Preparation And Muscle Growth-promoting Function Of Sheep MSTN Bivalent Nanobodies

Sheep Myostatin(MSTN) can negatively regulate muscle growth.Muscle growth function can be promoted by MSTN antibody blocking.In this study, we have been constructed sheep MSTN Nanobody libraries. We screened MSTN Nanobody gene by phage display technology. We build sheep MSTN bivalent Nanobody via mouse and sheep experiments on muscle growth promoting its role and functions. The further development of animal experiments is to promote muscle growth biologics foundation.The results are summarized as follows:1, phage library panning: We have the prokaryotic expression and purification of the MSTN antigen.We use ELISA principle panning which has built a library and get a Nanobody encoding gene by sequencing showed that consistent with the preliminary screening results.2, expression and purification of bivalent Nanobody sheep MSTN: Nanobody encoding gene would have been connected to bivalent Nanobody gene fragment. We constructed the recombinant prokaryotic expression vector and inducible expression in E. coli, using SDS-PAGE and Western Blot verification.Expression and purification of a large number of sheep MSTN monovalent and bivalent Nanobodies will be used for animal experiments.3, sheep MSTN Nanobodies promoting growth experiments on mice: mice weight evenly male rats were randomly assigned to the PBS, BSA protein, protein MSTN, sheep MSTN monovalent Nanobodies,sheep MSTN bivalent Nanobody groups. The right hind leg muscles of mice was injected protein, PBS for the control group. We injected every two days and weighed recording weight changes.Mice were long adductor muscle sliced and observed changes in the muscle fibers. The results showed that Nanobodies group of hindlimb muscle volume was significantly more than the control group, Nanobodies muscle group accounted for a significant increase in the proportion of the body and the bivalent weight gain is better than monovalent group. Histological muscle(10×10 magnification, observe the same area as the number of muscle fibers) showed that sheep MSTN Nanobodies promote increased muscle mass per unit area of the fiber.Promoting muscle growth was further verified in mice: Kunming mice were randomly divided into male rats, PBS, monovalent Nanobodies, bivalent Nanobody groups. The results showed that the bivalent Nanobody muscle group size and weight are significantly more than monovalent Nanobodies group.4, MSTN Nanobodies verification experiments on sheep: The 3-month-old nine sheep were randomly divided into three groups, the right hind injected twice weekly and weighed each injection 1mg. The results showed that the bivalent group and the monovalent group and PBS control weight gain was not significantly different. Muscle Histological observation showed that the number of bivalent and monovalent group compared with PBS muscle fibers increased and the difference is remarkable.In summary, this study successfully constructed MSTN bivalent Nanobody expression and purification of recombinant plasmids in E. coli.Animal experiments shows that sheep MSTN Nanobodies can increase body weight in mice and unit area muscle fiber number. Sheep also promoted on the number of muscle fibers per unit area, sheep MSTN bivalent Nanobody promotes muscle growth better than monovalent Nanobodies. Preparation of sheep MSTN bivalent Nanobody provides candidate materials for the development of animal muscle growth-promoting biological agents.