Isolation And Identification Of Orf Virus Gansu Prevalent Strain And Cloning, Expression Analysis Of Its Important Function Genes

【Objective】 Isolated an Orf virus strain from BeiShan district of Yuzhong county of Gansu province inChina which collected pustules and crusts of suspected sheep;5different ORFV ankyrin repeats genes werecloned and constructed eukaryotic expression vector of pEGFP-ORFV-ORF129to express ORF129gene inBHK-21cells. This will lay the foundation for the further study of the molecular mechanism of Orfvirus protein encoded by ORF129gene during virus infection.Besides, this study also constructedrecombinant expression strain GS115-pPIC9K-ORF040and expressed ORFV ORF040gene in the yaest,meanwhile predicted its sequence and structure, thus provide the theoretical basis for the research of biologicalfunction of G4L molecular and the development of ORFV molecule vaccine.【Methods】(1) Isolated an Orf virus strain from BeiShan district which collected pustules and crusts ofsuspected sheep through passaging in LT primary cell lines, amplified the B2L and F1L full length gene by thedesigning of specific primers, and constructed phylogenetic tree based on its nucleotide sequence and thecorresponding sequences of other strains both at home and abroad.(2) According to the published genesequences of ORFV-OV00strain(HQ694772) designed5different specific primers to amplify and clone5different ORFV ankyrin repeats genes of GSYZ,then analyzed their structures using bioinformatics.Based on it,chose ORF129as target and constructed eukaryotic expression vector of pEGFP-ORFV-ORF129,aftertransfecting BHK-21cells and obtain the expression system by identification.(3) Besides, this study alsodesign1specific primer to amplify ORFV ORF040gene, recombinant yeast expression strainGS115-pPIC9K-ORF040was constructed, expressed and purified the protein;Meanwhile, used bioinformaticssoftware to predict analysis its sequence and structure.【Results】(1) Successfully isolated an Orf virus strain from Yuzhong district which collected pustules andcrusts of suspected sheep through passaging in LT primary cell lines, called ORFV/GSYZ/China(abbreviationGSYZ), amplified the B2L and F1L full length gene by the designing of specific primers. The phylogeneticanalysis based on the B2L gene showed that the Xinjiang and Gansu2009strain were most closely related toORFV/GSYZ/China. Besides, isolates from New zealand and India also presented close relationship withORFV/GSYZ/China. However, isolates from Hubei, Liaoning, Shanxi and Taiwan were estranged from thevirus mentioned above. The result which based on sequence similarity demonstrated that ORFV/GSYZ/Chinamay be once prevalent in the regions of Xinjiang and Gansu province.(2) Successfully clone the5ORFVencoding ankyrin genes, among which ORF008was1548bp encoding515amino acids; ORF123was1578bpencoding525amino acids; ORF126was1494bp encoding497amino acids; ORF128was1506bp encoding501amino acids and ORF129was1551bp encoding516amino acids.The nucleotide homology was96.7%to98.4%with foreign ankyrin repeat proteins; The deduced amino acid sequence showed that all of these fiveankyrin have similar in structure, with a typical structure of6to9multiple ankyrin repeats motif at the aminoterminus; Secondary structure analysis showed that the alpha helices and beta folded at high levels, from69%to86.9%and41.4%to58.1%respectively, while the random coil content was only7.1%to9.1%;Thematching tertiary structure was not found by homology modeling comparison.The eukaryotic expressionplasmid pEGFP-ORFV-ORF129was constructed, the ORF129gene was stable expressed in BHK-21cells byfluorescence microscopy, RNA, genomic DNA level and Western-blot analysis.(3) The ORFV ORF040genewas cloned successfully, the gene was414bp, encoding protein with137amino acids; The eukaryoticexpression plasmid GS115-pPIC9K was constructed and successfully expressed in yeast induced by methanoland the purification of the protein with the weight of approximately16ku, the similar with the value of thetheoretical speculation; The G4L protein structure and subcellular localization prediction showed that theremight be8phosphorylation sites, among which5serine (position6,121,124,133and135),2 threonine(position28and68),1tyrosine(position78),the highest probability of the protein existed in thecytoplasm(65.2%), followed by the nucleus and mitochondria(both8.7%).【Conclusions】(1)This study successfully isolated an ORFV strain prevalent in Beishang region, calledORFV/GSYZ/China(abbreviation GSYZ).(2)Obtained5kinds of ankyrin repeat genes of GSYZ, and in whichtake ORF129gene as research target to express the gene in eukaryotic cells.(3)Successfully cloning andexpression of the ORF040gene of GSYZ in yeast, the expression product was purified and analysed bystructure prediction.This essay will lay a solid material foundation for the further study of ORFV infected host mechanism anddevelopment of molecular vaccine.