| Porcine contagious pleuropneumonia（PCP）is a highly infectious and lethal respiratory disease of pigs.19 serotypes of APP are known and cross-protection between serotypes is weak.Vaccination is one of the most effective prevention and control strategies at this stage under the trend of antibacterial restriction and prohibition,and APP subunit vaccine has good application prospects,but imported commercial subunit vaccine is expensive.Therefore,our laboratory pre-constructed p SUMO-Apx IAN-Transetta（DE3）,p SUMO-Apx IIAN-Transetta（DE3）,p SUMO-Apx IIIAN-Transetta（DE3）and p SUMO-Omp D-BL21（DE3）to express the recombinant proteins r Apx IAN,r Apx IIAN,r Apx IIIAN and r Omp D to develop a safe and efficient domestic APP subunit vaccine.The combination of E.coli genetically engineered strains and High Cell Density Cultivation（HCDC）technology can greatly improve the yield of recombinant proteins,reduce production costs,simplify the process and ensure the activity of vaccine antigenic proteins,which is an important link in the translation of laboratory technology to production applications.In view of this,this study optimized the high-density fermentation process of this subunit vaccine.The fermentation medium,inoculum volume,p H,loading volume,induction temperature,induction conditions,replenishment method,fermentation process and its parameters were experimentally explored and optimized in the shake flask stage and 30 L fermenter culture conditions,respectively,to establish a high-density fermentation process of recombinant E.coli APP subunit vaccine,laying the foundation for downstream purification and industrial production of the vaccine.The specific work is as follows:1.Screening of fermentation base media and optimization of fermentation media formulationsAfter clarifying the growth characteristics of recombinant p SUMO-Apx IIAN-Transetta,five media containing different inorganic salts were compared on the growth of recombinant p SUMO-Apx IIAN-Transetta,and the results showed that the density and dry weight of recombinant E.coli cultured in TB media were significantly higher than the rest of the media,therefore TB media was chosen as the basal medium for fermentation.On the basis of the shake flask fermentation medium,glycerol and glucose were selected as carbon sources to compare the effects of different carbon sources on the growth and protein expression of p SUMO-Apx IIAN-Transetta.The final high cell density cultivation fermentation medium was determined as 15 g/L Tryptone,10 g/L Yeast Extract,5 g/L Glucose,18 g/L K2HPO4-3H2O,3g/L KH2PO4,1 g/L Mg SO4,1 g/L EDTA and 1 m L/L Trace element solution.2.Optimisation of shaking flask culture conditionsThe Plackett-Burman test was used as the basis for a single-factor test to screen for three factors that had a significant effect on the fermentation of p SUMO-Apx IIAN-Transetta:induction time,induction temperature and p H,followed by a Box-Behnken response surface test to obtain the optimal fermentation conditions for this recombinant E.coli on shake flasks:medium initial p H 7.2,inoculum level 1%,filling volume 200 m L（1 L shake flask）,induction at 37°C for 6 h followed by the addition of IPTG,induction temperature 16°C,and end of induction culture 15.9 h.The soluble expression of the target protein r Apx IIAN was increased from 30.98 mg/L to 48.60 mg/L in the shake flask under the optimized conditions compared to the starting conditions.3.Optimisation of fermentation conditions and make-up in 30 L fermentersBased on the results of the shake flask experiment,we further explored the optimal process conditions and supplementation method for recombinant E.coli under 30 L fermenter conditions,and compared the effects of no supplementation and exponential constant p H flow plus supplementation on the growth and protein expression of recombinant bacteria.The optimum fermentation conditions and process parameters were:inoculum level of 1%,fermentation temperature of 37°C,4 h induction,induction temperature of 16°C,dissolved oxygen level of 20%and p H of 7.2.The wet weight of the bacteria was 77.90 g/L with exponential constant p H flow supplementation and only 25.92 g/L without supplementation,and the final supplementation strategy was determined to be exponential constant p H flow supplementation with glycerol and nitrogen.4.High cell density cultivation fermentation of four antigenic proteins of APP subunit vaccineUsing the optimised fermentation medium,all the components of the APP subunit vaccine were fermented on 30 L fermenters according to the optimal fermentation conditions and process parameters obtained on 30 L fermenters.The final wet weights of p SUMO-Apx IAN-Transetta,p SUMO-Apx IIAN-Transetta,p SUMO-Apx IIIAN-Transetta and p SUMO-Omp D-BL21 were able to reach 62.1 g/L,77.9 g/L,58.3 g/L and 76.3 g/L,respectively,and the final protein yields were 53.0 mg/L,68.6 mg/L,50.1 mg/L and 67.4mg/L,respectively,achieving a high density fermentation of each protein fraction of this APP subunit vaccine. |
