Cloning, Expression And Bioactivity Detection Of The Virulence Factor Of Staphylococcus Aureus In Dairy Cattle Mastitis

The animals collected in this experiment aimed at the dairy cows in two dairy cow farms and some scatteredly fed dairy cows, the bacteria from the milk were separated, cultivated, counted and identified through biochemical experiment. The drugs sensitivity test was made. The aim in this experiment was to analyse the mastitis. The bacteria separated from the milk in this test were staphylococcus, streptococcus, coliform, mycetozoan and microzyme. Most of bacteria isolated from the nomal milk were conditioned nosogenetic bacteria in the surroundings. But the most of bacteria separated from the milk of the mastitis were strong sogenetic bacteria, which caused evident hemolysis and positive concretionary enzyme reaction. The sum of the bacteria was higher than that in the nomal milk (p<0.01).To establish a method for detection of pathogenic Staphylococcus aureus in dairy cow mastitis, Polymerase-Chain Reaction (PCR) was used to amplify a sequence of Nuc gene. A pair of oligonucleotide primers special to the sequence of Nuc gene was designed according to the sequence of the Nuc gene, which encodes the thermostable nuclease of Staphylococcus aureus. DNA of the bacteria isolated from the milk of dairy cow with mastitis was used for template to identify the Staphylococcus aureus. The PCR products were detected by agarose gel electrophoresis analysis. The gene amplified was cloned into the pMD18-T plasmid, and the positive stain detected was sequenced by Songon. The Staphylococcus aureus revealed positive results of the presence of a 694bp specific fragment on gel, while other bacteria gave negative results demonstrating that the primers should be specific for Staphylococcus aureus tested.Coagulase is one of the most important indexes in detection of the pathogenic Staphylococcus aureus, which can enable the blood plasma of the rabbit or human to clotting. It conduce the pathogenic bacterium to resist the effect of the phagocytic cell and the germicide inside of the host. A pair of oligonucleotide primers special to the sequence of Coagulase gene was designed according to the sequence of the gene which encodes the coagulase of staphylococcus aureus. DNA of the bacteria isolated from the milk of dairy cow with mastitis was used for template to identify the Staphylococcus aureus. The PCR products were detected by agarose gel electrophoresis analysis. The gene amplified was cloned into the pMD18-T plasmid, and the positive stain detected was sequenced by Songon. The Staphylococcus aureus revealed positive results of the presence of a 761bp specific fragment on gel, while other bacteria gave negative results demonstrating that the primers should be specific for Staphylococcus aureus tested.Hemolysin is a kind of ectotoxin, which is the most important virulence factor of Staphylococcus aureus virulence factor, includingα,β,γandδ-toxin. Hemolysin has the hemolytic reaction effect on many kinds of mammal blood cell, and cause the toxic mastitis of mammary gland. To detect staphylococcus aureusαandβ-toxin involved in dairy cow mastitis, multiplex polymerase-chain reaction was used to amplify the gene ofα-toxin andβ-toxin. Two pairs of oligonucleotide primers special to the sequence ofαandβ-toxin gene were designed according to the sequences which encode theα-toxin andβ-toxin. DNA of the bacteria isolated from the milk of dairy cow with mastitis was used for template to identify the Staphylococcus aureus. The PCR products were detected by agarose gel electrophoresis analysis. Result showed that gene ofα-toxin were found in more than 80% of the Staphylococcus aureus with the 704bp specific fragment on gel, while theβ-toxin gene was found only in 59% isolates with the 496bp specific fragment.To detect staphylococcus aureus adhesins gene Fnbp A and Fnbp B involved in dairy cow mastitis, multiplex polymerase-chain reaction was used to amplify the gene of Fnbp A and Fnbp B. Two pairs of oligonucleotide primers special to the sequence of Fnbp A and Fnbp B gene were designed according to the sequences which encode the Fnbp A and Fnbp B. DNA of the bacteria isolated from the milk of dairy cow with mastitis was used for template to identify the Staphylococcus aureus. The PCR products were detected by agarose gel electrophoresis analysis. The gene amplified was cloned into the pMD18-T plasmid, and the positive stain detected was sequenced by Songon. The Staphylococcus aureus revealed positive results of the presence of 524bp and 642bp specific fragment on gel, while other bacteria gave negative results demonstrating that the primers should be specific for Staphylococcus aureus Fnbp A and Fnbp B gene tested. The detection of Staphylococcus aureus which were isolated from dairy cow with mastitis showed that adhesin gene of Fnbp A were found in more than 95% of the Staphylococcus aureus, while the Fnbp B gene was found only in 10% isolates.To detect staphylococcus aureus adhesin gene clfA and clfB involved in dairy cow mastitis, multiplex polymerase-chain reaction was used to amplify the gene of clfA and clfB. Two pairs of oligonucleotide primers special to the sequence of clfA and clfB gene were designed according