Abstract： Objective To construct a prokaryotic cloning vector of invasion- associated protein (iap) gene p60 of Listeria monocytogenes. Methods Under gradient PCR amplification conditions, Listeria monocytogenes 54002-4 strain iap gene was amplified with our in-house primers. BamH Ⅰ and Xho Ⅰ sites were induced to 5' and 3' terminals of iap gene, respectively. PCR products were then analyzed and collected by agarose gel electrophoresis. Purified PCR product was connected with the vector pMD18-T. The recombinant plasmid was transformed into Escherichia coli JM109 competent cells, with transformation effect observed after 1mmol/L IPTG induction for 4-6 h. Results Colorless bacterial colony was cultured and verified to produce positive recombinant plasmid pMD18-T-Iap by PCR and nucleotide sequencing. Conclusions PCR optimization conditions and methods are established through gradient PCR. Iap gene cloning vector is obtained after transformation.