Using a Relative Quantitative Proteomic Method to Identify Differentially Abundant Proteins in Brucella melitensis Biovar 3 and Brucella melitensis M5-90.

Brucellosis, caused by Brucella spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An investigation into the differential proteome expression patterns of wild and vaccine strains may help researchers and clinicians differentiate between the strains to diagnose and better understand the mechanism(s) underlying differences in virulence. In the present study, a mass spectrometry-based, label-free relative quantitative proteomics approach was used to investigate the proteins expressed by the wild strain, B. melitensis biovar 3 and compare it with those expressed by B. melitensis M5-90. The higher level of virulence for B. melitensis biovar 3 compared to B. melitensis M5-90 was validated in vitro and in vivo. A total of 2133 proteins, encompassing 68% of the theoretical proteome, were identified and quantified by proteomic analysis, resulting in broad coverage of the B. melitensis proteome. A total of 147 proteins were identified as differentially expressed (DE) between these two strains. In addition, 9 proteins and 30 proteins were identified as unique to B. melitensis M5-90 and B. melitensis biovar 3, respectively. Pathway analysis revealed that the majority of the DE proteins were involved in iron uptake, quorum sensing, pyrimidine metabolism, glycine betaine biosynthetic and metabolic processes, thiamine-containing compound metabolism and ABC transporters. The expression of BtpA and VjbR proteins (two well-known virulence factors) in B. melitensis biovar 3 was 8-fold and 2-fold higher than in B. melitensis M5-90. In summary, our results identified many unique proteins that could be selected as candidate markers for differentiating vaccinated animals from animals with wild-type infections. BtpA and VjbR proteins might be responsible for the residual virulence of B. melitensis M5-90, while ABC transporters and thiamine metabolism associated proteins may be newly identified Brucella virulence factors. All of the identified DE proteins provide valuable information for the development of vaccines and the discovery of novel therapeutic targets.

[Characterization of a recombinant goatpox virus expressing Orfv F1L gene].

OBJECTIVE: In order to establish the vaccine against the contagious ecthyma, we constructed and characterized recombinant goatpox virus expressing F1L protein of Orf virus. METHODS: The F1L gene was amplified and cloned into the vector pUC-TK12 carrying the LacZ gene and a bidirectional promoter. With the help of lipidosome, the recombinant plasmid pTL-F1L was transfected into the BHK-21 cells, which had been infected by Gpv. The aim is to make the Gpv and pTL-F1L recombined randomly and get the recombinant virus, which was defined as rGpv-F1L. The rGpv-F1L was screened by blue plaque, and then the F1L recombination and translation were identified by PCR, indirect immunofluorescence and Western blot. By the means of TCID50, we evaluated the physicochemical properties of rGpv-F1L. Female mice were immunized with the rGpv-F1L, and the specific antibodies levels in serum were detected by ELISA. RESULTS: We obtained rGpv-F1L, which was stably expressing F1L protein. The results of biological characteristics showed the rGpv-F1L was sensitive to acids, alkalis, organic solvents and ultraviolet. The activity of specific antibodies significantly increased in mice infected by rGpv-F1L more than Gpv (P < 0.01). CONCLUSION: In this research, we have successfully obtained the candidate vaccine, which is stably expressing F1L of Orf virus. Thereby the candidate vaccine with excellent antigenicity and biological activity provides new avenues for the prevention of contagious ecthyma and capripox.