Expression Of HFGF21 In Cordyceps Militaris Mycelium And Its Effects On Hypoglycemic And Lipid-lowering

As an important member of the FGF family,Fibroblast growth factor 21(FGF21)is a candidate drug with great application prospects effects to diabetes.However,repeated injections of FGF21 have seriously increased the suffering on patients.In order to develop an oral preparation of FGF21,The mycelium of Cordyceps militaris was selected as the bioreactor to express h FGF21,and the hypoglycemic and lipid lowering effects were studied through in vitro and in vivo experiments.1.The fungal binary expression vector p CB130-h FGF21 which contained GPD promoter was transformed to the protoplasts of Cordyceps militaris through PEG-mediated genetic transformation system.The effects of 0,350,400,450,500,550 mg/L hygromycin on the growth of mycelium were studied.The results showed that 450 mg/L was the critical concentration for hygromycin screening.And there were 8 positive transformants containing the target gene h FGF21 were finally obtained after genomic PCR,SDS-PAGE and Western-blot detection,the conversion rate was 12.3%.And the positive strain with the highest expression level of h FGF21 was named X06-49.2.In order to investigate the production of h FGF21 during the growth in mycelium,X06-49 was continuously cultured for 10 days.ELISA analysis showed the optimal culture time was 7 days when the content of hFGF21 was 1.8 mg/g in mycelium.Then it was serially passaged for 11 generations,and the content of hFGF21 of each generation in mycelium was detected by ELISA method.The results showed that the recombinant Cordyceps militaris militarum strain could stably inherited to 7th generation.3.Recombinant hFGF21 with His-tagged was purified by Ni-NTA nickel affinity chromatography column.The effects of rh FGF21 on glucose uptake in 3T3-L1 cell was detected by GOD-POD assay.The results showed similar effect to the commercial FGF21,rhFGF21 promoted glucose uptake in 3T3-L1 with a dose-dependent manner.The expression of related indicators in ERK pathway showed that rhFGF21 can activate phosphorylation of related proteins in ERK pathway.X06-49 stain and commercial FGF21 were stored at-20 ° C,4 ° C and 37 ° C for 24 months respectively.The activities of rh FGF21 were 95%,80%,and 48%,which were significantly better than the commercial FGF21,indicating that mycelium as a host can protect the activitiy of rhFGF21.4.In order to investigate the hypoglycemic and lipid-lowering effects of recombinant Cordyceps militaris mycelium which containing rhFGF21 on type ? diabetes,db/db mice were fed with mycelium for 60 days.The results showed that oral recombinant Cordyceps militaris mycelium effectively reduced blood glucose levels,increased insulin sensitivity,and improved insulin resistance in db/db mice.And it could relieve disturbance of blood lipid metabolism in diabetic mice through reducing blood triglyceride(TG),total cholesterol(T-CHO),free fatty acid(NEFA),low density lipoprotein(LDL-C)concentrations,increase high density lipoprotein(HDL-C)concentration.5.In order to investigate the effect of oral recombinant Cordyceps militaris mycelium on fatty liver and pancreas in db/db mice,the serum ALT and AST contents of the mice were measured,and mRNA expression of the fatty liver-related inflammatory factors in mice were detected by quantitative real-time PCR,including MCP-1,Cd11 b,F4/80,CD68 and TNF-a.And the histopathological sections of fatty liver tissue were observed.The results showed that oral recombinant Cordyceps militaris mycelium significantly reduced the content of blood ALT and AST(P <0.01),the expression of inflammatory-related genes in fatty liver(P <0.01)and the number of fat particles in fatty liver.At the same time,immunohistochemical results of pancreatic tissue showed that recombinant Cordyceps militaris mycelium can protect pancreatic tissue by reducing the apoptosis of ? cells.6.Acute toxicity and subacute toxicity tests were performed to evaluate the safety of recombinant Cordyceps militaris mycelium.Acute toxicity test confirmed that there were no death rats when the maximum dose was 15 g/kg/d.In the subacute toxicity test,rats were fed with high(10.5 g/kg/d),medium(1.05 g/kg/d),and low(0.35 g/kg/d)doses respectively.The results showed there were no effect about rat weight,relative organ weights,hematology index,blood biochemical index and histopathological of any organ in rats.It was proved that recombinant Cordyceps militaris mycelium was safe as an oral preparation.