to the sequences which encode the clfA and clfB. DNA of the bacteria isolated from the milk of dairy cow with mastitis was used for template to identify the Staphylococcus aureus. The PCR products were detected by agarose gel electrophoresis analysis. The gene amplified was cloned into the pMD18-T plasmid, and the positive stain detected was sequenced by Songon. The Staphylococcus aureus revealed positive results of the presence of 292bp and 205bp specific fragment on gel, while other bacteria gave negative results demonstrating that the primers should be specific for Staphylococcus aureus clfA and clfB gene tested. The detection of Staphylococcus aureus which were isolated from dairy cow with mastitis showed that adhesin gene of clfA were found in more than 90% of the Staphylococcus aureus, while the clfB gene was found only in 40% isolates.To clone and express the gene of fibronectin-binding protein (FnbpA), the staphylococcus aureu were isolated from the milk samples collected from dairy cows with peracute clinic mastitis. The DNA of the Staphylococcus aureus was extracted with classical technique to be template. The FnbpA gene was amplified by PCR and cloned into plasmid of pMD-18-T. The recombinant plasmid was transferred into competent E.coli. After the recombinants were screened and identified by restrition analysis, the cloned gene was sequenced. The FnbpA gene was cloned into pET-32a (+), with the inducement of IPTG, it is expressed at high levels in E.coli.BL21. Result showed that a specific protein band was found in SDS-PAGE with the molecular weight of approximately 80ku, which accorded with the anticipative result. The turnout of the protein was about 33.4% of the total protein of the bacterium analyzed with the BandScan5.0. To check the activation of the recombined protein, the bacterial adherence preventing experiment was carried out. Result showed that the annealed protein can prevent seventy-three point twenty four percent of the staphylococcus aureus adhere to the mammalian cell.A kind of peracute mastitis of dairy cow was found by us around Ji'nan in Shandong province of China, and the a-Hemolysin of staphylococcus aureus was suspected to be the pathogeny. To clone and express the gene ofα-Hemolysin (α-HL), the staphylococcus aureus were isolated from the milk samples collected from dairy cows with peracute clinic mastitis and were identified with biochemical test. The DNA of the Staphylococcus aureus was extracted with classical technique to be template. Theα-HL gene was amplified by PCR. The products were dispersed by gel electrophoresis, theα-HL gene was cloned into plasmid of pMD-18-T, and the recombinant plasmid was transferred into competent E.coli. After the recombinants were screened and identified by restrition analysis, the cloned gene was sequenced. Then theα-HL gene was cloned into pET-32a (+), with the inducement of IPTG, the a-Hemolysin is expressed at high levels in E.coli.BL21. To check the activation of the recombined protein, the purified protein was hypodermic injected into mice, and the necrosis was found in the side of injection. In the experiment of haemolysis of rabbit blood cell, the potency of the recombined protein was 2.26×104 HU/mg。Adhesins are considered the most important virulence factors during early phases Staphylococcus aureus infection. To clone the gene of cluming factor (clfA), the staphylococcus aureu were isolated from the milk samples collected from dairy cows with peracute clinic mastitis. The DNA of the Staphylococcus aureus was extracted with classical technique to be template. The clfaA gene was amplified by PCR with the special primers designed according to the clfaA gene published in the Genebank. The product of the PCR was cloned into plasmid of pMD-18-T. The cloned gene was sequenced. A specific band was found in approximately 990bp, which accorded with the anticipative result. The amplified clfA was 99% coincident with the gene of clfA in the Genebank. After digestion by restriction enzyme BamHⅠand XbaⅠ, the sequenced gene of clfaA was cloned into the pcDNA3.1 (+). The recombinant plasmid was transferred into competent E.coli and identified by restrition analysis, result exhibited that the eukaryotic expression plasmid was constructed rightly which to the DNA vaccine against the infection of the Staphylococcus aureus.As a kind of bacteriotropin, the antibody to the Fnbp can not only prevent the adhesion of the staphylococcus aureus to the cell, but also promot the removing of the body. Hemolysin is a kind of ectotoxin of Staphylococcus aureus, the anatoxine of which can partly protect goat from mastitis induced by Staphylococcus aureus. In this experiement, the engineered protein of rFnbp and rα-HL were mixed with adjuvant. The mice were vaccined with the protein vaccine and the eukaryotic expression plasmid pcDNA3.1+-Clfa, and the level of the antibody in the serum was tested by ELISA. The result showed that the specific antibody was found in all the serum of the mice vaccined with the protein. Through the initial test of aggression with thestaphylococcus aureus, the engineered protein vaccine could lower the infection rate and the death rate of the mice, which offered a new way to prevent the infection of Staphylococcus aureus.