MSTN Gene Cloning, Expression And Active Immunization

Myostatin (MSTN) is a negative regulatory factor of skeletal muscle growth and development, which plays an important role in the generation of muscle cells. In this study, we amplified full-length porcine MSTN gene through assembly-exons and porcine MSTN was expressed in E.coli and purified after construction the prokaryotic expression vector of MSTN; Besides, we constructed MSTN yeast expression vectors and expressed MSTN in yeast (S.cerevisiae); We vaccinated mice by oral feeding with yeast expression MSTN and subcutaneous injection with yeast expression MSTN and MSTN protein to verify that oral yeast expression MSTN can induce mice to produce antibodies against MSTN. The antibodies against MSTN blocked physiological function of MSTN so as to promote muscle growth in mice; A marker-free recombinant yeast strain with porcine MSTN gene was constructed, which lays the foundation of industrial production for the yeast expression MSTN.A. The full-length porcine MSTN gene was amplified by the method of assembly-exons from the pig genome, which was inserted into prokaryotic expression vector pET32a. The MSTN prokaryotic expression vector pET32a-MSTN was transformated into E.coli strain BL(DE3) and we optimized the condition of MSTN expression, expressed recombinant MSTN with His tag and purified MSTN fusion protein with nickel-chromatography.B.We amplified full-length chicken ovalbumin (OVA) gene by the method of assembly-exons from chicken genome and constructed yeast expression vector JMB88-OVA. MSTN gene digested from pET32a-MSTN was inserted with the same enzymes digested JMB88 and JMB88-OVA to construct the MSTN yeast expression vector JMB88-MSTN and JMB88-OVA-MSTN. The two MSTN yeast expression vector were transformated into yeast strain JMY1 and expression of MSTN and OVA-MSTN was induced by CuSO4 and confirmed by western blot. We immunized mice by oral feeding with yeast expression MSTN or OVA-MSTN and subcutaneous injection with yeast expression MSTN or OVA-MSTN and MSTN protein. Serum samples of immunized mice were analyzed with western blot to validate that oral yeast can cause to humoral immune to produce antibodies against MSTN. The antibodies against MSTN can bind MSTN protein in mice body and block the MSTN function. Body weight gain and weight of the major skeletal muscle increased significantly in experimental groups comparing to control group.C.After we investigated that it could promote muscle growth in mice orally immunized yeast expression MSTN. We designed and constructed a marker-free industrial yeast strain JMY12 integrated with MSTN cassette. Firstly we constructed the integration vector pBlue-kan-NTS5'3'-MSTN. The linearized integration vector pBlue-kan-NTS5'3'-MSTN was transformated into yeast JMY1 to integrate MSTN into yeast genome in which there was a marker gene KanMX4; Cre recombinase was transformated into the yeast integrated with MSTN and marker gene to delete KanMX4 gene at the use of Cre/Loxp system, and then we obtained the marker-free yeast strain integrated with MSTN gene, named JMY12; Expression of MSTN in JMY12 was verified by western blot.This study demonstrated that mice fed with yeast expression MSTN could produce anti-MSTN antibodies so that the functions of MSTN would be neutralized and it promote the muscle growth in mice; we constructed a marker-free yeast expression MSTN.