The Production Optimization Of Fermentation Recombinant Human Tumor Necrosis Factor For Injection（rmh-TNF）

The recombinant E.coli. expressing rmh-TNF was compressively tested. Overall control tests proved that the recombinant E.coli.was correctly constructed,and that the expression level of target protein was no less than 30%. The bacterial strain showed high stability and typical characteristics of E.coli.,which was suitable for establishing the Strain bank of recombinant E.coli..The fermentation conditions of rmh-TNF expressed in E.coli. were optimized. Time of inducing, expression times, p H and p O2 on the growth of E.coli. were investigated by single factor method. The results showed that the optimum conditions for fermentation were as follows: the OD value of 0.8-1.0 for introduction, the expression time of 3.5h, the p H value of 6.8-7.2, and the p O2 value higher than 40%.According to the characteristics of secondary metabolites of rmh-TNF, fed-batch medium was optimized. Orthogonal design in detail was applied to study the impact of a variety of different Tryptone and Yeast Extract on fed-batch medium. The result indicated that 100 g of Tryptone and 50 g of Yeast Extract were optimal medium formula of fed-batch.Through the study of the fed-batch technics, it was determined that the fermentation unit which was better than normal when the high-temperature sterilized fed-batch medium was added in running after inducing.Based on the optimized fed-batch technics, the average rmh-TNF expression level can be maintained at about 255.8mg/g, which is 41.5% higher than that of the original process.