Preparation And Identification Of Polyclonal Antibody For VP2 Structure Protein Of MVC Bocavirus

Object Minute Virus of canine(MVC) is one member of Boca virus family in the family Parvoviridae.The VP2 protein as a structural protein of bocavirus,which is the main component to consist of capsid, it is an major immunogenic antigen in tiny virus which has coagulant activity, and it is also the key influence factor to decides host range and interreaction between host and virus. Therefore, it is good contribution to further research infection function of VP2 in MVC and reveal the molecular mechanisms of infection and pateogenes is in MVC by constructing prokaryotic expression vector of VP2 protein in MVC and preparing polyclonal antibody of VP2.Method In the previous work,the research group has successfully gotten the infectious MVC clone vector p IMVC,and VP2 gene segment by PCR technology to construct the prokaryotic expression vector p ET-32a(+)-VP2. After PCR, the gene sequencing is identified, recombinant plasmid is transformed into E.coli by IPTG induction expression. The group immunize New Zealand rabbits with purified protein and detect antibody titer and its specificity. The antibody titer is detected by ELISA. By western blotting and immunofluorescence staining, antibody specificity is identified after MVC infect Walter Reed Dog(WRD).Result PCR and sequencing identification proved that the group has successfully constructed the prokaryotic expression vector p ET-32a(+)-VP2. After transformation and IPTG induction, recombinant plasmid successfully expressed a target protein band whose relative molecular weight(Mr) is about 39 k D. The result agreed with expectations. ELISA method examination shows that the titer of polyclonal antibody is 1∶200 000.Conclusion pET32a(+)-VP2 is successfully constructed, which is VP2 prokaryotic expression vector of MVC, and its corresponding soluble protein is expressed. The high effectVP2 polyclonal antibody is obtained, which has high specificity, which laid a foundation for further research of VP2 gene’s function in MVC.