The Distribution Of Staphylococcal Enterotoxin Genes From Milk In Inner Mongolia Area And The Expression Of The SEI Protein

The test aims to understand the current distribution condition of Staphylococcal enterotoxin genes in Inner Mongolia area,and provide basic information of assessment of pollution risk of Staphylococcal enterotoxin and development of Staphylococcus aureus(S.aureus)vaccine.Using PCR method,the 16 kinds of enterotoxin genes of 121 strains of S.aureus isolated from milk samples of clinical mastitis in dairy cows in Inner Mongolia area were detected.The results showed that121 strains of S.aureus carried enterotoxin gene.The sei,selm and selj genes with higher proportion were 121(100%),81(66.94%)and 64(52.89%),respectively.But see、seh、selo and selp were not detected.A total of 59 strains(48.76%)carried traditional enterotoxin genes(sea~see).A total of 121 strains(100%)carried newly enterotoxin genes(seg~selq).There were 118 strains(97.52%)carrying 2 and more than 2 kinds of enterotoxin genes at the same time,and the combination was very complex.These showed that the situation of clinical mastitis caused by S.aureus is complex in Inner Mongolia area,and prevention and treatment are difficult.S.aureus strains isolated from clinical mastitis samples in Mongolia area contain staphylococcal enterotoxin i gene(sei),so it is necessary to study the staphylococcal enterotoxin I(SEI).In order to study the antibody preparation of SEI and the Vaccine preparation of S.aureus,the protein expression of SEI was studied in this test.According to the gene sequence of SEI in GenBank(accession number: AB060537.1),a pair of specific primers containing restriction sites were designed.The sei was amplified from S.aureus using PCR technique,and then was sub cloned into vector Trans1-TI to construct a recombinant plasmid pET32a-sei.The expression protein was purified by IPTG and purified by nickel column.The expression of fusion protein was verified by Western blot analysis using His labeled monoclonal antibody and staphylococcal enterotoxin 1 monoclonal antibody.The results showed that the sei was cloned successfully,the fragment size was 634 bp,and the fusion protein was highly expressed.The recombinant protein with molecular weight of about 45 KDa was successfully identified by SDS-PAGE.A single target band was obtained,and the purity of recombinant protein was higher.Western-blot showed that the recombinant protein had good immunogenicity.The results showed that the expression system Trans1-T1 / BL21 could efficiently express staphylococcal enterotoxin I.