Prokaryotic Expression Of Lsiteria Monoyctgoenes Iap, ActA, PlcB Genes, Analysis Of Immunogenicity Of Their Products And Development Of Polyclonal Antibodies Against P60

Objectives: Listeria monocytogenes(LM) is the most important bacterium in Listeriaspp because it can cause severe food-borne infections in humans and animals，It cangrow in the temperature as low as4℃，so LM is one of the most virulent strains inReady-to-eat food and frozen food．The infection of LM involves a variety of virulentsuch as LLO、ActA、plcA/B、p60、PrfA、Internalin and P104.In this paper，we clonedand prokaryoticly expressed p60、 ActA、 plcB from LM， identified theimmunogenicity of these recombient protein，pre pared polyclonal antibody againstp60．To provide basis for the further studies on the development of monoclonalantibody against protein p60of Listeria monocytogenes and the establishment of theimmuno-capture with magnetic beads methods.Methods: Designing the primers of actA，plcB，iap gene by biological software; Thegenes was amplified by PCR according to the templete of LM DNA,and constructedinto cloning vector pMD18-T.The recombinant plasmid was transformed into E.coliDH5α to clone,identified by enzyme digestion and DNA sequencing;Recombinant ofcorrect products and prokaryotic exoression vector PET28a（+）was constructed andtransformed into E.coli BL21then DNA sequencing.The recombinant protein wasexpressed through ITPG induction. Recombinant E.coli BL21were sonicated byultrasonic oscillation, to ananlyse the soluble expression by SDS-PAGE. Therecombinant protein was purified by Ni2+-chelating affinity columnchromatography;identifing the immunogenicity by Western blotting and ELISA. Thepurified recombinant p60was used as antigen for immunization of BALB/c;testingthe binding affinity between polyclonal antibody and LM by ELISA.Results: It was demonstrated by agarose gel electrophoresis and DNA sequencingthat the recombinant cloning and expressing vectors of actA，plcB，iap gene weresuccessfully constructed, getting99%homology in sequence which was published inGene Bank.p60and ActA were solubly expressed.plcB was insoluble Western blottingand ELISA showed that the recombinant protein p60and ActA had immunogenicityfor mice and rabbit.plcB binded well with mice anti-LM serum. The p60antiserum were obtained with a titer as high as1∶8000after the third immunization of miceand had low cross reaction with L.welshimeri and L. innocua.Conclusion:The virulent gene actA，plcB，iap of LM were successfully expressed.Analysis of immunogenity demonstrated that combinant protein p60was binding wellwith both mice and rabbit anti-LM serum.The polyclonal antibodies against p60wasprepared preliminarily, with ideal titre and low cross reaction.