| At the end of 2019,the epidemic of pneumonia transmitted by New Coronavirus erupted in China,which lasted for two years.During this period,the virus has mutated many times,especially the delta mutant that appeared in the first half of 2021 has the strongest pathogenicity and is widely spread.In order to control the development of the epidemic,we studied the downstream fermentation technology and purification process of Pichia pastoris engineering strain,which expressed the RBD protein of COVID-19 delta strain.The quadruple tank was used to explore the fermentation process.The conditions of medium p H,culture temperature,induction time and induction mode were studied.The fermentation conditions were determined as follows:（1）the culture temperature was 30 ℃ and the induction temperature was 25 ℃;（2）The p H of the medium was set to 6.5;（3）The feed is supplemented with glycerol / methanol at the flow acceleration rate of 5g / L / h（glycerol: methanol = 4:1）;（4）The induction time was 48 hours.The purification process of RBD protein in fermentation broth is explored.Through the experiments of various chromatography methods,the process routes of Butyl hydrophobic chromatography,CM cation exchange chromatography and DEAE anion exchange chromatography are determined.Ammonium sulfate is added to the fermentation supernatant of the engineering bacteria until the final concentration is 15%,and the sample is loaded to the butyl hydrophobic chromatographic column balanced with 15% ammonium sulfate-10 m M PB in advance,then the impurities are washed with 5% ammonium sulfate-10 m M PB,and the target protein is eluted with 10 m M PB.After the eluent of the target protein of hydrophobic chromatography is replaced with 10 m M PB by ultrafiltration and desalting,it is transferred to CM cation exchange chromatography column.After the equilibrium solution is returned to the baseline,the target protein is directly eluted with 10 m M PB of p H11.0 The CM chromatographic eluent is desalted by ultrafiltration and changed to p H 7.0,then DEAE chromatography is conducted,and the flow through peak is collected to obtain the vaccine stock solution.On the basis of small-scale test,three batches of pilot level fermentation and purification research are carried out.Three batches of engineering bacteria fermentation broth were successfully prepared in a 40 liter fermentor,and the expression of target protein RBD in each batch of fermentation broth was not less than 1g / L.The purity of RBD protein in the three batches of purified stock solution is more than 95%,and the yield is more than 40%.Endotoxin,residual host protein and residual host DNA meet the requirements of Chinese Pharmacopoeia.The pilot study showed that the fermentation and purification process was stable and suitable for scale-up production in the future.The purified RBD protein were formulated into a vaccine for mouse immunogenicity test.Neutralizing antibodies were obtained after immunization with0/2/4 on a Wednesday needle.The results showed that neutralizing antibodies after 2and 3 immunization showed good neutralization effect on the South African strain and the delta strain COVID-19,and the neutralization of the delta strain was better.The fermentation and purification technology of COVID-19（Delta）RBD expressing Pichia pastoris was systematically studied,which laid the foundation for the development of the new recombinant protein vaccine. |
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