Antibodies produced in animals vaccinated using live attenuated vaccines against Brucella spp. are indistinguishable using current conventional serological tests from those produced in infected animals. One potential approach is to develop marker vaccines in which specific genes have been deleted from parental vaccine strains that show good immunogenicity and vaccine efficacy. Corresponding methods of detection for antibodies raised by the marker vaccine should also be developed. A specific fragment of the bp26 gene of Brucella melitensis M5-90 was cloned into vector pQE32 to construct the recombinant plasmid (pQE32-rΔbp26). It was used to transform Escherichia coli M15 (pREP4) host cells, which expressed the rΔbp26 protein. Subsequently, the recombinant protein was purified by immobilized metal affinity chromatography and size-exclusion chromatography. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified rΔbp26 protein was represented by only one band, with a molecular weight of 14 kDa, and it showed good antigenic specificity on western blot and enzyme-linked immunosorbent assay (ELISA). The purified rΔbp26 protein was intended to be used as an antigen to develop a novel ELISA to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains.

Background: Transmissible gastroenteritis virus (TGEV) could cause swine enteric infection, which is characterized by vomiting, severe diarrhea and dehydration, and the mortality in suckling piglets is often high up to 100%. Vaccination is an effective measure to control the disease caused by TGEV.Methods: In this study, cell-cultured TGEV HN-2012 strain was inactivated by formaldehyde (FA), β-propiolactone (BPL) or binaryethylenimine (BEI), respectively. Then the inactivated TGEV vaccine was prepared with freund's adjuvant, and the immunization effects were evaluated in mice. The TGEV-specific IgG level was detected by ELISA.Results: The results showed that the FA group (n=17) developed earlier and stronger TGEV-specific IgG level, while the BEI group could produce much longer-term IgG level. Lymphocyte proliferation test demonstrated that the BEI group showed a stronger inducibility of spleen lymphocyte proliferation. The BEI group got higher positive rates of CD4+ and CD8+ T lymphocyte subsets of peripheral blood lymphocyte than that of the FA and BPL groups through flow cytometry assay. The BPL group had the highest positive rate of CD4+IFN-γ+ T lymphocyte subset, while the positive rate of CD4+IL-4+ T lymphocyte subset was got the best in FA group. Moreover, there were no obvious pathological changes between the vaccinated mice and control group by the macroscopic and histopathological examination.Conclusions: These results indicated that the three experimental groups both induced cellular and humoral immunities, and the FA group had better effect on humoral immunity, while the BEI group showed its excellent effect on cellular immunity.