Abstract: Myostatin(MSTN), an important regulator of embryonic myogenesis and adult skeletal muscle growth, regulated the muscle development by controlling the proliferation and differentiation of myoblast. To explore the biological function of MSTN in sheep fibroblasts, we had amplified the MSTN gene from sheep skeletal muscle by reverse transcription PCR (RT-PCR), deleted the stop codons TGA and cloned into the eukaryotic expression vector pAcGFP-N1 by directional clone. So, the fusion protein recombinant plasmid pAcGFP-MSTN had been constructed. After the restriction enzyme digestion of XhoⅠ/SacⅡ and sequencing, the plasmid had been transfected into the sheep fibroblasts by lipofectamine. We also observed the fluorescence expression under the microscope, examined the transcription and translation of the expression vector pAcGFP-MSTN by RT-PCR and western blotting. The results showed that we had successfully cloned sheep MSTN gene, designed the enzyme site of XhoⅠ and SacⅡ at the both ends of MSTN open reading frame by PCR, and constructed the fusion protein expression vector pAcGFP-MSTN. We could observe the green fluorescence after 24 h of the transfection of the recombinant plasmid pAcGFP-MSTN. We also had amplified the transcription product of 1138 bp by RT-PCR. The targeted protein 78 ku was also detected by western blotting. The results would provide the important foundation to detect the regulation mechanism of MSTN in fibroblasts and adipocyte differention.

Key words: myostatin; sheep fibroblasts cell; eukaryotic expression vector