Expression And Purification Of SEP For Enterotoxin P In E.coli And Preparation Of Chicken IgY

Staphylococcus aureus enterotoxin(SEs)is an effective exotoxin synthesized by S.aureus during the logarithmic or exponential to a stable stage.S.aureus can produce many kinds of enterotoxins,including S.aureus enterotoxins A,B,C,D,E,F,G,H,I,J,K,L,M,N,O,P and other enterotoxins.S.aureus enterotoxin P(SEP)is one of the main factors leading to food poisoning.Food,especially meat and dairy products,can be contaminated by S.aureus due to improper treatment or storage at high temperature.SEP poisoning symptoms are rapid,patients nausea and vomiting,with or without diarrhea.In view of the importance of SEP detection in food safety,this study subcloned SEP gene to E.coli expression vector,expressed,purified,immunized chicken.The aim of this study is to establish suitable reagents for SEP detection based on immunoassay.The main research results of this paper are as follows:(1)The SEP gene was amplified by using PUC18-SEP as template DNA and primer F(containing EcoRI)and primer(including SalI site)as primers.The amplified products were separated by EcoRI and SalI enzyme digestion,agarose gel electrophoresis,and then connected with the pET28a carriers treated with corresponding restriction enzymes,and pET28a-SEP recombinant vector was obtained.The sequence of sub clone SEP gene was consistent with that of a template and NCBI database.(2)E.coli Rossita containing the recombinant plasmid pET28a SEP was cultured at 37?and 220rpm to OD₆₀₀?0.6,and then induced overnight at 16?and 220rpm with 1mmol/l IPTG.After ultrasonic crushing,the cells were collected by centrifugation and analyzed by SDS-PAGE.The findings revealed that after the cell was split,SEP protein was primarily found in the supernatant as a soluble protein,and only a small amount of SEP protein was deposited.(3)The protein band(32.6k Da)with the largest amount of induced expression was detected by using the commercial SEP antibody.The results show that the molecular weight of SEP protein(29.77k Da)is basically the same as that of SEP protein-containing 6xhis-thrombin-t7tag.(4)The supernatant of the above-mentioned cell lysate was purified by Ni NTA affinity chromatography.SDS-PAGE analysis showed that the protein with a molecular weight of32.6k Da was purified and retained.(5)According to SDS PAGE results,there are still some trace heteroproteins in the purified SEP protein by Ni NTA.Therefore,Sephadex G-100 was further purified,and then the selective precipitation was carried out with 50%saturated ammonium sulfate,which further improved the purity of the recombinant protein Sep.(6)after purification by Ni-NTA affinity chromatography,the molecular weight of32.6k Da protein was cut from SDS-PAGE gel and identified by tandem mass spectrometry.The amino acid residues of 8peptide segments were determined by tandem mass spectrometry,which were consistent with the sequence of recombinant SEP protein.(7)The egg laying hens were immunized three times after the purified SEP protein was mixed with complete Freund adjuvant,15 days apart.Dot blotting analysis showed that IgY activity was the highest after the second injection of antigen.