞,运用Western-Blot技术鉴定蛋白表达情况;最后用MDRV感染过表达番鸭Viperin的293T细胞,通过RT-PCR和qRT-PCR检测病毒在细胞中的增殖情况。结果表明,Viperin基因在感染MDRV的293T细胞、MDEF细胞和雏番鸭脾脏中表达量均升高;MDRV P10基因在过表达番鸭Viperin基因的293T细胞中的表达量显著低于对照组(P<0.01),证实番鸭Viperin蛋白具有抑制MDRV增殖的作用。

In order to investigate the expression of Viperin gene of Muscovy duck after Muscovy duck reovirus（ MDRV） infection,and the antiviral effect of Viperin against MDRV,we first used RT-PCR to detect Viperin expression in MDRV infected 293 T cells,Muscovy duck fibroblasts（ MDEFs） and Muscovy ducks. Then,we amplified the full-length open reading frame（ ORF） of Viperin gene from spleen of Muscovy duck by RT-RCR,and successfully subcloned it into eukaryotic expression vector p Flag-CMV-5 a. The recombinant plasmid was transfected into 293 T cells,and the protein expression of Viperin was verified by Western-bloting.Finally,Viperin overexpressing 293 T cells were inoculated with MDRV,and the virus replication was analyzed by detecting viral P10 expression. The results showed that the Viperin expression was up-regulated both in vitro and in vivo after MDRV infection. In addition,the expression of P10 gene was significantly lower in Viperin overexpressing cells than that in control cells（ P 0. 01）,suggesting that Viperin plays a key role in inhibiting MDRV infection.