Preparation Of Recombinant Myostatin And Effects Of Active Immunization Against Myostatin On Growing Pigs

Myostatin is a negative regulator of skeletal muscle growth. It plays an important role during myogenic diferentiation. The full fragment DNAs for myostatin of Hampshire pig was cloned and myostatin proteins were expressed in E.coli expressing system in this project. The aim of the present project is to provide the material preparation for utilizing myostatin gene and producing myostatin vaccine that can increase the muscle mass through blocking the physiological function of myostatin. At the same time, this project can also provide technical support for the research of expression of heterologous gene in E.coli.This experiment was conducted to gain the recombinant myostatin by technique for gene engineering. The specific primers were designed and produced according to myostatin gene sequence. EcoRⅠand SalⅠrestriction enzyme cut sites and protective bases were added at the ends of the primer respectively. The full fragment gene was cloned by RT-PCR from pig skeletal muscle, PCR product was cloned into the pMD18-T vector. Sequencing analysis showed that the nucleotide sequence was the same as the published myostatin cDNA sequence by Voelker. The recombinant plasmid pMD18-T-M was identified with EcoRⅠand SalⅠrestriction enzyme, the fragment were collected by agarose gel fraction method. After purification the fragment was ligated to the pET-30a express vector, the recombinant pET-30a-M express vector was constructed. After transformation, the engineered strain E.coli BL21(DE3)/6×His-M was expressed by the induction of IPTG. The specific approximate 48ku band was obtained by analysis of SDS-PAGE. The result indicated that myostatin protein had been expressed successfully in E.coli expressing system. Through the expression conditions optimizing, the optimizated condition was IPTG 0.4mmol/L,and induced 6h. Induced the recobinant expression stain by IPTG, the product was fuse expressed as inclusions. The expression product was purified by passing the Ni affinity chromatograph collumn using the recombinant protein with a tag of 6×His. The purity of myostatin was very high when multiple ultrasonication and washing was carried out. The two method both gained excellent effect.Oil-emulsion vaccines were prepared using purified myostatin protein by which 27 growing pigs (DLY) with body weigh of 25±1.5kg were allotted randomly to three groups by weight. Every group included three replicates of three pigs each. Pigs of each group were injected every 14 days with physiological saline or oil-emulsion vaccines. Primary inoculation was on day 1 and booster inoculation were on day 15 and 29. 1 of each replicates was slaughtered on day 49. Compared to the control group, the obviously phase effect on growing performance were observed in both two MSTN treatment-group pigs. In the 3-4 weeks, the good effect was occurred. The feed efficiency of low dosage group was better than the control group. In the 5-6 weeks, for both two MSTN treatment groups, there was obviouse good effect on the average daily gain and the feed efficiency. The lean percentage of high dosage MSTN treatment group increased of 3 percents. For both two MSTN treatment groups, there were obviouse effect on the backfat thickness, rib longissimus muscle area, lean percentage. Active immunization against myostatin had certain efects on pork quality. In the MSTN treatment groups the water loss of pork lower compared with control pigs. The muscle shear force was higher in MSTN treatment pigs. Active immunization against myostatin had no efect on the pH of pork and pork color.To summarize, animals immunized with the myostatin fusion proteins expressed in E.coli, was shown to yield antibody and had positive efect on muscle growth, laying foundation for further study towards developing genetically-engineered myostatin vaccines.