Fermentation And Purification Technology Of Recombinant Human Serum Albumin

HSA synthesis in the liver is the most abundant protein content in plasma,mainly transport fatty acids,amino acids and other nutrients,at the same time to maintain the balance of blood osmotic pressure,stability within the plasma environment.It is clinically used for the treatment of shock,burns and blood loss due to massive bleeding,and is also one of the country’s reserve drugs.Albumin is mainly obtained from human plasma by low temperature ethanol method and column chromatography.At present,there are mature techniques for the production of recombinant human serum albumin through multiple expression systems,among which pichia pastoris is widely used.In this study,GS115-p PIC9k-hsa-5-4 strains were used as expression strains constructed in the laboratory.In view of the short fermentation time on the organic medium BMGY tank and the large amount of pigment in the fermentation medium,the wet weight of BMGY bacteria was attempted to be replaced by inorganic medium BSM with 500 g/L,and the expression of target protein was 11.46 g/L induced 192 h,both higher than that of BMGY medium.BSM with different salt concentration was selected to further optimize the medium,and it was found that 1/4 BSM could not meet the nutrient requirements of long-term fermentation.1/2BSM medium could meet the long-term high-density fermentation,and 17.47 g/L yield was obtained after196 h induction.In order to ensure that the target protein would not be degraded during storage,heat treatment was used to basically inactivate the protease in the fermentation broth for subsequent purification.The p H of the top sample and elution in Blue Sepharose affinity chromatography was optimized,and the final p H of the top sample and elution was determined to be 5 and 7,respectively.The purity of the target protein reached 94.67%.Phenyl Sepharose hydrophobic chromatography can further remove the pigment and other impurities in the product,increasing the purity to 97.348%,and the total recovery of the two-step purification is over 61%.The specific affinity between the cona affinity filler and polysaccharide was used to separate the target protein from the polysaccharide impurities,and the change of polysaccharide content before and after cona affinity chromatography was detected by anthrone-sulfuric acid method.The polysaccharide content in the final solution was 2.41mg/L,lower than6.59mg/L in p HSA under the same conditions.In order to further improve the purity of target protein,remove conformation,incorrect albumin in p HSA antigen immunity in mice,by hybridoma technology to screen for a 7H5 strains hybridoma cell lines,produce monoclonal antibody has strong affinity with p HSA,by ELISA detection antibody titer of 10^-5,Western blot to verify the effectiveness of the purification of single resistance combined with recombinant human serum albumin is characteristic.The antibody was coupled with an agarose activated by hydrogen bromide to prepare an HSA specific Immunoaffinity Chromatography column with a recovery of 95.95%from r HSA purification.The highly sensitive Pichia Pastoris Host Cell protein Kit was used to detect the content of Host Cell protein,and the purity of the target protein was calculated as 99.9666%,with a total recovery of 40.98%.