Objective      To construct Burkholderia pseudomallei (B. pseudomallei) BPSL1549 gene knockout mutant strain

and establish efficient B. pseudomallei virulence gene knockout platform.       Methods      Primers were designed to amplify upstream and downstream homologous arms of BPSL1549 gene

and then the arms were linked to pK18mobSacB suicide plasmid. The recombinant suicide plasmid was introduced into B. pseudomallei BPC006 from Escherichia coli S17-1λpir by conjugation. BPSL1549 gene in wild strain was knocked out by homologous recombination

and the target gene knockout mutant strain was selected by sucrose screening. PCR and Western blotting were used to identify BPSL1549 gene knockout mutant strain. Mouse model was established to evaluate the phenotypic variation of the mutant strain.       Results      BPSL1549 gene homologous arm fragments of B. pseudomallei mutant strain (△BPSL1549) had a lack of 600 bp compared with the wild strain. Western blotting showed that the mutant strain did not express BPSL1549 proteins

indicating that B. pseudomallei BPSL1549 gene knockout mutant strain was successfully constructed. In the animal experiments

the virulence of the mutant strain was significantly lower than that of the wild strain (P0.05).       Conclusion      B. pseudomallei BPSL1549 gene knockout mutant strain is successfully constructed by homologous recombination

which improves B. pseudomallei gene knockout platform and evaluation system.