Characterization, Expression And Extraction Of Recombinant Humor Tumor Necrosis Factor α(rhTNFα) On Large Scale

The thesis was a part of the pilot study of the production process of rhTNF-a, expressed by E. Coli. The main part of the thesis was about the expression and extraction of rhTNF-a.First, we extracted plasmid pTNF320 from E. Coli BL 21. Then we linked the TNF cDNA cut from the plasmid with the vector pUC19 and then determined the sequence of TNF cDNA. The results showed that it was consistent with those reported in the literature. So the plasmid pTNF 320 was transferred into fresh E. Coli BL 21 and thus fermentation was performed.It was found that the vector pTNF 320 was not suitable according to the protein concentration and TNF activity, therefore TNF cDNA was cloned into the vector pET-21a, so a new vector pET/TNF was obtained. The cell output using new vector was almost the same as that of the original one, however, protein concentration and TNF activity were increased.Bead mill was used in the cell disruption because of the large amount of cells. Then we optimized the factor of stirring-speed, cell concentration, and velocity of fluid during the disruption, using protein concentration and TNF activity as criteria.The kinetics of cell disruption in batch and continuous operation was studied. The efficiency of different operations and influence of high velocity, multi-cycle were also examined.Based on the hear-tolerance of TNF, the disruption, heat treatment, and precipitation of 40-65% ammonium sulfate were integrated. An optimal process was achieved.