Differetial Analysis Of MicroRNA Expression From Host Cells Infected With Orf Virus And Preparation Of Antibody Against VIR Protein Of Orf Virus And Cellular Localization Observation Of VIR Protein

Contagious ecthyma, an important zoonotic infectious disease, is caused by the Parapoxvirus orf virus(ORFV), Contagious ecthyma of outbreaks and widespread popu Larity not only caused serious economic losses,but also severely threatens the health of people.micro RNA is a kind of about 22 nucleotides long noncoding single-stranded RNA molecu Les, have regulate function in gene transcription. In the aspects of embryonic development, cell differentiation, cell apoptosis, hematopoietic function and occurrence of disease,micro RNA play an important role. Interferon resistance gene(VIR gene) from ORFV has important role for viral replication.This gene,the conservatism gene of ORFV,has the function of interferon resistance and encoding early interferon resistance protein.This experiment with sheep skin fibroblasts(GSFs) infected and non-infected by ORFV as the research object, which explore and analyze micro RNA from GSFs infected by ORFV using the solexa high-throughput sequencing technologies. The results show that the experiment successfully constructed the difference of micro RNA expression spectrum from GSFs infected and non-infected by ORFV, of which 509 were significantly up-regu Lated and 169 were significantly down-regu Lated. Micro RNA target gene GO enrichment analysis show that the main difference between micro RNA from infection group of GSFs cell and micro RNA from non-infection group of GSFs cell is micro RNA from infection group widely exist in many enzymes and ribonucleotide binding activity in the aspect of gene molecu Lar function. Micro RNA from infection group of GSFs cell major related to the cell membrane, cracking, vacuole and lysosome in the aspect of the cell position.Micro RNA from infection group of GSFs cell major related to cell lipid metabolic process in the aspect of involved in biological processes. Significant analysis by micro RNA target genes KEGG pathways, and comparison from the enrichment of infection and the infection group KEGG pathways show that micro RNA from the interactions process of ORFV and GSFs cell widely distributed in the ECM-receptor interaction,Lysosome, Focal adhesion, Leukocyte transendothelial migrtion, Hedgehog signaling pathway,Peroxisome,Bile secretion, Regu Lation of actin cytoskeleton and Retinol metabolism. In order to further study the biological characteristics of VIR protein from orf virus(ORFV), VIR gene was amplified from ORFV and cloned into p ET-28 a and p EGFP-N1 respectively. The recombinant plasmids were named as p ET-VIR and p EGFP-VIR. BALB/c mice were immunized with the purified VIR protein to produce polyclonal antibody.Testicular cells of ovine were transfected with p EGFP-N1. The expression of VIR protein in ovine testicular cells was observed with laser confocal fluorescence microscope.The results indicated that the expressed VIR protein, with about 27 KDa molecu Lar weight, mainly existed in a soluble form. The prepared polyclonal antibody against VIR protein reached a titer of 1 ∶ 218, show a good antibody specificity. Laser confocal fluorescence microscope showed that the localization of VIR protein was mainly distributed in cell nucleus.The experiment analyze the difference of micro RNA expression spectrum from GSFs infected and non-infected by ORFV, of which 509 were significantly up-regu Lated and 169 were significantlydown-regu Lated. In the process of interaction between ORFV and GSFs cell, micro RNA not only influenced the activity of many enzymes,participate in ribonucleotide binding activity and cell lipid metabolic process,but also may be involved in the ECM-receptor interaction, Lysosome activity, Focal adhesion, Leukocyte transendothelial migrtion, Hedgehog signaling pathway, Peroxisome,Bile secretion, Regu Lation of actin cytoskeleton and Retinol metabolism. The experiment successfully prepared polyclonal antibody against VIR protein and the localization of VIR protein was mainly distributed in the nucleus of sheep testicular cells.