The Cloning,Expression And Bioinformatic Analysis Of ORF132 Gene Of Lumpy Skin Disease Virus

Lumpy skin disease(LSD)is an acute,sub-acute disease in cattle,caused by lumpy skin disease virus(LSDV),which belongs to the genus Capripoxvirus of the.family Poxviridae.LSD is characterized by fever,nodules on the skin,loss of milk production,mastitis and infertility,it causes significant economic loss and affects the development of international trade and cattle industry.So,it is defined as a notifiable disease by the World Organization for Animal Health(OIE),and listed "List? " by China Inspection and Quarantine(CIQ).It is currently endemic in most African countries and has recently spread out of Africa into the Middle East region,especially in Vietnam.To prevent the risk of LSDV spreading to China,it is necessary to establish and reserve inspection methods and vaccine research.In this study,LSDV-ORF132 gene of hypothetical protein was cloned and expressed in prokaryotic expression system,its secondary structure,antigenicity and function of the protein was preliminary predicted and analysised.The results were shown as follows:1.Cloning of ORF132 gene of Lumpy skin disease virusAlthough the Capripoxvirus genus are very closely related,ORF132 gene is an unique of LSDV in virulence and host range,and lacks homology to other known proteins.The viruse was proliferated in lamb testicular cells to facilitate follow-up study.Primers contained restriction endonuclease of EcoR ? and BamH ? were designed referring the published LW132 gene sequences of Neethling vaccine LW1959 in GenBank.The fragment gene was amplified by PCR,purified and cloned into pMD-19-T,named plasmid pMD-19-T.The plasmid was identified by restriction endonuclease of EcoR ? and BamH ?,plasmid PCR and sequencing.The results showed the sequence size was 534bp,the content of GC was 25.7%.Compared with AF409138,homology is above 99%,LSDV-ORF132 is highly conserved.The study revealed that the LSDV-ORF132 gene could be used as a diagnostic tool for the differential diagnosis of Capripoxvirus.2.Bioinformatic analysis of deduced amino acids of LSDV-ORF132 geneThe amino acid sequences of protein were deduced by published LSDV-ORF132 gene for analysis and prediction,it encods 177 amino acids,a molecular weight of 20.63 kD,and 59.8 percent chance of insolubility were predicted by intemet.The transmembrane regions and signal peptide were analyzed by using CBS server of Tekniske University of Denmark;The secondary structure?hydrophilic?surface accessibility and antigenic index of extracellular factor were analyzed by the software DNAstar.The result:No signal peptide,the transmembrane regions was possible to locate within N-terminal No.4?21;there are 3 ?-helixes,7 ?-sheets,6 tums,high hydrophilic and surface accessibility,the most possible B cell epitopes for extracellular factor of LSDV-ORF132,which located within or nearby its N-terminal No.35-42,61-65,71-76,95-99,107-114 and 120-125,were predicted.Using BLAST P,phylogenetic analysis showed that LSDV was diverged from other Capripoxvirus species SPPV and GTPV.Just considering the specificity and linear epitopes,LSDV-ORF132 can be used as a highly expressed antigen candidate for further researches about developing diagnostic reagents and genetic engineering vaccine.3.Prokaryotic expression of protein encoding ORF132 gene of LSDVThe protein-encoding ORF132 gene of LSD was cloned and sub-cloned into pET-32-a(+)or pGEX-6p-1 to establish prokaryotic expressed plasmid pET/pGEX-ORF132,identified with enzyme digestion and PCR.then the positive clone was transformed into Eoli BL21(DE3)and expressed by IPTG induction.The SDS-PAGE analysis showed that the fusion protein had a molecular weight of 39.97 kD and 47.45 kD respectively and mainly existed in form of inclusion bodies.The expressed protein were transferred to PVDF membrane,was recognized by His/GST-tag McAb..Using semidry SDS-PAGE protein bands were transferred to PVDF membrane,GST/His-tag McAb(1:1000)from mouse as the primary antibody,rabbit anti-mouse IgG-HRP(1:2000)for second antibody,DAB chromogenic solution to proceed Western blot.Two bands about 47.45 kD,and 39.97 kD appeared positive reaction at the obvious ribbon.This study provides material for the study on ORFs functionality and proteomics of lumpy skin disease virus.LSDV-ORF132 can be used as a candidate antigen for the development of more effective and safer vaccine.