| Myostatin was a secreted protein that acted as a negative regulator of skeletal muscle mass. During embryogenesis, myostatin was expressed by cells in themyotome and in developing skeletal muscle and acted to regulate the final number of muscle fibers that are formed. During adult life, myostatin protein was produced by skeletal muscle, circulated in the blood, and acted to limit muscle fiber growth. But unfortunately, the precise molecular mechanism by which MSTN was regulated was still unknown.The biological functions of myostatin had raised the possibility that targeting the myostatin pathway may be an effective strategy for increasing muscle growth not only for a variety of clinical applications, but also for animal production. To improve muscle mass, we explored the potential of recombinant MSTN protein as vaccine in our study. Firstly, to obtatin enough protein of MSTN and its polyclonal antibody for further study, the fragment of MSTN gene was amplified and cloned into the expression vector Pet-30a. Induced by IPTG, MSTN protein was obtatined. The protein was purified by metal chelate affinity chromatography and its purity and content were detected by thin-layer scan analysis and Bradford assay. Thin-layer scanning showed that its purity reached at least 90%. Rabbits were immunolized by MSTN protein. The titer and specificity of polyclonal antibody were detected by indirect ELISA assay and Western blotting. The titer of antiserum was as high as 1:250000, with very high specificity detected by Western blotting. We concluded that after IPTG induction and metal chelate affinity chromatography purification, recombinant MSTN protein with high purity and its specific antibody have been obtained.Secondly, we detected the effect of immunizing mother mouse with recombinant MSTN protein on the body weight of neonatal mouse. The indirect ELISA assay was used to detect the titer of antibody against MSTN. Twety-four female KUNming mice were used in our study. Half of these was immunized by recombinant MSTN protein, another half was regared as control groups. After a period of immunization, the mice were mated with male mice. Results showed that the titer of mother mouse in experiment group elevated to 1:250000, but that of control group didn't change significantly before pregnancy. The body weights of mice of experiment group didn't change compared with that of control group. But during the evening of pregnancy, the increase of body weight of experiment group exceeded that of control group significantly. After the complication of pregnancy, the difference of mother mice in two groups disappeared again. However, the body weight of neonatal mice in experiment group was significantly higher than that of control group. We concluded that immunize mother mouse with recombinant MSTN protein could increase the body weight of neonatal mouse.Thirdly, to enhance the immunogenicity of MSTN, we also fused MSTN gene with the gene of hepatitis B virus core antigene. The DNA fragments encoding MSTN was amplified by PCR and fused to the gene of HBV core antigene at the position of N-terminaI,C-terminal and internal respectively. After the complication of fusion, the fused genes were inserted into the plasmid Pet-30a to constract expression vector pET-30a-c(+)-SM/ HBcAg ( N ), pET-30a-c(+)SM/ HBcAg ( C ) andpET-30a-c(+)-SM/ HBcAg (M) respectively. The fused genes were expressioned in E.coli, and the recombinant proteins were also purified by metal chelate affinity chromatography and its purity were detected by thin-layer scan analysis. Thin-layer scanning showed that their purity all reached over 90%. These evidences showed that the expression vectors for MSTN-HBcAg fusion genes were successfully constructed, the fused proteins were expressed in E.coli., and the fusion proteins were purified.Lastly, it had been previously shown that modification of thyroid hormone levels had a significant impact on skeletal muscle, predominantly through a direct regulation involved in thyroid hormone receptors. Nevertheless, little was known...
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