Functional Characterization of Human Myostatin C-Domain Recombinant Protein and Production of Myostatin Polyclonal Antibody

摘要

Myostatin (GDF-8) is a member of the Transforming Growth Factor-Beta (TGF-β) superfamily that is highly expressed in skeletal muscle and myostatin loss of function leads to doubling of skeletal muscle mass while dysregulated myostatin activity is associated with a number of metabolic disorders including muscle cachexia, obesity and type II diabetes. In this study, to further investigate the application of the myostatin protein, researchers constructed and expressed recombinant myostatin protein and its polyclonal antibody. The C-domain of human myostatin gene was cloned into the prokaryotic expression vector pET-32a-c (+) to express His-tagged myostatin-C protein and was expressed in Origami B DE3) induced by IPTG. After purification, the recombinant protein was used to raise the anti-myostatin polyclonal antibody. In addition, the immunogenicity of the recombinant myostatin-C protein was detected by mice immunized with the protein. The results showed the purity of the recombinant myostatin-C protein is >90%. The final concentration is 3.5 mg mL-1. It reacted with the myostatin antibody with a strong specific reactive band. The titer of anti-myostatin serum was determined to be 1:50,000. Immunization of mice with recombinant protein could significantly increase the body weight of neonatal mice and themselves. These results proved that researchers obtained a high-level expression of the recombinant myostatin-C protein as well as high titer of rabbit polyclonal antibody. It retaining antigen-binding activity can be employed for some therapeutic use. This special polyclonal antibody could provide a good tool for further studying structural and functional characterization of myostatin protein.