Study On Anti-RNA Virus Activity Of Duck Mx Protein In Vitro

Mx proteins are interferon(IFN)-induced dynamin-like GTPase with Antiviral activity found in all vertebrate species examined so far.At present,the research on the antiviral activity of Mx protein in animals such as mouse source,fish source and chicken source has been deepened.However,the viral activity of duck Mx against RNA virus is still uncertain,so the anti-RNA viral activity of duck Mx needs further evaluation.In this study,human embryonic renal epithelial cells(HEK293T)were infected with vesicular stomatitis virus(VSV),Newcastle disease virus(NDV),type 1 duck hepatitis A virus(DHAV-1)and type 3 duck hepatitis A virus(DHAV-3)respectively.After three generations of blind passage,TCID50 of them was determined,TCID50 of VSV,NDV,DHAV-1and DHAV-3 were 10-5.164/0.1m L,10-6.181/0.1 m L,10-7.748/0.1 m L and 10-9/0.1 m L respectively.The RNA of four viruses in 293 T cells was extracted and reverse transcribed into c DNA.The target genes were amplified by PCR.Four standard plasmids of RNA virus were prepared,and standard curves were made using standard plasmids as templates.Real-time fluorescence quantitative PCR(RT-q PCR)assay for viral load analysis was successfully established through repeatability,sensitivity and specificity detection.The lentiviral shuttle plasmid p LV-m Cherry-Mx-2166 with mcherry red fluorescent tag was synthesized,The p LV-m Cherry-Mx-2166 plasmid was transiently transfected into 293 T cells using liposome 2000 transfection reagent,and the transfection dose and time were optimized.Western blot results showed that the expression of duck Mx protein was better than that of other time pointswhen 293 T cells were transfected instantaneously with 2 ug/well p LV-m Cherry-Mx-2166 for 24 hours.On this basis,293 T cells were infected with four RNA viruses,the expression level and viral load of duck Mx were detected by Western blot and RT-q PCR at 6h,9h,12 h,24h and 36 h after infection,the results showed that transient overexpression of duck Mx could significantly inhibit the replication of VSV,NDV,DHAV-1 and DHAV-3 viruses in vitro compared with control group.Three pairs of si RNA primers si RNA-Mx1,si RNA-Mx2,si RNA-Mx3,and negative control primer si RNA-NC were designed and synthesized according to the conserved coding region of duck Mx gene.The si RNA-Mx2 with stable interference effect was screened by Western blot and RT-q PCR.The optimum concentration of si RNA-Mx2 for VSV and NDV was 40 pmol/well,and 50 pmol/well for DHAV-1 and DHAV-3,and the interference efficiency was more than 50% comparing with the non-interfering group.The results showed that si RNA-Mx2 significantly inhibited the activities of duck Mx against VSV,NDV,DHAV-1 and DHAV-3 viruses compared with non-interference group.In this study,overexpression and RNA interference were used to reveal that duck Mx has antiviral activity against VSV,NDV,DHAV-1 and DHAV-3 after overexpression in 293 T cells.This study will provide an important theoretical foundation for research the antiviral function of duck Mx protein and antiviral mechanismdeeply.