Production of egg yolk antibody (IgY) against recombinant canine parvovirus VP2 protein.

Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection. Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The isolate was defined as CPV-2a (JN403045) subtype by sequencing. The VP2 ORF was inserted into pET-32a by T4 ligase and introduced into the E. coli Bal21. VP2 protein was produced by the induction of the E. coli Bal21 containing pET-32a-VP2 with isopropyl-β-D-1-thiogalactopyrannoside (IPTG). Expression of the recombinant VP2 protein (rVP2) fused with His-tag was analyzed by SDS-PAGE and detected by western blotting using anti-His monoclonal antibody. The rVP2 was purified by Ni+-affinity purification chromatography under denature condition and dialyzed against PBS. The concentration of the rVP2 was determined by Bradford method. After immunizing the chickens with rVP2, anti-VP2 IgY was isolated by PEG 6000 precipitation and analyzed by SDS-PAGE. The activity and specificity of the IgY antibody were analyzed by indirect ELISA and western blotting. SDS-PAGE analysis showed that the rVP2 fused with His-tag had a molecular of 85 kDa, accompanied with the low molecular fractions around 50 kDa and 40 kDa. The rVP2 could be recognized be the anti-His monoclonal antibody. The IgY antibody isolated by PEG 6000 method and analyzed by SDS-PAGE showed the IgY mainly contained two parts, 23 kDa and 67 kDa, which corresponded to light and heavy chain, respectively. In additional, some lower bands around 40 kDa were presented on the gel. The anti-VP2 IgY reached to 1:40960 after the fourth immunization. The anti-VP2-IgY could recognize the VP2 specially in Western blotting, while no reaction was seen with the low molecular. Discussion: The emergence of the low molecular proteins in 50 kDa and 40 kDa in the pellets of the bacterial lysates may be due to the degradation of the rVP2 by endogenous proteases in E. coli. The fact that IgY could recognize the entire VP2 fraction suggests the lost of the antigenic sites of the low molecular proteins.