Detection Of Drug Resistance Of Staphylococcus Aureus From Animals And Expression Of SEA Gene And Preparation Of Monoclonal Antibodies Against SEA

Staphylococcus aureus is a common pathogen which causes pustular and toxin related diseases.About ten serotypes(A,B,C,D,E,F,G,H,I and J) of enterotoxin have been described according to their antigenic differences.Among them,serotype A is proved to be the most virulent and causes most cases of food poisoning.Methicillin resistant staphylococcus aureus(MRSA) is multidrug resistant pathogen responsible for human and animal infection.Vancomycin treatment is proved to be effective. Because of the difficulty of therapy,it is vital significant for deeply study on mechanism of drug fast,the detection of MRSA exactly and promptly,and preventing its disseminateion.A total of 45 samples collected from Jiangsu province were isolated,cultured and identified,All the 41 strains were identified to be Staphylococcus aureus by biochemistry test,catalase test and coagulase test.Among them 41 strians were proved to be antibiotic resistant by K-B method;meanwhile,methicillin resistant staphylococcus aureus(MRSA) were tested by cefoxitin method,oxacillin method and PCR.The drug resistance rate of 41 strains was in the range of 7.3%～75.6%, indicating Staphylococcus aureus isolated from clinic samples were multi-drug resistant.However,all the strains tested were sensitive to Vancomycin.Specificity and sensitivity of PCR reached 100%.Sensitivity and specificity of cefoxitin method were 100%and 96.4%respecitively.Sensitivity of oxacillin method was 96.3%,specificity was 89.6%.Cefoxitin method was easy to perform with high degree of specificity and sensibility.In a word,Cefoxitin method was more suitable for clinical diagnosis.In order to further understand the biology characteristic of SEA,two pairs of primers specific to SEA was designed and synthetized according to the published sequence of SEA gene.Following the gene SEA was amplified from standard strain ATCC-13565 by PCR.The PCR product fragment was subcloned into expression vector pET-28a(+). The resulting plasmid contained the SEA gene was renamed as pET-28a-SEA.It confirmed that BL21(DE3) E.coli which contained pET-28a-SEA was able to express large quantities of a 30kD fusion protein by SDS-PAGE analysis and Western blotting.The fusion protein pET-28a-SEA was immunized the 6-8 weeks old BALB/c mice for 4 times.Then the spleen cells of immunized mice were fused with SP2/0 myeloma cells by a routine method,and the specific antibody was detected by indirect-ELISA and Dot-ELISA methods.The hybridoma cells strain B6 and G2 were obtained,which could stably secrete monoclonal antibody specific for SEA after subcloned for 3 times. Further more,indirect-ELISA by using the monoclonal antibodies was performed to detect SEA,and made a basis for further setting up a SEA-testing method which had high specificity and sensibility,moreover could be operated easily and quickly.