Expression, purification of recombinant flounder MRF4 protein in Escherichia coli and analysis of its polyclonal antibodies

MRF4 is one of muscle regulatory factors and plays critical roles during skeletal muscle development. The muscle development is important for the fish growth which is an important economic factor for the fish culture. To analyze the function of MRF4 in fish, the founder MRF4 antibody was prepared. The flounder MRF4 was cloned, ligated into prokaryotic expression vector pET-30b and expressed in strain E. coli BL21 (130). The recombinant flounder MRF4 fusion protein was soluble and purified with cobalt IMAC resins. To prepare MRF4 polyclonal antibodies, rabbits were immunized with the soluble protein and the increasing level of antibodies was determined by Western blot. Also, the endogenous flounder MRF4 was recognized by the anti-serum. The result further proved the existence of the anti-MRF4 antibody in the anti-serum, which will be useful for studies on the function of flounder MRF4.