Isolation And Identification Of Sheeppox Virus Gansu Strain And Cloning, Eepression, Immunocompetence Of Membrane Protein-encoding ORF23 Gene

Capripox is a severe and highly contagious disease in sheep or goats caused by sheeppox virus or goatpox virus.In this study one strain of sheeppox virus was isolated from the skin tissue of suspected capripox sheep in Jingtai, Gansu Province, and identified by cell culture, polymerase chain reaction detection (PCR) and animal regression experiment. The membrane protein-encoding ORF23 gene of enveloped extracellular virus (EEV) was cloned and expressed in prokaryotic expression system; the expression product was examined by the immune methods.The results were shown as follows:1. Isolation and identification of the CapripoxThe virus isolated from pathological tissues of sheep caused classical and regular cytopathic effect (CPE) on primary sheep testis cell and hyperthermia with skin eruptions appeared over the less wooly parts in sheep which was intradermaly inoculated with the virus. The glycoprotein-encoding ORF118 gene was cloned by PCR from genomic DNA which was extracted from the pathological tissues and cell cultures. The results of sequence analysis and structural prediction of ORF118 gene suggested that the ORF118 glycoprotein is completely conserved among sheeppox field strains and has one signal peptide which was ecoded by the initiative 35 amino acids. The transmembrane domain at 21 and 38 flank was predicted accoding to the amino acid sequence of ORF118 gene.2. Cloning, Expression, Biologic activity of membrane protein-encoding ORF23 of sheeppox Gansu strainThe membrane protein-encoding ORF23 gene of sheeppox was cloned and sub-cloned to pET-30a (+) to establish prokaryotic expressed plasmid pET-SPV23 which was transformed into Eoli. BL21 (DE3) and identified with enzyme digestion and PCR. The positive clone was expressed by IPTG and the product expressed was analyzed by SDS-PAGE. The target protein was expressed in the form of inclusion bodies with a molecular weight of 43kDa. The expressed protein was recognized by standard positive serum of sheeppox virus.This study provides foundation for further researches about developing diagnostic reagents and genetic engineering vaccine.