Bioinformatics analysis of Brucella secretory protein BspC,preparation of polyclonal antibody and screening of interacting protein
Objective To investigate the mechanism of BspC protein in Brucella infection.Methods The biological function of BspC protein was analyzed by online prediction software.pET-28a-BspC prokaryotic fusion expression vector was constructed by chemical synthesis method and transformed into Escherichia coli BL2 receptor cells.BspC protein was expressed and purified by IPTG induction.The purified protein was used for animal immunization and antiserum was collected.The antiserum titer was detected by ELISA.Antibodies were purified and identified by Western blot.The cDNA library of RAW264.7 cells and the decoy plasmid of BspC protein were constructed,and the self-activation and toxicity of the decoy plasmid were detected,and the host proteins interacting with BspC were screened,and the interacting proteins were verified by backcross verification experiment.Results The BspC protein had signal peptide and 6 antigenic determinant,and the secondary structure was mainly α-helix.The pET-28a-BspC vector was successfully constructed,and the size of the recombinant protein was about 16.7 ku after induced expression.The titer of rabbit antiserum was 1∶512 000 by ELISA.Western blot analysis showed that the prepared polyclonal antibody could bind specifically to BspC protein expressed in vitro.Seven positive clones were sequenced by NCBI BLAST,and potential interacting host proteins FDX1,DNAJA1,HSPA5 and PTPN2 were obtained.Conclusion This study successfully prepared BspC polyclonal antibody and screened the interaction protein of BspC protein,which laid a foundation for further study on the function and mechanism of BspC protein during Brucella infection.
Brucellasecretory protein BspCbioinformatics analysispolyclonal antibodyyeast two-hybridinteracting protein
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