| Sheeppox, which is caused by Sheeppox virus (SPPV) and characterized by pox lesions in the skin,is one of the important diseases of sheep, and mainly affects international trade of livestock andlivestock products. The genetic sequencing analysis of SPPV has suggested the34th open reading frameencodes orthologue of VV E3protein. The previous studies showed E3protein coded by VV couldinhibit the activity of antiviral molecule (e.g PKR) in interferon system. The inhibition of SPPV E3protein on PKR hadn’t been reported. In this study, we focused on the anti-IFN ability of SPPV and themodulation on anti-viral signal pathway mediated by PKR. The mian studies were as following:1. The development of qPCR assay for detection of SPPVFor research purpose, we developed qPCR assay for detection of SPPV, and tested its specificity,sensitivity and repeatability. The results showed developed qPCR assay was specific (the qPCR assaycould differentiate between SPPV and ORFV), sensitive (the limit detection was102copies/μL), andreproductive (coefficient of variation was less than5%).2. Bioinformatic analysis of SPPV E3SPPV E3L was analylized by bioinformatics software, and the result suggested SPPV E3L genewas534bp, and encoded177amino acids. SPPV E3contained two conserved domains: Z-alpha andDSRM. The subcellular localization prediction implied SPPV E3protein largely localized in nucleusand cytoplasm. Compared with VV E3protein, Z-alpha domain on N-terminus of SPPV E3protein wasvaried, but DRRM on C-terminus was more conserved. These results indicated SPPV E3protein couldpossiblely inhibit the activity of PKR.3. Expression, purification of SPPV E3protein and production of polyclonal seraThe SPPV E3L was amplified and cloned into the pGEX4T-1vector to express the GST-taggedrecombinant protein in BL21(DE3) plysS. Recombinant protein was purified with GST binding resin.The purified recombinant protein mixed with freund’s adjuvant was used to produce polyclonal serawith BALB/c mice, and antibody titer of the produced sera was about1:100000. The results suggestedthe antisera could be used to detect SPPV E3in the follow-up experiment.4. SPPV interferon sensibility assay and the study of modulation effect of SPPV E3protein on PKRVero cell monolayers in12-well plates were treated24h at37℃with or without0-5000units/mlof human IFN-β. Cells were infected with SPPV and incubated for1h at37℃. After Virus suspensionwas discarded, cells were cultured in cell culture maintenance medium containing differentconcentration of IFN-β. The virus yield was tested with developed qPCR, when the cells with virus butno IFN-β exhibit complete CPE. The results implied the replication of SPPV was inhibited markedlywith100units/mL IFN-β. This suggested SPPV was sensitive to IFN-β.The Vero cell monolayers in6-well plate were transfected with pcDNA3.1(+)-SPPV E3L andincubated at37℃for48h, and then the vero cells were transfected with different concentration (10,20, 50μg/mL) of polyI:C to activate PKR. Following incubating at37℃for8h, the total protein wasextracted with RIPA lysis buffer. Western-blot assay suggested phosphorylation level of PKR wasup-regulated with the increasing concentration of polyI:C, and the expression of SPPV E3protein incells did not reverse this. The results showed SPPV E3protein did not inhibit the activation of PKR. |
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