[Preparation and characterization of monoclonal antibodies against FlhF protein of Campylobacter jejuni].

OBJECTIVE

To develop a prokaryotic expression system for Campylobacrer jejuni FlhF protein and prepare monoclonal antibodies(mAb) against this protein.

METHODS

The C. jejuni flhF gene was amplified and inserted into the expression plasmids, pET-30a(+) and pGEX-6p-1. Then the rHis-FlhF protein and rGST-FlhF protein were expressed and purified by affinity column chromatography. They were subsequently used as immunogen and detecting antigen to screen antibodies produced by hybridoma cells against these proteins. The titers of the mAbs were measured by indirect ELISA, and the specificities of the mAbs was evaluated by Dot-ELISA and Western blot analysis.

RESULTS

The recombinant expression plasmids pET-30a(+)-flhF and pGEX-6p-1-flhF were successfully constructed, and the fusion proteins rHis-FlhF and rGST-FlhF were produced at sufficient quantities. Two hybridoma cell lines were screened, designated 2C12 and 7A9, which secrete mAbs against FlhF stably. Their immunoglobulin subclasses were both IgG1. The titers of the ascites fluid were 1×10(9); and 2×10(7); respectively. Western blot analyses confirmed that the two mAbs both reacted with the rHis-FlhF fusion protein with good sensitivity. The Dot-ELISA results demonstrated that the two mAbs reacted specifically with Campylobacrer jejuni.

CONCLUSION

The mAbs against Campylobacrer jejuni FlhF protein with high specificity were successfully prepared.