Preparation and characterization of monoclonal antibodies against cytolethal distending toxin protein of Campylobacter jejuni

Abstract:

Abstract:［Objective］To express Campylobacter jejuni cytolethal distending toxin B protein (CdtB) in a prokaryote to prepare monoclonal antibodies (mAbs) against the protein，and to study their antitoxic effects.［Methods］The C．jejuni cdtB gene was amplified and inserted into the expression plasmids pET-30a(+) and pGEX-6p-1．The purified rGST-CdtB protein was used as the immunogen to screen hybridoma cells for mAbs against the protein．The mAb titers were determined with an indirect enzyme-linked immunosorbent assay (ELISA)，and their specificity with a Dot-ELISA and western blotting analysis．We determined the antitoxic properties of the mAbs in CaCo-2 and HD-11 cells．［Results］Recombinant expression plasmids pET-30a (+)-cdtB and pGEX-6p-1-cdtB were successfully constructed，and fusion proteins rHis-CdtB and rGST-CdtB expressed，respectively．Five hybridoma cell lines，designated 1F3，1F5，2E4，2E11，and 2F2，were screened for the stable secretion of mAbs against CdtB． The immunoglobulin subclass of 2E11 was IgG2b and that of the other mAbs was IgG1．The mAb titers in the ascites fluids were 1:1 108 on indirect ELISA．Dot-ELISA demonstrated that the five mAbs reacted specifically with C．jejuni．Western blotting analysis confirmed that the five mAbs reacted well with the rGST-CdtB fusion protein．The mAbs significantly reduced the adhesion and invasion capacities of the bacterium in CaCo-2 cells (P＜0.01)．［Conclusion］The successful preparation of five mAbs specific for the CdtB protein will allow further study of the biological characteristics of CdtB and the pathogenesis of C．jejuni．