Profiling Antibody Resposes Against Brucella Melitensis By Protein Microarray

Brucellosis is one of the most serious human zoonoses, which is caused by the facultative bacteria Brucella. Vaccination and accurate diagnosis play important roles in prevention and control of brucellosis. However, for the present, the related researches are focused on only a few antigens. There should be more unknown antigens that can be used in diagnosis and vaccine development. Many outer membrane proteins, secreted proteins and virulence-related proteins can induce immune response and antibody. Therefore they are usually be used as target antigens. Fruits of genomics and proteomics research facilitate our researches on these proteins, and a new research strategy appeared:finding candidate antigens by analyzing genome sequence and proteome. In the present study, we chose proteins according to our previous study about outer membrane and secretion proteome, and virulence proteins found in literature. These proteins were expressed, purified and used to fabricate protein microarray, which is then used to analyze their antibody profiles, finding the candidate proteins for diagnosis and vaccine development.According to the characteristics of diagnostic and protective antigens, outer membrane and serected proteins were chosen for candidate antigen screening. According to the results of secretion proteome and outer membrane proteome,91 proteins were selected.64 virulence genes were chosen according to related literatures. Flagellar proteins are important antigens. Brucella does not have flagella, however, it have flagella related genes. Therefore, in the present study,29 flagellar genes were selected. With those proteins expressed previously in our lab, a total of 207 proteins were selected for in vitro expression and antigenecity analysis.With the sequence of the selected genes, specific primers were designed, with attB linker sequences were added at their 5'terminus. In addition, a pair of universal primers attB1 and attB2, which was used to add the attB sequence, was designed. Products of the target genes were PCR amplified with specific primers in the first round, which was then used as template to amplify with universal primer to add the recombination sequences. Of the 207 genes,176 were successfully amplified. The PCR products were purified and cloned into enry plasmid by BP reaction.156 genes were cloned into entry clones, which were confirmed by DNA sequencing. By comparing expression of 4 proteins in 3 expression plasmid, plasmid pHXGWA was chosen for subsequent protein expression. Entry plasmids were isolated and LR cloned into expression plasmid, generating expression clones. Expression analysis showed that 123 genes were successfully expressed.The purified proteins and IgG of different animal species were used to print aldehyde modified glass slides to make protein microarray. By optimizing the blocking buffers, concentration of antibody and second antibody, the reation condition of protein microarray was defined. Then, the sera from rabbit immunized with different methods, mouse and that of patients were analyzed by the microarray. These proteins showed different profiles among these sera. A total of 37 proteins showed antibodies in one or more of these sera, and 18 of them was the newly identified antigen. Of these proteins induced antibodies, several of them could be chosen as candidate ones for diagnosis and vaccine research.