Research On Function Of Host ASC Gene In The Inflammatory Process Of The Orf Virus Strain Isolated From Guangdong Province

Orf disease, also named contagious ecthyma, is a zoonotic disease caused by the orf virus(ORFV) that manily affects sheep, goats, wide small ruminants and humans. The typical lesions are characterized by erythema, papule, pustular and scab formation occurr in the epithelium of lips, mouth muzzle, nostrils, teats, and oral mucosa. ORFV is widely distributed that all sheep-breeding countries existing the incidence and prevalence of this disease. In addition to traditional sheep industry such as northeastern or northwestern China, Shandong, Guangdong, Yunnan and other regions also apperared in the incidence and prevalence of this disease. Althogh ORFV is limited harmful on the bodies of human, its wide spread and high infection rate could cause great economical loss to animal husbandry. ORFV can escape host immune response by prducing anti-inflammatory protein and cause the host repeated infection. The increase of Orf outbreaks may be associated to the lack of effective prevention, control measures and understanding of immune characteristics and pathogenesis of ORFV. Apoptosis-associated speck-like protein(ASC) is the adaptor of inflammasome and plays key role in inflammasome medicated against pathogenic microorganisms. Currently, there are no detailed about the function of ASC during ORFV infection, which is the negative factor for further research on the immune characteristics and pathogenesis of ORFV. Therefore, we conducted the following study: 1. Isolation and Identification of orf virus GDZC strainsThe virus strain was isolated from crust tissues of black goat with Orf like-clinical symptoms from goat farm in Zengcheng city of Guangdong province by passage in ovine fetal turbinate cells(OFTu). The virus was identified as orf virus using cell morphology of lesions, TCID50 test and polymerase chain reaction(PCR) method. The results showed that OFTu cultures infected with crust tissues displayed cytopathic effects at four blind passages. The titer of the fifth passages virus was 108.65TCID50/m L. The specific orf virus ORFV011, ORFV059, ORFV109, ORFV110 and ORFV127 genes were amplified. Phylogenetic analysis indicated that the isolated orf virus had high homology with the FJ-DS and FJ-GT strains. Above all, one orf virus srain had been successfully isolated and named ORFV-GDZC strain. 2. Amplification of Hainan black goat ASC gene and sequence analysisHainan black goat ASC gene was amplified by RT-PCR and the sequence was deposited in Gen Bank(accession no.: KM576770), which analyzed with bioinformatics softwares. The results showed that the cloned open reading frame(ORF) of ASC gene of Hainan black goat contained the entire ASC gene coding deduced proteins with 196 amino acids and its deduced molecular weight is 22.1 k Da. The nucleotide sequences of ASC were aligned using the software Meg Align(DNAstar, Madison, WI). The nucleotide homology of ASC among Hainan black goat and Bos taurus, Homo sapiens, Sus scrofa, Macaca mulatta, Mus musculus, Rattus norvegicus, Xenopus laevis, and Danio rerio were 95.2%, 81.3%, 83.0%, 79.8%, 75.4%, 75.1%, 49.2%, 43.7%. The ASC gene is conservative in mammalian but significantly different with amphibian, with only 50% homology. 3. Prokaryotic expression of Hainan black goat ASC and the preparation of the polyclonal antibodyDouble enzyme digestion and nucleotide sequence analysis showed that expression plasmid p ET32a(+)-ASC was successfully constructed. The recombinant His-tagged ASC was efficiently expressed and SDS-PAGE showed that the purified protein band was consistent with the predicted size, which is approximately 40 k Da. Western blot detection using His-tag antibody and our prepared Ig G showed that the fusion protein was the His-tagged ASC. 4. Construction of overexpression plasmid and RNA interference plasmid of ASCUsing the ASC gene from p MD18-T-C-ASC plasmids to design the specific primer and construct the p IRES2-EGFP-ASC eukaryotic expression plasmid. The recombinant p IRES2-EGFP-ASC was transfected into OFTu cells by liposomes. The expression of the recombinant p IRES2-EGFP-ASC in the OFTu cells was observed by fluorescence microscope and IFA. The results showed that the overexpression plasmid of ASC gene was successfully constructed. Oligonucleotide primer of ASC gene was designed by biology software and ligated into the p Super. Inhibit ASC expression in OFTu cellls using RNA interference vectors. The results from Real-Time PCR demonstrate that the suppression efficiency of ASC m RNA reach 60% after trancfected sh RNA-ASC plasmid within 24 h. 5. Reserch on the role of ASC during the inflammation induced by ORFV infection in host cellsBy using the Real-Time PCR to monitor ASC gene and host cells proinflammatory cytokines expression within 24h(1h, 2h, 3h, 4h, 6h, 12 h, 24 h) after ORFV infection we found that the level of these genes expression increases rapidly in the early of viral infection. The results indicate that ORFV activate the host immune response. For further study the ASC gene function during ORFV infection, we over-express or inhibit ASC gene expression in OFTu cells using eukaryotic expression plasmid. After the virus infection, the Real-Time PCR assay demonstrate that ASC plays a key role in the process of host antiviral response.