Panning And Identification Of Sheep Myostatin Nanobody Library

Inorder to explore a way of promoting the growth of sheep, this study intend to inhibit the function of sheep myostatin by injection of MSTN nanobody. Using phage display technology to build the sheep myostatin nanobody gene library, high specificity of MSTN nanobody fragments were acquired after four rounds of affinity panning. Expressing and purifing MSTN nanobody in Escherichia coli,then to verify whether direct injection of MSTN nanobody into cells and mice muscle can enhance growth. The main results are as follows:1. Inducing the sheep MSTN protein. To determine the demand of antigen concentration for ELISA. The MSTN mature peptide was induced by IPTG and purified by Ni-NTA column, using ELISA and Western blot assay detected that the antigencity, determined affinity antigen protein concentration needed for panning at the same time.2. Panning and ifentification of sheep MSTN nanobody library. After infecting the original antibody library with the helper phage propagation liquid, separated to obtain the recombinant phage antibody display library. The titer of the phage display library achieved 1.8×1012 pfu/m L, moreover, after four rounds of enrichment and affinity panning through MSTN antigen, acquired a positive clone with high specificity and high affinity. The blast results showed that the sequencing really is camel VHH sequence.3. Inducing and expressing MSTN nanobody in Escherichia coli. The nanobody’s gene sequence was cloned and built into two kinds of prokaryotic expression vector, then it was used to transform E.coli BL21, induced by IPTG and purified with a His-Ni-NTA column and confirmed the affinity, specificity with Western blot and ELISA assay.4. Studing the effects of MSTN nanobody on growth performance of mice. And an MTT colorimetric assay was used to determine the MSTN nanobody had no toxic effect on C2C12 cells. The experimental group of mice injected by quantitative nanobody to compare the change with other groups. In addition, we performed histological and morphometric analysis of quadriceps after intramuscular injection of nanobody.The results showed the weight of mice injected by MSTN nanobody displayed a significant difference compared with the control group. Furthermore,the myofiber sizes were showed significant increase after injected nanobody over the PBS(p < 0.05) and control group(p < 0.01) which suggested the MSTN nanobody has obviousely promoting effect on the growth of muscle mass.In summary, the study showed that through four rounds of affinity panningsuccessfully developed a nanobody derived from the camel against myostatin,expressed MSTN nanobody in Escherichia coli and verifed it had no cytotoxicity on the muscle cells, whats more, it could promote mice muscle growth. This study provided a candidate material R&D biological agents to promote animal growth.