| Canine parvovirus infection is a contagious disease of dogs caused by canine parvovirus (canine parvovirus, CPV). It can cause dogs with acute enteritis, neutropenia, vomiting, puppies myocarditis and other diseases. Canine parvovirus is a single-stranded linear DNA virus, its major structural protein are VP1and VP2. The whole length of VP2gene is1755bP that decides the CPV antigen type and invade host specificity and encodes CPV antigen decided to clusters and induces neutralizing antibody.In this study, two CPVs were isolated from two suspected CPV-infected dogs faeces and intestinal contents and named as the DN7strains and strains DN8. Two isolates and vaccine strains, SL VP2gene were sequenced to anlayze nucleotide sequence and amino acid sequence homology. Results showed that nucleotide sequence homology between the nucleotide sequences of two CPV strains were99.5%, the homologies between DN7, DN8and vaccine strain of C154were98.6%,99.1%. respectively. Compared with domestic isolate BJ02007strain the highest nucleotide homology were99.5%and100%, respectively. with isolates V154nucleotide homology as the lowest, which were99.0%and99.4%, respectively. DN7, DN8, with foreign isolates from South Korea DH426had the highest nucleotide homology, which were99.4%,99.8%, respectively. Separation with strain2c (b) of the Japan nucleotide Nucleotide homology minimum, were99.0%,99.4%, respectively. It showed that the two isolates belonged to the CPV-2a by the evolutionary tree.In order to improve the speed of clinical detection of canine parvovirus, a CPV loop-mediated isothermal amplification (LAMP) method for detection was established. Designing and synthesizing two pairs of primers for the conserved regions of CPV gene, on the reaction conditions of the CPV LAMP detection method to optimize the choice, then using the optimized reaction conditions for specificity test, sensitivity test and the detection of clinical samples. Results showed that LAMP detection method established by the positive clinical samples showed the characteristic ladder by electrophoresis, and negative clinical samples amplified products showed no amplified bands. The minimum detectable DNA of the canine parvovirus is0.11×10-4μg/μL, which is more sensitive than PCR for100times, and is1000times of rapid antigen detection test strips of canine parvovirus. LAMP and PCR test results of the clinical samples was100%. Data Showed that the detection of LAMP method was simple, rapid, which helped to increase the CPV detection speed and accuracy.By the epitope tandem, selecting a linear epitope of canine parvovirus, repeated series, and transformed into a prokaryotic expression system, we obtained the CPV VP2gene of B-cell linear epitopes of recombinant proteins. The recombinant protein of CPV isolates DN7, canine parvovirus live vaccine antigen were used for immuning7months old Hyline for three times. The egg yolk antibody was extracted by the chloroform extraction method. With indirect ELISA for detecting antibody titer, three antigens with different titers were acquired, the highest value of egg yolk antibody ELISA titer were1:1024,1:4096, and1:2048. Vitro neutralization test results showed that the yolk antibody stock solution had weak capacity to CPV, but yolk antibody concentration in multiples the increased capacity to CPV, also strengthened the cytoprotective effects. Therefore, F81cells inoculated with CPV does not produce any lesions within36h, but began to show lesions slowly after48h.The results of this study showed that the isolated two strains of canine parvovirus, and established of CPV VP2gene LAMP for rapid detection method. The tandem epitope of recombinant protein yolk antibody has the neutralization effect to canine parvovirus at certain level, and can delay its effects on cell infection, which could provide experimental reference for clinical application. |
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