The Cloning and Fusion Expression of Musclin from Rat and Preparation of Its Polyclonal Antibody

Abstract: Musclin protein is exclusively secreted in myocyte, involved in the homeostasis of glucose and lipid metabolism, and could function as an autocrine and paracrine factor in skeletal muscles. Musclin mRNA is almost exclusively expressed in skeletal muscles, regulated by nutritional changes and hormonal factors, especially insulin, and augmented in the skeletal muscle of obese insulin-resistant mice. Musclin protein can inhibit insulin-stimulated glucose uptake and glycogen synthesis in myocytes. The observations suggest that musclin protein could be related to the development of diabetes mellitus. In order to further understand the physiological significance of musclin protein and its clinical application, the prokaryotic expression, purification of musclin and preparation of polyclonal antibody were done in this paper.The gene fragment encoding Musclin was amplified by RT-PCR from total RNA of rat skeletal muscle, and was cloned into prokaryotic expression vector pGEX-5x-3, The DNA sequence of cloned gene had 1% difference from that of musclin in GeneBank, while the amino acid sequence of protein was the same with Musclin in GeneBank.E.coli BL21 was transformed with recombinant plasmid pGEX-5x-3-musclin. The transformant was induced to express the recombinant protein by IPTG A 37kDa recombinant protein was identified in SDS-PAGE analysis, and further proved by Western blot. The recombinant protein exists two expression forms: soluble and insoluble, and the soluble form was purified by GSH-Sepharose Resin, the purified recombinant protein was used to immunize rabbit to prepare the polyclonal antibody. By the indirect ELISA and Western blot analysis and comparing the preimmune serum with antiserum, the recombinant protein could induce efficiently the high level and specific antibody IgG, the antibody titer is up to 1:32 000. These results suggested that the recombinant protein antiserum could be used to detect the musclin as its specific antibody…
Key words: musclin; clone; prokaryotic expression; polyclonal antibody

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