| Canine parvovirus (CPV) is a member of the feline parvovirus (FPV) subgroup and is classified into autonomous parvoviruses of the family Parvoviridae (genus parvovirus, subfamily parvovirinae). CPV is an important pathogen in domestic dogs and some wild carnivore animals. It causes an acute hemorrhagic gastroenteritis, leucopenia, nausea and occasionally fatal myocarditis in young puppies. At early 1967,We got a minute virus of canine (MVC)isolate and named CPV-1.Then America scholar Eugster and Nairn found CPV from Canine feces, which is very different from CPV-1.That CPV is we common called CPV-2.After the initial appearance in 1978,antigenic drift was continuously observed. CPV-2 spread worldwide within a few years after that (during 1978–1981) and then the original type 2 was replaced by new genetic and antigenic variants, type 2a (CPV-2a), type 2b (CPV-2b) and type 2c(CPV-2c).This study is aimed to develop the epidemic and variation of CPV virus. Through viral isolation from the a case of CPV disease in pets clinic hospital in NanJing, we obtained a isolation which was designated CPV through a series of systematic identification assays, such as morphological, physicochemical, biological and molecular virology assay. Virion is cubical symmetry, about 20-24 nm in diameter, without envelope. This strain can grow in CRFK cell line and produce cytopathic effects with the characteristics of CPV, swelling, rounding, broken, droping. The strain resist the chloroform treatment and are stable in pH3.0 and 56℃, the viruse can agglutinate RBC of pig, but not of human, chicken, guinea pig. Which highest hemagglutinin is 128, and could be neutralized by the positive CPV serum that preserved before. Also, it was confirmed by PCR amplification by special primer of CPV. What is more, the isolation virus can infect canine. The CPV isolation named CPV/NJ01/06.According to the CPV sequences from GenBank,We design two pairs of primes to amplify the 5'and 3'of the CPV/NJ01/06 genome. which covering the two ORFs of CPV in the whole of 4600 bp.Analysis of hydrophobicity and antigenicity of CPV's nonstructure protein NS1 and capsids VP2, We found that there was N terminal and C terminal hydrophobicity in NS1 segment, Beside It was in the main form ofαhelix, In the another side ,2 segments were high-hydrophobicity regions in VP2 protein of CPV/NJ01/06; one could be a potential transmembrane domain at the inner of VP2; the other could be a potential signal peptide at the N terminal. Refering to some another CPV VP2 seqences; we construct a phylogenetic tress of typical CPV VP2 nucleotides of the world. Comparing to the far distant of CPV isolations from The USA, New Zealand, There is evidently that CPV/nj01/06 is close to the CPV isolations from China, Vietnam, Taiwan (Province) and Japan.Besides, CPV/nj01/06 belongs to CPV-2a by contrasting CPV amino acid sequences in key position.A pair of specific primers is synthesized according to the CPV VP2 sequence published by Genbank. The fragment is amplified by PCR under the template CPV/nj01/06, then cloned into the expression vector pGEX-6p-1, and transformed into component bacteria BL21 (DE3). SDS-PAGE analysis showed that the recombinant protein had a molecular weight of approximate right after IPTG induction, the fuse-protein harvested mostly at the condition of 1.0mmol/L IPTG, 37℃,6h induction. Total proteins collected from the lysate by sonicated after centrifugation, and was in the form as insoluble fraction. Western blot analysis showed that the recombinant protein have constructed in the right way, which exited low molecular weight protein. Both proteins have CPV virus antigen. |
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