| Bovine mastitis is one of the most common and harmful diseases in dairy industry.Staphylococcus aureus is one of the main pathogenic bacteria and the mastitis caused by it is difficult-to-treat.Enterotoxins are a series of virulence factors produced by Staphylococcus aureus with superantigen activity.They are also closely related to the pathogenicity of Staphylococcus aureus causing bovine mastitis.Multiple epidemiological investigations have shown that the enterotoxin gene seu is found in bovine mastitis samples.Based on the prevalence of the gene seu,this study cloned seu gene,expressed and purified SEU protein.Then an enzyme-linked immunoassay method to measure SEU was established and the effect of SEU on bovine mammary epithelial cell function was explored.The main research contents are as follows:(1)Seu gene was cloned.Seu gene of Staphylococcus aureus isolated from bovine was cloned into T vector.Bioinformatics analysis indicates that the molecular weight of SEU protein is about 30.53 k Da which is composed of 261 amino acids with the molecular formula C1365H2095N355O415S13.The theoretical PI of SEU is 6.66,and the total average hydrophilicity is-0.675,which means SEU is a basic hydrophilic protein.SEU is a structurally stable protein with the half-life in vitro more than 10 h and the instability coefficient 33.97.SEU protein was an extra-membrane protein that do not contain transmembrane region.(2)SEU protein was expressed and purified.The fragment of seu gene was ligated with the p ET-30a(+)and transformed into expression strains.The appropriate expression conditions are as follows: the expression strain: BL21(DE3)p Lys S,inducer: 0.5 mmol/L IPTG concentration,induction time: 4 h,temperature: 25℃.The recombinant SEU was purified by Ni2+-NTA affinity chromatography using 100mmol/L imidazole elution buffer.(3)The level of SEU protein was measured by an ELISA system.Using the protein SEU as an antigen,the polyclonal antibodies were prepared.They wereemployed for detection of SEU in the ELISA system.The results showed that the optimal antigen coating concentration was 2.5 μg/m L and rabbit polyclonal serum dilution was 1:6000.The optimal dilution and reaction time of the enzyme-labeled antibody were 1:2000 and 40 min.The wells were coated with phosphate buffer at37℃ for 2 h and blocked with 5% skim milk for 2 h.The sensitivity was 0.195 μg/m L,and with a detection concentration range of 0.195~16 μg/m L.Intra-and inter-assay variability are both less than 4% and the recovery rates of milk were over 80%.Using this method,the change of secretion of SEU in pure milk and skim milk contaminated with seu positive strains was studied.The results revealed that the concentration of SEU in milk increased rapidly within 0~36 h and the SEU concentrations were 0.44μg/m L and 0.50 μg/m L at the 120 th hour,respectively.(4)The effect of SEU on proliferation and secretion of bovine mammary epithelial cells was investigated.In this study,the effects of SEU on the proliferation and secretion of IL-6,IL-8 and TNF-α were detected by CCK-8 and ELISA,respectively.The results showed that the cell viability of the SEU-treated group decreased with SEU treatment at the 4 th and 24 th hour.In addition,SEU increased the levels of IL-6 and TNF-α in the cell supernatants,and moreover the level of IL-6was significantly increased with 10 μg/m L SEU challenge.However,SEU did not change the level of IL-8.In summary,we cloned seu gene,expressed and purified SEU protein,developed an ELISA system to detect SEU and investigated the effect of SEU on proliferation and secretion of bovine mammary epithelial cells.These results provide basic information for further research on the role and mechanism of SEU in bovine mastitis. |
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