Rapid verification of the effect of fim family genes on the motility of Acinetobacter baumannii based on homologous recombination / 安徽医科大学学报

ABSTRACT

Objective @#To use linear PCR fragment containing antibiotic resistance cassette to carry out homologous recombination and replacement of target gene fragment of Acinetobacter baumannii to achieve rapid gene knockout and functional verification．@*Methods@#Acinetobacter baumannii Ab4294 was used as the research object，and the upper (901 bp) and lower ( 1 028 bp) reaches of fim gene cluster (4 980 bp in length) were amplified by PCR , which was used as the recombinant homologous arm．Kanamycin antibiotic resistance cassette (KanR) was ampli- fied from pUC57 plasmid．The above three fragments were connected by overlapping extended PCR technique，and the connected fragments were transformed into wild Acinetobacter baumannii strains．The gene deletion mutant was screened，and the plasmid complement strain was constructed．The phenotype of the obtained strains was identi- fied，and the function of fim gene cluster was explored. @*Results @#A mutant strain of Acinetobacter baumannii Ab4294 with deletion of fim gene cluster was successfully constructed by homologous substitution of linear PCR frag- ment containing antibiotic resistance cassette．Compared with the wild strain，the growth curve of the deletion strain had no significant difference ，and the rubbing ability significantly decreased ，and the phenotype recovered after complementing the gene cluster．@*Conclusion @#The fim family genes of Acinetobacter baumannii Ab4294 is success- fully knocked out by homologous substitution of linear PCR fragment containing antibiotic resistance cassette，which encodes the product involved in the motile movement of Acinetobacter baumannii.