Expression and purification of InternalinA from Listeria monocytogenes and preparation rabbit polyclonal antibody against InternalinA

Abstract:

[Objective] To clone and express protein InternalinA (InlA) from Listeria monocytogenes (Lm) and prepare the polyclonal antibody against InlA. To provide basis for preparing the immunomagnetic beads which separated Lm efficiently and specifically. [Methods] Use biological software to design the primers of inlA gene, amplify the inlA gene from Lm by PCR and construct the gene into prokaryotic expression vector PET28a(+), then transform the gene into Escherichia coli BL21 and express optimally; purify the recombinant product InlA by nickel affinity chromatography, identify the recombinant protein by mass spectrometry analysis, and test the immunogenicity by ELISA. The purified protein is used as antigen for immunization of rabbit to prepare polyclonal antibody and the binding affinity between polyclonal antibody and Lm is determined. [Results] The recombinant InlA protein with relatively molecular mass of 92 kD was over-expressed in E.coli BL21. The InlA antiserum were obtained with a titer as high as 1:100 000. The antibodies had no cross reaction with other pathogenic microorganisms except Staphylococcus aureus. Immunofluorescent staining showed that it only binds to the cell surface of Lm but not L. welshimeri. [Conclusion] The polyclonal antibodies which binds to the cell surface of Lm specifically are prepared successfully. These results would provide basis for preparing the immunomagnetic beads which separated Lm efficiently and specifically.