Preliminary Detection Of Recombinant Human Fibroblast Growth Factor 8a Pilot Process And Pharmacodynamics

Fibroblast Growth Factor8 isoform a(FGF8a),a member of Fibroblast Growth Factor family, has a general role of other family members: promoting cartilage formation, activating the epithelial cells,promoting wound healing and so on. Comparing with other family members, FGF8a is expected to become a new cell growth factor, which not only stimulates normal cell differentiation, but also evades the risk of cancer. The efficiency and safety of FGF8a will be focused amomg the research.In this study, "four factors four level" orthogonal experiment was designed to explore the fermentation conditions, in which the culture medium pH, induction time, final concentration of IPTG and induct temperature would be considered to identify the optimal fermentation parameters. Then fermentation parameters are controlled according to the results of shake flask experiments. Protein content is analyzed by SDS-PAGE. To simplify the process of protein refolding, pre-experimental purification methods are optimized. Then rhFGF8a protein activity is calculated through MTT method. Chickembryo chorioallantoic membrane (CAM) experiment is used to observing the angiogenesis in CAM capabilities in FGF8a's pharmacodynamics. Then the relationship between rhFGF8a and gap junction protein Cx43 (tumor suppressor gene) was researched. Whether rhFGF8a up-regulates the expression of Cx43 gene is analyzed by the western blotting method.The results showed that, the optimal parameters are medium pH value is 7.0, OD600 value is 0.5, IPTG eventually concentration is 0.5 mM, inducing temperature is 30 degrees by orthogonal experimen. In the fermentation process parameters are controlled: (1) the air flow is set 10L/min first, 15L/min is set after induced.Dissolved oxygen is controlled at 40%. (2) Temperature is controlled at 37 degrees first, 30 degrees is set after induced. (3) pH value is set at 7.0.(4) Stirring speed is controlled between 105rpm and 500rpm. (5) After 3 hours the fermentation cultivation are joined at 250ml/h speed.20 grams wet bacteria per liter fermentation solution could be obtained, and the yield rate of expressed products was about 30% of total host proteins which is identified through SDS-PAGE. The dosage of oxidized glutathione and reduced glutathione are decreased to 0.1 times after optimizing purification conditions, and the yield of purified rhFGF8a is 57%. By MTT method, the purified rhFGF8a plays a role in promoting the growth of fibroblast 3T3 cells, and its biological activity is 22000IU/mg. CAM experiments show that the rhFGF8a has weak capacity on angiogenesis. And compared with bFGF and EGF, the rhFGF8a can up-regulate expression of Cx43 gene through western blotting.