Analysis of the Differences of Chemokine-binding Protein Gene Between Ovine Contagious Pustular Dermatitis Virus Oral and Visceral Infection Strains and Their Prokaryotic Expression
[Objective]This study was aimed to clone and analyze the chemokine-binding protein(CBP)gene of the oral and visceral strains of Ovine contagious pustular dermatitis virus(ORFV),and provide basic data for the subsequent study of the immune escape mechanism of CBP in ORFV.[Method]Specific primers were designed using the DNA extracted from oral scabs and small intestine of sick lambs as templates,PCR amplification,cloning and sequencing were performed,and the genetic and evolutionary relationship of CBP gene and the physicochemical properties and structural characteristics of the expressed protein were analyzed.Recombinant plasmids PGEX-4T-1-CBP-K and PGEX-4T-1-CBP-N were constructed using the prokaryotic expression system pGEX-4T-1 and transformed into Escherichia coli Rosetta(DE3)competent cells,and the protein expression was detected using SDS-PAGE.The antigenicity of the protein was analyzed by Western blotting.[Result]The total length of CBP gene in ORFV oral infection strain was 873 bp,and the total length of CBP gene in ORFV visceral infection strain was 846 bp,and the latter had 24 bp base loss at 217 bp.The phylogenetic tree showed that oral and visceral infection strains did not belong to the same evolutionary branch,and the genetic distance was far away.The CBP gene of the oral infection strain strain was closely related to the Jilin isolates in China.The CBP gene of the visceral infection strain was closely related to that of the Malaysian strain.The CBP gene of the oral infection strain encoded 291 amino acids,while the CBP gene of the visceral infection strain encodes 282 amino acids.The protein secondary structure was mainly composed of alpha-helix and random coil,the tertiary structure had 5 differences,mainly alpha-helix and beta sheet.The prokaryotic expression plasmids pGEX-4T-1-CBP-K and pGEX-4T-1-CBP-N were successfully constructed.CBP fusion proteins were expressed mainly in the form of inclusion bodies with a molecular mass of about 60 ku in Escherichia coli Rosetta(DE3)competent cells.[Conclusion]In this study a 24 bp deletion in the visceral infection strain compared to the oral infection strain.The prokaryotic expression plasmids pGEX-4T-1-CBP-K and pGEX-4T-1-CBP-N were successfully constructed,which could express CBP fusion protein in Escherichia coli,the results of this study could provide reference for revealing the role of CBP in the interaction between ORFV and host.It could also provide a basis for finding new prevention and treatment methods of ovine contagious pustular dermatitis.
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