Cloning And Expression Of Myostatin And Leptin Gene From Guangxi Bama Mini-Pig And The Immunation Effect Of Recombined Fusion Protein In Animals

Myostatin, also known as growth and differentiation factor 8, is a secreted glycoproteins and member of the transforming growth factor-βfamily that negatively modulates muscle satellite cell proliferation and inhibits muscle cell differentiation. Due to its properties, the naturally occurring mutations related to loss of function in the highly conserved myostatin gene lead to dramatic increase of muscle mass.Leptin is one of the most important adipose derived peptipe hormone which plays a key role in regulating energy intake and energy expenditure, including appetite and metabolism.Upon the binding to the long form leptin receptor to initiates multiple intracellular signal transduction pathways, leptin inform the brain about the status of the body's energy store.In this study, primers were designed according the myostatin and leptin gene sequences released in GenBank. The total RNA purified from Guangxi Bama Mini-pig's muscle and fat tissues, respevtively, was used to amplify myostatin and leptin cDNA by RT-PCR. The amplified segments were ligated into pMD18-T vector after purification and then the recombined vector was transformed into E.coli DH5αfor sequencing. The results indicated that the cloned myostatin and leptin cDNA contained 1128bp and 441bp, which included the coding sequence of entire length of myostatin and leptin mature peptides, respectively. Myostatin and leptin nucleodite sequence of Guangxi Bama Mini-pig shared 99.7% and 99.1% homologue to the previously released pig myostatin and leptin nucleodite sequence in Genbank, respectively.The cloned myostatin or leptin mature peptide cDNA was cloned into BamHI and EcoRI or BamHI and HindIII sites of expression vector pRSETA to construct the recombined plasmid pRSETA-MSNT₇₅ or pRSETA-Leptin. The recombined prokaryotic expression vectors then respectively transformed into BL₂₁(DE3)LysS to express myostatin or leptin fusion proteins after confirming the open reading frame was corrected. The proteins expressed were confirmed by SDS-PAGE and western blotting analysis. The expressing condition optimization indicated that the best cultivation temperature, IPTG concentration and inducing time for the fusion protein expression was 37℃, 1 mmol/L and 7h (myotatin) or 4h (leptin), respectively.A sufficient amount of recombined myostatin or leptin fusion protein expressed in inclusion body form was denatured in guanidine hydrocloride (6mol/L) solution and purified by 50% Ni-NTA chromatography. The purified recombined fusion protein was refolded by dialysis and then homogenied with mineral oil to form immunogen used for animal immunization trail.Animals including mouse, Bama Mini-pigs and chickens were inoculated with the above prepared immunogens against myostatin. No antibody against recombined myostatin protein was detected in the immunized animal and no significant difference in body weight gain was observed between the immunized animals and the non-immunized animal (p>0.05). Immunizing mouse with immunogen against leptin did not affected significantly on the average body weight gain(P>0.05), but significantly drcrease animal's feed intake (P<0.05).It is concluded that immunizing animal with recombined myostatin protein did not yieded antibody and had not positive effect on the growth of animal, but immunizing animals with recombined leptin protein had positive effect on the performance of animals. The study lays the foundation for further study towards developing genetically-engineered myostatin and leptin vaccines.