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| Bioinformatic Analysis and Immunogenic Study of Brucella ManB Protein |
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Abstract Brucella is a Gram-negative facultative intracellular bacterium that is the causative agent of various animal and human brucellosis. The manB gene of Brucella, which encodes mannose phosphate mutase, is an important component of Brucella virulence factor. In order to analyze the biological function and immunogenicity of ManB protein of lipopolysaccharide, this study constructed the prokaryotic expression vector of Mannose phosphate mutase manb gene and predicted the bioinformatics of ManB protein and identified its immunogenicity. The structure and function of ManB protein was analyzed by bioinformatic tools. The manB gene of Brucella was amplified by PCR and ligated with pET-28a expression vector and then transformed into Escherichia coli BL21 (DE3). The expression of target protein was detected by SDS-PAGE and its reaction was detected by Western blot. Then the immunogenicity was analyzed by mouse immunoassay. Bioinformatics analysis showed the protein had no transmembrane domain and had no signal peptide. Its secondary structure was mainly α-helix. The three-dimensional structure of the protein was constructed by Phyre2 Server. SDS-PAGE analysis showed the fusion protein was about 56.3 kD. Western blot showed the protein could react with sheep serum against Brucella, and indicated it possessed reactogenicity. Indirect ELISA results showed that the mice (Mus musculus) in the experimental group produced antibodies in 7th, 14th and 21st d after immunization. The antibody level increased with time and reached the peak in 21st d. The ManB fusion protein had good reactogenicity, immunized mice were able to induce high level of antibody, and had good immunogenicity, which could provide new materials for the development of new vaccine of Brucella in the future.
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Received: 15 May 2017
Published: 04 February 2018
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