Study On Cloning And Expression Of VP2 Gene Of Canine Parvovirus And Antigenicity Of Its Recombinant Protein

Canine parvovirus (CPV) infection is a kind of serious contagious transmission disease, which is caused by CPV and spreads very rapidly. And its rate of infection and death are also quite high. So it caused big damage in dogs, economic animals and wild animals. Nowadays, there is no remedial method for its infection. Clinically, preventive measures against CPV are mainly dependent on attenuated vaccine, however, there are some certain negative problems existing. Therefore, we need develop a new safe and effective vaccine against CPV. There were major epitopes in VP2 protein of CPV, so scientists are interested in VP2 protein. The contents of this study includes four parts as follows:1. Development and application of nested PCR for the detection of CPVTotal DNAs were extracted from stools of dogs with suspected CPV infection clinically. According to the published sequence of CPV from GenBank, two pairs of nested PCR primers were designed based on conserved VP2 gene. In compare to standard sequences published in GenBank, sequence analysis showed that their homology were 99.3～100%. In addition, the results showed nested PCR had high sensitivity, stability and reproducibility, which could be considered as a method for diagnosis and molecular epidemiological studies of CPV.2. Cloning and sequence analysis of VP2 gene of CPVBased on CPV genome published in GenBank, specific primers were designed to amplify VP2 gene by PCR from positive samples identified by nested PCR. The sequencing results showed the length of VP2 gene generated in this study was 1755bp, in comparison to foreign and Chinese strains using DNAStar software, its homology was from 98.4% to 99.8%. It indicated we got the candidate gene of CPV vaccine.3. Study on prokaryotic expression and immunological activity of VP2 of CPVRecombinant prokaryotic expression plasmid (pGEX-4T-VP2) was successfully constructed. Expressed protein induced using IPTG was analyzed by SDS-PAGE and Western-blotting. The results showed that the 90kDa-size fusion protein was recognized and had good reactogenicity.4. Study on the optimation prokaryotic expression condition, protein purification and immunological activity of VP2 of CPVIn order to get the best express condition of VP2 gene in E.coli identified by SDS-PAGE, the induction temperature and time and the inducer concentration were optimized. Moreover, the GST-VP2 fusion protein was purified by Glutathione Sepharose 4B affinity column.Mus musculus albus were injected using purified protein through vena caudalis, ELISA results showed the antiserum titers of GST-VP2 fusion protein gradually became higher with the time of immune. The above data indicated that VP2 recombinant protein had good immunological activity and could be considered as a candidate of CPV genetic engineering subunit vaccine.