Construction Of E. Coli BL28-rSEA And Study On The Fermentation Conditions Of This Strain To Produce RSEA

Staphylococcus aureus is a kind of important bacteria, which can cause food poison and hospital pyogenic infection. The virulence ability is attributed to staphylococcal enterotoxins (SEs), which are produced by Staphylococcus aureus. SEs belong to a group of single strand with similary structure and virulence. They have different antigenicity. And their molecular mass are between 24-32 KDa. Among them, staphylococcal enterotoxin A (SEA) is the most frequent SEs implicated in staphylococcal food poisoning.In this paper, through genetic engineering technique, we constructed recombinant Escherichia coli (E. coli) strain expressing SEA. Then fermentation conditions and medium of recombinant E. coli strain were optimized. The results lay a foundation with large-scale batch fermentation for production of rSEA from this strain. And main results were as followed.1 Construction of recombinant E. coli strain expressing SEA and purification of rSEASEA gene was cloned from wild Staphylococcal aureus strain (ATCC 13565) into the prokaryotic expressed vector pET28a(+) possessing histiding-tag. Then recombinant vector translation into E. coli BL21(DE3). The strain were named E. coli BL28-rSEA, which can express rSEA. rSEA was purified by Ni-NTA His Bind Resin affinity chromatography purification column and SDS-PAGE showed its molecular mass was about 28 KDa.2 Optimization of fermentation conditions in recombinant E. coli strain expressing rSEAAn indirect competitive enzyme-linked immunosorbent assay used for quantitative analysis of rSEA production was established initially. The feasibility of expression of staphylococcal enterotoxin A in E. coli BL28r-SEA induced by lactose was investigated. The influences of culture conditions, including inoculation, pH, the point of induction, duration of induction phase and lactose concentration were analyzed. Results showed that optimal fermentation conditions for producing rSEA were followed as,2% inoculation, pH 7.0,37℃and 0.6 mmol/L (fimal concentration) lactose being added at mid-phase of cell growth (OD600=0.6). rSEA yield induced by lactose for 7 h was about 10.6 mg/L.3 Optimization of medium in recombinant E. coli strain expressing rSEAEffects of medium components such as for producing rSEA were evaluated by Plackett-Burman design. Then optimal region of media composition was accessed by the steepest ascent method, and concentrations of tested factors were optimized using Box-Behnken strategy and RSM. Results showed that optimal medium compositions for producing rSEA were followed as (g/L):glucose 2.00, peptone 23.00, Na2HPO4·12H2O 21.00, NaH2PO4·2H2O 4.00, yeast exact 2.60 and glycerol 36.48. On this optimal medium condition, the amount of rSEA produced by E. coli BL28-rSEA could up to 33.17 mg/L, which was about 1.84 times of that using original media without optimization and nearly 9 times of wild Staphylococcus aureus.