The Expression, Purification And Bioactivity Evaluation Of Recombinant Human Bone Morphogenetic Protein 10

Heart failure(HF)is the end stage phenomenon of heart disease development.Drug side effect is one of the major factors involved in this process.Due to the irregeneratable features of heart,drug research for cardioprotective agent is an important part of drug design work.We found that Bone morphogenetic protein 10(BMP10)promotes and regulates embryonic development and postnatal growth and development of heart tissue.In this thesis,highly active human BMP10 was obtained by biological preparation,and the protective effect of rhBMP10on drug-induced heart failure was evaluated by in vivo models.The main results are as follows:(1)Plasmid containing human BMP10 cDNA sequence was constructed,and stably transfected into CHO-S cells,and a CHO-BMP10 monoclonal cell strain stably expressing rhBMP10 was obtained.The EcoRI restriction site(GAATTC)in the human BMP10 cDNA sequence was synonymously mutated to GAGTTC and the cDNA was inserted into the multiple cloning site of the pMH3 plasmid.The expression of rhBMP10 was verified by dot blot and western blot.The CHO-BMP10 monoclonal cell strain obtained set a milestone for the subsequent studies herein.(2)CHO-BMP10 cell line was suspend acclimated,fed-batch cultured,and the culture supernatant was purified by Q column and Gel filtration column,and the biological activity was evaluated in vitro.After suspension domesticated for 10 d,the density of suspension cultured CHO-BMP10 cells can be doubled within 24 h.In the fed-batch culture,the maximum density of CHO-BMP10 cells reached 9.1×10~6 cells/mL,and the cumulative concentration of rhBMP10in the culture supernatant was 35.3 mg/L at the end of the culture.The purity of rhBMP10obtained after purification was over 95%,and the yield was 49.8%.The structural characteristics of the purified rhBMP10 were analyzed by Coomassie blue staining,whose results included four bands at~116 kDa,~68 kDa,~43 kDa and~26 kDa,which were full-length dimers and partially cleaved dimers,propeptide monomer and growth factor domain dimer,respectively.The feasibility of using furin in vitro digestion to obtain N-terminal uniform growth factor domain dimer was verified.Using a set of BRE responsive elements(BRE)isolated from the Id1 gene,a pGL6-BRE-Luc plasmid capable of specifically responding to BMP10 stimulation was constructed.The biological activity of the rhBMP10 we expressed was thus verified.Through batch-feed culture,and two-stage temperature control,a large quantity of the highly bioactive rhBMP10 abundant for further in vivo experiments was generated.(3)The mouse heart failure model was induced by DOX administration and its cardiac function and morphological changes were examined.The ejection fraction(EF)of the DOX model mice was 73.1±3.8%,which was lower than that of the control group(84.6±2.4%)and the rhBMP10 treatment group(82.6±5.6%);the Masson’s staining of the tissue samples of the DOX model mice showed large area of myocardial fibrosis,and the minimum muscle fiber diameter was 11.3μm.However,the myocardial tissue of rhBMP10 treatment group showed no large-area fibrous tissue,and the minimum muscle fiber diameter was 12.3μm.The ratio of cPARP in myocardial tissue of DOX model mice was higher compared to other groups.The percentage of apoptotic cardiomyocyte in TUNEL assay was 0.196%,which was significantly higher than that of 0.014%in the control group and 0.036%in the rhBMP10 treatment group.In addition,the levels of serum lactate dehydrogenase,creatine kinase,cTroponin I,and Myoglobin were significantly elevated in the DOX model mice than those in the control and rhBMP10-treated mice.Real-time PCR results showed that the transcription levels of mitochondrial function-related genes including Ppargc1a,Esrra,Sdha and Nudfa3 were significantly down-regulated in the myocardial tissue of the DOX model mice,while the transcription levels of the above genes in the rhBMP10-treated group were significantly higher than those in the DOX model mice.The above results show that rhBMP10 can alleviate cardiac damage caused by DOX.In this study,a pMH3 plasmid encoding human BMP10 cDNA was constructed and stably transfected into CHO-S cells,and a CHO-BMP10 cell line stably expressing rhBMP10 was obtained.The product was cumulated through fed-batch culture,and the purification conditions of rhBMP10 were explored,its structural characteristics were analyzed.A DOX-induced mouse heart disease model was constructed by high-dose multiple injection of DOX.Echocardiography,Masson’s staining,real-time PCR,western blot and TUNEL were used to demonstrate that rhBMP10 can attenuate the cardiotoxicity induced by DOX.