The Study On The Virulence Factors Phenotype And The Genetype Of Staphyloccocus Aureus And Which Contribute To Apoptosis In Mammary Epithelial Cells

The experiments of Shandong bovine mastitis detection,we separated129 strains of staphylococcus aureus.We design related primers according to staphylococcus aureus virulence factor,then test thermostable nulear , coagulase,Alphatoxin,Betatoxin,mec-a, qac-a ,pvl gene with the method of PCR. We attack the mammary epithelial cells with alpha hemolysin,then we tested IL2,IL6,TNF-α,to explore the injuries and the influence of cell apoptosis caused by alpha hemolysin.Hope our work can made a system identification genetype,get a molecular epidemiology material of S. aureus for bovine Mastitis aureaus.1. Staphylococcus aureus various of virulence factors genetype detectionDesign related primers according to staphylococcus aureus virulence factors,then test thermostable nuclease,coagulase, Alphatoxin,Betatoxin,mec-a, qac-a ,pvl gene with the method of PCR. The typing method for Staphylococccal hemolysin phenotype and genes with PCR was developed.The isolates withα- hemolysis was 56 in 129 isolates , account for 43.4%;43 showed beta-hemolysis , account for 34.11%; Another 29 isolates showed no hemolysis phenotype, and it account for 22.48%. The isolates with hla gene account for 34.88%, hlb gene account for 42.60%. Strains with Pvl genes found 46, it has a detectiong rate of 35.66%; coa has a detectiong rate of 30.23%; Nuc has a detectiong rate of 24.03%; mec-a has a detectiong rate of 19.38%; qac-a has a detectiong rate of 10.08%.In the hemolytic phenotype ,α- hemolysis are more than beta-hemolysis;however the hlb gene detection rates is higher than the hla gene. The distribution of hemolysin phenotype and hemolysin genes is not a corresponding relationship of Staphylococcus aureus isolates from raw milk of cows . Found 46 strains with Pvl gene, a detection rate of 35.66% with no significant differences with Staphylococcus aureus get from human.Coagulase gene has a detectiong rate of 30.23%, consistent with various reports. Nuc gene has a detectiong rate of 24.03%, it can be used as a added method detection of pathogenic aureaus. We found 13 strains with mec-a gene, this shows in shandong have appeared MRSA strains and there are antibiotics strains the phenomenon of abuse and disorderly use. Found 13 strains with qac-a gene,this shows some of the cattles in shandong province exist the the disinfectant nonstandard use in dose and various disinfectant.Test the virulence factors with the method of PCR, ,the test result is significant. Also should noticed the PCR detection results and biochemical test results is not always a corresponding relationship of Staphylococcus aureus.Some gene have a high detection rate, may be the gene was silent, causing the corresponding proteins do not express.But for the higher gene detection rate than biochemical phenotype detection, we shound find the cause,increasing the number of testing.2. Study the injuries and apoptosis in Mammary epithelial cells by alphahemolysin of Staphyloccocus aureus.Alphahemolysin was expressed and purificated from gene engineering strain, then attacked the mice mammary epithelial cell.In order to find the differences of various genetype of Staphyloccocus aureus.Alphahemolysin we obtained concentration was 940μg/ml.In the mammary epithelial cells experiment,we used it to attacked the cell,then we tested the IL2,IL6,TNF-α.The experimental group and matched group of TNF-α.First,TNF-αincreased ,and then they declined, 3 groups is the higherst, 5 groups have a rising trend. The experimental group and matched group of IL2 and IL6 also shows a first high and secong low, 3 groups is the higherst. The IL2,IL6 and TNF-α.in 3 groups presented in a statistical significance for comparison with the blank control challenged group.Compared with the control group, found that alpha hemolysin can cause some certain damage to the mice mammary epithelial cell.In the experiment we hope we can established a genetype technical platform of S.aureaus, provide control data for the injury by various different type aureaus.Made a preliminary studies of the damage and apoptosis effect in the mammary epithelial cell by alpha hemolysin of staphylococcus aureus. Provided the data support for the research of cows mammary epithelial cell damage mechanism by alpha hemolysin.