The Composition of Capsid Proteins and Development of Subunit Vaccine of Waterfowl Parvoviruses

• Main Authors: Chien-Hua Chen, 陳建華
• Other Authors: 張伯俊
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/18858031777148443990

碩士 === 國立中興大學 === 獸醫微生物學研究所 === 94 === Waterfowl parvovirus infection is caused by goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) and might lead to severe economic losses. The conventional method for the detection of waterfowl parvovirus was using polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) or nucleotide sequence analyses to distinct GPV from MDPV. In this study, we designed primers that can directly differentiate GPV from MDPV using a single PCR reaction. Although live vaccine is available for the control of GPV and the vaccine can elicit neutralizing antibody against the virus, it is still impossible to differentiate field-infected animals from vaccinated ones. To enable this, the capsid protein (VP3) and the N-terminal of protein VP1 (VP1N), and nonstructural proteins (NS1) of GPV and MDPV were expressed in E. coli. These whole-cell proteins were used as subunit vaccines to vaccinate adult ducks and sera of vaccinated ducks were used in passive immunization of ducklings. The results showed that these sera were not able to provide any protection against the challenge of MDPV. We then used purified NS1, VP1N, and VP3 of MDPV as subunit vaccines to vaccinate adult ducks and sera of vaccinated ducks were used in passive immunization of ducklings. The results showed that these sera were able to provide protection against the challenge of MDPV. It was found previously by monoclonal antibody that the fourth capsid protein (VP4) with MW of 65 kDa was the most antigenically reactive one and could be a candidate for subunit vaccine. In this study, monoclonal antibody was prepared by recombinant capsid protein VP3 of GPV, and then used in Western blotting assay to investigate the composition of GPV capsid protein. The capsid protein VP1, VP2, VP3 and a protein about 50 kDa were recognized by the monoclonal antibody. The size of the 50 kDa protein is much smaller than the VP4 found in GPV but is closed to the 51 kDa protein found in MDPV. This 50 kDa protein was subjected to LCMSMS analyses and only one peptide sequence conformed to the VP1 at residues 607-617 of GPV and MDPV was found. Moreover another peptide identified by the monoclonal antibody was found to be a part of GPV capsid proteins. It is possible that this 50 kDa protein was part of GPV capsid proteins. Further experiments is necessary to see if the 50 kDa protein could be developed as a subunit vaccine.