Cloning and expression of an oiter membrane protein gene in Riemerella anatipestifer and immunity of the recombinant protein in ducklings

• Main Authors: Yen-Hua Liao, 廖彥華
• Other Authors: Chao-Fu Chang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/85338211313700765197

碩士 === 國立臺灣大學 === 獸醫學研究所 === 89 === Riemerella anatipestifer (RA) is the causative agent of infectious serositis in water fowl. As a serious disease in the world, RA infection causes severe losses in waterfowl industries. There are 21 serovars which have little or no cross-protection. The development of an effective vaccine against RA infection is therefore urgently needed. In this study, the purpose is to clone, express and characterize the protective antigen genes of RA for a subunit vaccine development. A 0.7 kb open reading frame was a target gene, which has the same characteristic as outer membrane protein by amino acid sequence analysis. A pair of primers were designed from 0.7 kb open reading frame and it amplified product was 200 bp. After having used this primers to react with the 19 different RA strains, it was found that this gene exists in all of these RA serovars. This gene was ligated with pET32a express vector, and this recombinant vector was transfered into BL21(DE3) E. coli. A 52 kDa recombinant fusion protein was over-expressed with 1mM IPTG induction. With Western blotting in RA recovery duck serum, this recombinant fusion protein was analyzed. The finding was that the recovery serum reacted strongly to recombinant fusion protein. This suggested that the recombinant fusion protein has antigenicity. After being immunized with the recombinant fusion protein twice, the ducks were challenged with serovar 2 live RA (RA 27) via artificial inoculation. The mortalities of immunized groups and control groups were 83% (10/12) and 66% (8/12) respectively. The recombinant protein thus demonstrated no protective effect. We therefore confirm that this recombinant fusion protein is unable to protect the ducks from the RA infection.