| Canine parvovirus-2(CPV-2) disease is highly contagious and often fatal in canine withsymptoms of hemorrhagic diarrhea and myocarditis. Detection and treatment of CPV-2infection based on antibodies generated from mammalian, such as mice monoclonal antibody,may remain cross-reactivity and high price. IgY technology has been recognized as apromising alternative to generate a large amount of qualified high specific antibody forimmunodiagnostic and immunotherapy with the advantage of phylogenetic distance betweenchickens and mammals. The purpose of this study was to produce the IgY antibody againstCPV-VP2protein expressed in E. coli, which could be developped into the diagnostic kit andantibody drug for the CPV infection. The study was divided into three parts:1. Amplification and analysis of the CPV-VP2sequence. A pair of primers was designedby primer prime5.0software based on the reference sequence on the GenBank andcommercially synthesized to amplify the VP2genes. The amplified fragments (1755bp) wereinserted into the pMD18-T vector, then introduced into DH5α. Plasmids of the cultured DH5αwere extracted and digested with restriction endonuclease BamH I and Xho I after blue/whiteselection. The positive clones were selected for sequencing and analyzing by DNASTARsoftware. To examine the phylogenetic relationship of the CPV obtained from Shaanxi, a treebased on the nucleotides sequence of the full length of VP2genes retrieved from the GenBankwas constructed by MEGA5.0software. Results showed that the sample isolated fromShaanxi was defined as CPV-2a subtype (JN403045). The VP2sequence alignment of theisolated strain exhibited more than92.4%nucleotide and99.5%amino identity with CPV-2astrains from foreigns. The unique Ile-324mutation in the VP2of the sample isolated fromShaanxi was detected, as compared with a Tyr-324in the reference CPV strains. Thephylogenetic tree constructed from the VP2genes showed that the Shaanxi strain and theCPVs isolated from Korean, Japan and China Taiwan province formed a cluster, indicating aclose relationship.2. Expression and purification of the CPV-VP2protein. The pMD18-T-VP2and pET-32awere both digested with BamH I/Xho I and ligated with T4DNA ligase to yield expressionplasmid pET-32a-VP2. The pET-32a-VP2was introduced into Bal21(DE3) pLysS andinduced with IPTG after identifying by enzyme analysis and PCR reaction. The concentration of IPTG and the induction time were optimized subsequently. After analysized by SDS-PAGEand Western blot, the VP2protein was purified by Ni+affinity column. The results showedthat the VP2protein (89ku) was expressed in an inclusion format when induced with0.75mmol/L IPTG for5hours. In addition to the target VP2protein, two bands,50ku and40ku,were expressed in the insoluble fraction analysized by SDS-PAGE. The VP2protein (89ku)and the lower molecular weight band (50ku) were purified by Ni+affinity column andexhibited perfect reactivity by Western blot.3. Production of IgY antibody against VP2protein. Anti-VP2IgY was produced byimmunizing the lay hens with recombinant VP2protein after dialysis. The IgY antibody wasextracted from eggs by PEG6000method and analysed by SDS-PAGE. The titer of IgYantibody against VP2protein was monitored by indirect ELISA. Specificity of the IgYantibody against VP2protein was identified by Western blot. Results showed that the IgYantibody consisted two parts,65ku and19ku.The titer of the anti-VP2IgY increased to1:40560after the fourth immunization and may react with the VP2protein the specificity byWestern blot.In this study, the special IgY antibody against CPV-VP2protein expressed in E. coli wasproduced and could be developped into the diagnostic kit and antibody drug for the CPVinfection. |
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