Yang Y.,Jilin University |
Yang Y.,The Eleventh Institute of the Academy of Military Medical science of PLA |
Yin J.,Jilin University |
Guo D.,Heilongjiang Bayi Agricultural University |
And 3 more authors.
FEMS Immunology and Medical Microbiology | Year: 2011
Due to drawbacks of live attenuated vaccines, much attention has been focused on screening Brucella-protective antigens as subunit vaccine candidates. Here, an immunoproteomic assay was used to identify the immunogenic soluble proteins of Brucella melitensis 16M. In the present study, 27 unique immunogenic proteins were identified from the two-dimensional electrophoresis immunoblot profiles by liquid chromatography tandem MS (LC-MS/MS). From this set, the gene encoding one immunodominant protein of interest, S-adenosyl-l-homocysteine hydrolase (AdoHcyase), was expressed in Escherichia coli. The recombinant AdoHcyase induced a strong antibody response in BALB/c mice, and the polyclonal antibody could recognize a band of approximately 52kDa in the immunoblots of soluble protein extracts from five Brucella strains. rAdoHcyase significantly stimulated the production of interferon-γ and interleukin-2, and induced a high level of protection against B. melitensis 16M challenge at 4 weeks postchallenge. Our results indicated that rAdoHcyase could be a useful candidate for the development of subunit vaccines against B. melitensis. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.
PubMed | The Eleventh Institute of the Academy of Military Medical science of PLA
Type: Journal Article | Journal: Molecular immunology | Year: 2011
In order to screen immunogenic candidate antigens for the development of a brucellosis subunit vaccine, an immunoproteomic assay was used to identify immunogenic proteins from Brucella melitensis 16 M soluble proteins. In this study, a total of 56 immunodominant proteins were identified from the two-dimensional electrophoresis immunoblot profiles by liquid chromatography tandem mass spectrometry (LC-MS/MS). Two proteins of interest, riboflavin synthase alpha chain (RS-) and Loraine synthase (LS-2), which are both involved in riboflavin synthesis, were detected by two-dimensional immunoblots using antisera obtained from Brucella-infected human and goats. LS-2, however, is an already well-known vaccine candidate. Therefore, we focussed our studies on the novel vaccine candidate RS-. B. melitensis RS- and LS-2 were then expressed in Escherichia coli as fusion proteins with His tag. The humoral and cellular immune responses to the recombinant (r)RS- was characterized. In response to in vitro stimulation by rRS-, splenocytes from mice vaccinated with rRS- were able to produce -interferon (IFN-) and interleukin (IL)-2 but not interleukin (IL)-4 and interleukin (IL)-10. Furthermore, rRS- or rLS-2-vaccinated mice were partially protected against B. melitensis infection. Our results suggested that we have developed a high-throughout, accurate, rapid and highly efficient method for the identification of candidate antigens by a combination of immunoproteomics with immunisation and bacterial challenge and rRs- could be a useful candidate for the development of subunit vaccines against B. melitensis.