Cloning And Eukaryotic Expression Of The Coding Sequence Of Porcine Myostatin

Myostatin, a member of the transforming growth factor- P (TGF- P )superfamily,has been shown to be a negative regulator of myogenesis. Mice and catties possesing mutant MSTN allels display a 'double muscling' phenotype characterized by extreme skeletal muscles of hypertrophy and/or hyperplasia. Myostatin is translated as a pre-propolypeptide. Removal of the signal peptide and the subsequent cleavage at a conserved RSRR proteolytic processing site separates the bioactive carboxyl terminal region from the amino terminal of latency-associated protein, both of which are further processed and secreted.In this study , the cDNA fragment of myostatin was amplified by reverse transcription and Polymerase Chain Reaction (RT-PCR) and a nested PCR from porcine skeletal muscle of JunMu-1, and then was cloned into pMD18-T vector and subsequently subjected to restriction endonuclease analysis and sequencing. The result indicated that the coding sequence of myostatin was conserved .In order to express the coding sequence of porcine myostatin (MSTN) in eukaryotic system, two special primers(MSTNENPl,MSTNENP2) were designed according to the myostatin cDNA gene including TTA TCA at the end and two restriction endonucleases(BamH I ,EcoR I) sites. Using these primers , PCR was proceeded to amplify the 1095bp coding sequence of myostatin.The PCR products were purified in 1% agarose gel electrophoresis and recovered by Agrose Gel DNA Extraction Kit, and then inserted into the pMD18-T vector between the sites of BamH I and EcoR I .The ligated products were transformed to the competence E.coli DH5 a .The recombinant positive clones were screened by a -complementation, identified by restriction endonucleases digestion, PCR and sequencing.The cloning plasmid pMD-18-T-MSTN and pcDNAS.l vector were digested by restriction endonucleases BamH I and EcoR I simultaneously. The eukaryotic expressionplasmid pcDNA3.1-MSTN was constructed by inserting the coding sequence of myostatin from pMD18-T-MSTN into the multi cloning sites of eukaryotic expression vector pcDNAS.l. The recombinant plasmid pcDNA3.1-MSTN was confirmed as mentioned above and introduced into the COS-7 cells by lipofectamine. COS-7 cells were cultured in DMEM supplemented with 10% NBCS(New born calf serum) until approximately 40% confluence and then were transfected with pcDNA3.1-MSNT and lipofactamine. After 68h, the expression the coding sequence of myostatin was detected by RT-PCR using the total RNA as templat which was extracted from the transfectant COS-7 cell. On the other hand, the expression of the coding sequence of porcine myostatin was also detected by SDS-PAGE and then transferred to nitrocellulose membrane followed by detection with primary antibody(rabbit anti porcine myostatin), secondary antibody (HRP.goat anti rabbit IgG) and DAB Staining. The results showed a 43kDa special band which was transfected recombinant plasmid pcDNA3.1-MSTN.