Study On Fermentation Technology,Gel Preparation And Pharmacodynamics Of Long-acting Recombinant Human Platelet-derived Growth Factor

Background and Objectives:Wound healing is a complex and orderly biological process in the body after tissue damage under the action of external factors.It involves the regeneration of various tissues,granulomat tissue hyperplasia and scar formation.Traditional treatment methods include anti-infection,enhancement of body nutrition and protection of local wounds.The treatment effect is good for minor wounds,but for more serious wounds,the traditional treatment methods not only increase the cost and period of treatment,but also is not conducive to the recovery of the patient.Therefore,there is clinical significance and social value to search for the key active molecules to promote wound healing according to the biological laws of wound healing in vivo and realize mass production.Platelet-derived growth factor(PDGF)is a kind of cytokine secreted by platelets at the site of injury.It plays an important role in nerve regeneration,angiogenesis and wound repair.As a tissue repair factor in wound healing,PDGF can be used in the treatment of various wound surfaces and has extensive clinical application value.However,currently listed PDGF drugs lack long-term protective function,poor stability in vivo,short half-life and easy to be degraded by protease,leading to poor efficacy after use by patients.Therefore,the research of PDGF protein drugs with long-term function is of great significance.This study integrated PDGF B chain(PDGF B)with human immunoglobulin(Ig G)Fc fragment and recombinant expression in Pichia,established pilot fermentation and purification process,preparated the gel dosage forms for external use.The meaning of this study is to improve the half-life of drugs in vivo,reduce the dosage of drugs,shorten the time of wound healing,lay a foundation to realize large-scale production and industrialization development.Methods:1.To construct the p PICZɑ-PDGF-B-Fc expression vector,and to identify the recombinant Pichia pastoris strain;2.Optimize the methanol-induced fermentation process of PDGF-BB-Fc recombinant protein,including p H value,DO value,tank pressure,temperature,ventilation rate and other factors;3.Microfiltration and ultrafiltration were performed by tangential flow filtration technology,and Protein A affinity chromatography was used to purify the target protein.Protein purification process system was established;4.CCK8 was used to detect the proliferative effect of purified recombinant PDGF-BB-FC on NIH3T3 cells in vitro;5.Preparation the gel of recombinant PDGF-BB-FC protein;6.The effect of recombinant PDGF-BB-FC protein gel on wound healing was detected in the animal model of skin injury rats.Results:1.The results showed that the target gene was successfully integrated into the yeast genome and could successfully express the recombinant PDGF-BB-Fc protein;2.The optimal fermentation conditions were explored,and the optimal conditions for the expression of target protein were determined as 30℃,p H 6.0 of the medium,36 hours,and the expression level of target protein was 50.2 mg/L;3.The purification process system was optimized.Protein A affinity chromatography was used to purify the target protein,and citric acid eluent was used for elution to obtain PDGF-BB-Fc recombinant protein with purity over 90%;4.Recombinant PDGF-BB-Fc protein can promote the proliferation of NIH3T3 cells in vitro,there are no statistical differences in effect with commercial PDGF-BB;5.The recombinant PDGF-BB-Fc protein gel with stable physical and chemical properties was successfully prepared with carbomer as the matrix,heparin sodium and human serum albumin as the protective agent;6.Recombinant PDGF-BB-Fc protein gel can control wound infection and accelerate wound healing in the process of wound healing,and positively correlate the concentration of PDGF-BB-Fc with wound healing effect during the wound healing process.Conclusions:1.Build yeast engineering bacteria expressing PDGF-BB-Fc.2.Establish a recombinant PDGF-BB-Fc trial fermentation process system.3.A recombinant PDGF-BB-Fc protein gel with stable traits and good biological activity was successfully prepared.