Screening And Preliminary Study Of Staphylococcus Aureus Promoting Agglutination Gene

Objectives:The Tn917 transposon mutant library was constructed by using plasma agglutination reduction method to screen and identify the related genes,and the screening genes were verified.To construct a mutant strain of Staphylococcus aureus quinone cytochrome oxidase A subunit gene deletion,the biological function of Staphylococcus aureus was preliminarily discussed.Methods:1.Using the transposon Tn917 temperature sensitive plasmid was transformed into Rn4220 was modified,and then transferred to Newman standard strains,the erythromycin and high temperature induced transposon in Staphylococcus aureus,Staphylococcus aureus caused by random mutation a transposon mutant library,using the turbidimetric and agglutination screening agglutination ability decreased mutant;application of resistance marker gene and mutation identification method to save the application of bioinformatics prediction of gene function.2.The screened genes were verified by gene knockout technique: gene fusion through PCR connection on the homologous fragment,using site-specific recombination Gateway cloning into plasmid PKOR1 based on temperature sensitivity,transformed into Escherichia coli DC10 B modified defective homologous recombination plasmid and the plasmid into Newman strain,subculture in chloramphenicol and under high temperature,and then screened by reverse tetracycline,using genomic sequencing and PCR identification of mutants.The test tube agglutination technique was used for identification.3.The function of ubiquinone cytochrome oxidase gene was preliminarily explored through bacterial growth curve assay,antioxidation experiment,biofilm formation experiment and animal experiment.Results:1.The transposon mutant library was successfully constructed,and 82 of the mutant strains were screened by observing the decrease of plasma agglutination ability.The transposon insertion sites of 76 mutant strains were identified,including 13 genes,including 4 genes related to agglutination,accounting for 30.8% of the screened genes.2.Knockout of the genes which were significantly decreased and unreported were verified.It was confirmed that the ubiquinone cytochrome A subunit(cydA)mutation could not induce plasma agglutination.3.The growth of cydA gene deletion strain was not significantly affected by aerobic condition,the hemolytic ability of bacteria was significantly weakened,and the antioxidant capacity decreased significantly,but it had no obvious effect on biofilm formation.In the skin abscess model,the ulcer area of the mutant mice was significantly reduced.In the bacteremia model,the survival time of the mutant mice was obviously prolonged.Conclusion:The transposon mutagenesis library constructed in this study can be used for the screening of specific phenotypic related genes of bacteria.Besides the known effect of Staphylococcus aureus on promoting plasma agglutination ability,it is screened and confirmed that the mutation of A subunit of ubiquinone can significantly affect plasma agglutination.The cydA gene plays an indispensable role in the pathogenesis of Staphylococcus aureus and is expected to be the target of the new vaccine.