Cloning,expression and antisera preparation of internalin a from Listeria monocytogenesis

Objective To clone and express protein InlA from Listeria monocytogenes and prepare the specific antibodies against Listeria monocytogenes.Method InlA gene of Listeria monocytogenesis serotype 4b was amplified by PCR technology,plasmid pGEX-4T-Li was constructed and expressed by molecular cloning method.Fusion protein InlA was purified by affinity chromatography and correspondingly,the antiserum was prepared by immunization.Result Fusion protein InlA with molecular weight of 110 kD was expressed,the titre of prepared antiserum was 1∶1600.Three InlA MAb clones were obtained with a titer as high as 1∶1×105 ~1∶3×105 in ascities.The antibodies had no cross reaction with other pathogenic microorganisms.Conclusion Expression of the specific antigen from Listeria monocytogenes e.g.InlA and preparation of the polyclonal antibodies was a valid method to eliminate the cross-reaction from other species,and thus was beneficial to improve the specificity for the detection of Listeria monocytogenesis.