Detection And Expression Of Main Enterotoxin Genes From Staphylococcus Aureu Isolated From Raw Milk And Antibodies Preparation Against Main Enterotoxin

Staphylococcal enterotoxin (SE) is the main ceausation that results in foodpoisoning and staphylococcus gastroenteritis. At present, SE usually can be dividedinto18genotypes, and different detectable rate of SE gene relates on the isolatedplace of strain and host sources. The distribution of SE genotype of56Staphylococcus aureu isolated from raw milk in Hefei is not clear yet. In this study,ten different10SE genes from56isolates were detected by PCR so as to determinetheir major genotype firstly, then SEA and SEG protein successfully expressed usingthe prokaryotic expression system and the specific antibodies against SEA and SEGwere prepared. Our studies provide a foundation for developing immunological assayfor analysing SE and guiding significance to the security of dairy products andpreventing the food poisoning of SE.In order to determine SE major genotype of56Staphylococcus aureu isolatedfrom raw milk in Hefei, ten pair of primers were designed and synthesized accordingto the published different SE gene sequences in GenBank and10SE genes （SEA,SEB, SEC, SED, SEE, SEG, SEH, SEI, SEM, SEN）form56isolates were amplified byPCR. The result showed that53of56isolates carried at least one of SE genes and thepositive rate of SE gene in isolates was94.64%. The deteetion rate of SEA, SEB, SEC,SED, SEG, SEH, SEI, SEM and SEN was41.07%,16.07%,19.64%,3.57%,91.07%,5.36%,17.86%,5.36%and24.43%respectively, no SEE gene was be detected. Twoor more SE genes were detected in SE positive strains, which composed of49kinds ofSE genotypes, and SEA-SEG was the major genotype. These results suggested that weshould establish a sensitive, rapid and specific immunological assay to strengthen thedetection of the SEA and SEG from raw milk in Hefei.For the purpose of obtaining recombinant SEA and SEG protein, two pairs ofspecific primers were designed according to the SEA and SEG gene sequences ofStaphylococcus aureus ATCC25923, then full-length SEA gene (774bp) and themature peptide SEG gene (702bp) were amplified by PCR and were cloned into thethe pAML-c4X plasmid to construct recombinant plasmids pAML-c4X-SEA andpAML-c4X-SEG. The soluble recombinant SEA and SEG protein were purified byaffinity chromatography. The purified SEA protein with98.32%purity,1.0mg/mLconcentration and SEG protein with97.69%purity,1.5mg/mL concentration all couldserve as detected antigen and immunogen.In order to prepare anti-SEA antibody and anti-SEG antibody, rabbits were immunized with purified recombinant SEA or SEG protein emulsified in Oil adjuvantA5fully three times. On the14th day after the second immunization, anti-SEAantibody titer was1:6553600detected by indirect ELISA and was1:64by argorsedeposion diffution reaction, anti-SEG antibody titer was1:3276800detected byindirect ELISA and was1:32by argorse deposion diffution reaction. They could beused as antibodies for detection after purified.