Abstract: Objective To construct virulence gene recombinant plasmid vectors in five food-borne pathogenic microorganisms of Escherichia coli O157∶H7, vibrio parahaemolyticus, staphylococcal aureus, salmonella, and shigella, identify their expression products, and explore the feasibility of directly detecting virulence genes of pathogenic microorganism by marking the corresponding antibody with high throughput magnetic fluorescent nanoparticles.Methods Specific virulence genes were selected for primer design from pathogenic islands in five food-borne pathogenic microorganisms, respectively. Firstly, DNA fragments were extracted from five target bacteria and gene fragments were obtained by using PCR and electrophoresis. Then, respective recombinant plasmids, constructed by pET-23a, pET-28a and pET-32a with the gene fragments, were transformed into E?coli DH5α to extract plasmid vectors for enzyme and sequencing identification and transformed into E?coliBL21(DE3). At last, the expression of target proteins were induced by 0.1 mmol/L IPTG and analyzed by SDS-PAGE. Results Recombinant plasmid vectors pET-23a-eaeA, pET-28a-tdh, pET-28a-enterotoxin B, pET-32a-invA and pET-28a-ipaH of E?coli O157:H7, vibrio parahaemolyticus, staphylococcal, salmonella, and shigella were successfully constructed. The eaeA gene fragment of 969 bp, tdh gene fragment of 567 bp, enterotoxin B gene fragment of 798 bp, invA gene fragment of 1 041 bp, and ipaH gene fragment of 771 bp were cloned. The result showed that recombinant plasmid vectors were consistent with target gene sequence. Plasmid expressing proteins 37.5 kDa (eaeA), 26 kDa(tdh), 34.5 kDa (enterotoxin B), 41 kDa (invA), and 32 kDa (ipaH) were detected in E?coliBL21 (DE3) strain. Conclusion The virulence gene recombinant plasmids were successfully constructed in five food-borne pathogenic microorganisms and efficiently expressed in prokaryotic cells, which laid foundation for obtaining corresponding virulence genes of antibodies and diagnostic reagent application.