Expression And Function Of Aap Gene Of Staphylococcus Epidermidis Isolated From Southern Xinjiang

Staphylococcus epidermidis is one of the important pathogenic bacteria of recessive dairycow mastitis and its pathogenicity is mainly through the biofilm formation. At present, biofilmformation by staphylococcus epidermidis mainly depends on extracellular polysaccharideadhesions, extracellular DNA and related accumulation-associated protein. The Aap protein is animportant protein adhesion molecule in staphylococcus epidermidis cell wall surface, about90%of the staphylococcus epidermidis contains aap gene, which expression is associated with biofilmformation. So the aap gene can be looks as important targets of biofilm formation bystaphylococcus epidermidis, this will bring the control and prevention of cow mastitis disease andeffective treatment plan or provide theoretical guidance.First of all, look staphylococcus epidermidis as the research object, which harm the healthydevelopment of dairy farming in Xin jiang, PCR amplification the aap characteristic gene anddetect the biofilm formation ability. Characteristic gene segments of aap are expected about486bp, throughe PCR amplification,15strains of staphylococcus epidermidis strains were containingthe aap gene, accounted for26.3%of all strains. Use a semi-quantitive microtiter plate methodreceived of10strains of biofilm formation positive strains, and accounted for18.2%of all strains,50%of the biofilm positive strains are aap positive (aap+) strains, and the ability of biofilmformation is generally higher than the aap negative (aap-) strains. On the basis of the aboveexperiment screened biofilm positive, aap positive (aap+BF+) and biofilm positive, aap negative(BF+aap) strain, at the same time, for the three main component of biofilm, test the BF+aap+andBF+aap-strains biofilm formation dependent types, the results show that the biofilm formation bystaphylococcus epidermidis were contain extracellular DNA, protein, and polysaccharide, whichrelated to biofilm formation by staphylococcus epidermidis in this region and its inhibition ratewere99.6%,82.6%and99.6%respectively. Protein, extracellular DNA and polysaccharide are thethe important components in biofilm of cow breast inflammatory caused by staphylococcus epidermidis. Therefore this study base on the three components in staphylococcus epidermidisbiofilm in this region and carried on the thorough research and exploration.Secondly, in order to further reserch the aap gene’s function, construct the prokaryoticexpression vector and induced expression of aap gene: aap gene were obtained through the PCRamplification of clinical isolates staphylococcus epidermidis, application of gene recombinationtechnology to construct prokaryotic expression vector pET32a (+)-aap, after transformation to thehost bacterium DH5a, transformation to Ecoli BL21again, and carry on induce expression ofrecombinant proteins. The results showed that the0.75mmol/L IPTG induction for4h can obtainthe highest expression of recombinant proteins. Recombinant protein molecular weight is about180KDa, consistent with the expected size. Using nickel ion affinity chromatography topurification Aap protein. Found that when the concentration of imidazole is40mM in washingbuffer and200mM in the elution buffer can achieve the purpose of protein. In order to furtherverify the accuracy of the protein, Use Western blot, because in the aap genetic prokaryoticexpression vector respectively introduces the histidine labels at the upstream and downstream, inabout180KDa, Aap protein can effectively combine with histidine label antibody, proved theaccuracy of the protein.Finally, by semi-quantitative method to detect the influence on biofilm formation by Aapprotein, and based on the three components of biofilm tested its effect on the biofilm formation byAap protein respectively. The experiment proves the Aap protein have good inhibitory to standardstrains and clinical strains biofilm formation, and its inhibition began at the initial adhesion period,when its concentration achieve1ug/mL or0.4ug/mL can completely suppress ATCC35984andclinical strains biofilm formation. ATCC35984genome DNA processing by Aap protein told thatAap protein inhibition ATCC35984biofilm formation not by degradation its eDNA. By TLCanalysis the whole-cell polysaccharide and extracellular polysaccharide components, told Aapprotein has no effect on the polysaccharide production type. Fluorescent tags the Aap protein, andobserve under the microscope, find tagged Aap protein contact with cells, and wrap around it,making it appear fluorescent green. By the flow cytometry instrument statistics found that with the increase of Aap protein, fluorescence intensity enhance, the number of positive cells which werestaining by FITC increased gradually.When the concentration of Aap protein achieve1.0ug/mLATCC35984cells tinctorial yield increased by95.91%than the control group, which can bespeculated that Aap proteins can inhibition of the biofilm formation by package the ATCC35984cell.Above all, Aap protein has a strong inhibitory to biofilm, so the aap gene can looks as apotential candidate gene to inhibit the cow breast inflammatory caused by staphylococcusepidermidis biofilm infection drugs, can be used as bio-control of dairy mastitis disease treatmentand help to further research, at the same time, this study in order to further study the function ofaap gene may provide the theoretical basis.