| Myostatin, also known as growth differentiation factor-8, is a member of the TGF-βSuper-family. Myostatin has a negative regulatory role on skeletal muscle growth, development, differentiation and can inhibit cell proliferation and differentiation, regulate the muscle weight, and inhibit intramuscular fat deposition. The loss of MSTN activity will lead to hypertrophic animal muscle tissue, more muscle fibers, larger the diameter and less subcutaneous fat. A large number of studies have shown, the animals with MSTN mutations have widespread skeletal muscle and improved meat quality. Screening individuals that have MSTN mutations, we can cultivate superior animal breeds with high yield meat. Inhibiting the expression of MSTN gene in vivo can improve muscle atrophy and provide new means for the treatments of muscular dystrophy or other muscle wasting diseases. Therefore, it is important to produce MSTN preparation applying genetic engineering for in-depth study of the biological effects of MSTN and its mechanism and the development of promoting muscle growth vaccines. On the basis of construction of the MSTN mature region of prokaryotic expression vector previously, by optimizing expression conditions, we got MSTN sterling successfully and then used them for cell culture and immunology. Besides, we identified the structure of the the expressed products using bioinformatics methods. The specific contents are as follows:1. Identification of remaining strains:The remaining strains are identified by PCR, restriction enzyme digestion and sequencing. The strains which can express stably are screening by determinating the content of expression products applying SDS-PAGE and Western-blot.2. Optimizing expression conditions of strains:The experiment is designed applying response surface design methods, and the best experimental conditions are obtained. Then the expression content in various conditions are compared, and the response surface between the two factors is established according to the expression content. Finally, the optimal conditions for protein expression are obtained by calculating:The incubation temperature is 34℃; the OD value of cell concentration is 0.6; the concentration of IPTG is 0.5μg/mL; the induction time is 6h. Under the optimal conditions,87 mg expressed protein can be obtained per liter bacterial liquid.3. The effects of MSTN fusion protein on Hela cells:Respectively adding 0.5μg/mL,1μg/mL,2μg/mL,4μg/mL,8μg/mL,16μg/mL expression products into DMEM culture medium, the effects of expression products on proliferation and cell cycle of Hela cells are dectected by MTT and Flow cytometry classification. The results showed that expression products can inhibit the proliferation of Hela cells and terminate the cell cycle at G2 phase. With the concentration of MSTN fusion protein increasing, the apoptosis of Hela cells is increased.4. The role of MSTN fusion protein on skeletal muscle of mice development:The mice were immunized with the concentration of 0.5 mg/mL,1 mg/mL,2 mg/mL of MSTN inclusion body protein or soluble protein respectively, and the serum antibody levels and skeletal muscle cross-sectional area were detected after 4 weeks. The results showed that the titers of serum antibody after immunized (320±09.54,400±244.95, 320±109.54,240±89.44,200±122.47,300±115.47) were higher than the control group, The titers of serum antibody which immunized with solution protein (320±109.54, 400±244.95,320±109.54) was higher than the group immunized with inclusion body (240±89.44,200±122.47,300±115.47), the gap was not obvious between solution protein and inclusion; mid-dose group enhance cross-sectional area of skeletal muscle increasing, high-dose group inhibit development of skeletal muscle.5. Amino acid sequence of Myostatin was analysised by bioinformatics. The result showed that Myostatin mature region is hydropbic, no signal peptide, no transmembrane. However, the sequence contains a TGF-βfamily signature which can regulate proliferation and differentiation of cell.
|
PDF Full Text Request