Preparation And Application Of Anti-chicken Myostain Polyclonal Antibody

Myostatin(MSTN)is also called Growth differentiation factor-8(GDF-8).It is mainly expressed in skeletal muscle and plays negative role in muscle growth and development.The mutations in the MSTN gene could result in the“double muscling” phenotype in animals,and reduced fat mass.Therefore,it is an important economic trait in livestock and poultry.Anti-MSTN antibody is an indispensable part in studying the mechanism related to regulation of MSTN on muscle development,which involves cell cycle,myoblast proliferation and differentiation,and related signal transduction pathways(Smad,BMP,etc.).Currently,available commercial GDF-8 antibodies are only suitable for humans and mice,and no anti-chicken MSTN antibody is available.The preparation of anti-chicken MSTN polyclonal antibody will lay a foundation for studying the mechanism related to regulation of chicken MSTN on muscle development.Chicken muscle tissue was collected and the RNA was extracted.The MSTN gene was amplified by RT-PCR,and p MD18T-MSTN cloning plasmid was constructed by TA cloning.Bioinformatic analysis of chicken MSTN was conducted by taking advantage of online tools.The MSTN fragment was removed from p MD18T-MSTN,subcloned to pET28a-Sumo vector to construct a prokaryotic expression vector pET28a-Sumo-MSTN The recombinant plasmid was transformed to Rosetta-gami2(DE3)to induce Sumo-His-MSTN fusion protein expression through addition of IPTG and then the fusion protein was purified by affinity chromatography.A His-tagged antibody was used for Western blotting to validate immunogenicity of Sumo-His-MSTN fusion protein.New Zealand white rabbits were immunized by Sumo-His-MSTN fusion protein,and,rabbit anti-chicken MSTN polyclonal antibody was prepared.ELISA was used to determine antibody titer,Western blot was used not only to verify the specific binding reaction of antibody to Sumo-His-MSTN fusion protein,but also to determine antibody sensitivity.Chicken MSTN polyclonal antibody was used as the first antibody in western blot assays,to determine the abundance of MSTN expression in different tissues of chicken embryos on the same embryonic day(ED),and the expression profile of MSTN in pectoralis major and leg muscle from chicken embryos on different embryonic days,and finally the subcellular localization of MSTN was determined by immunohistochemical staining.The results showed that:(1)Chicken MSTN consists of 1 128 bp nucleotides,and encoding a total of 375 amino acids;(2)MSTN protein is a hydrophilic protein with transmembrane region,signal peptide,containing and14 epitopes,which cover the whole open reading frame,and the secondary structure of MSTN is mainly random crimp;(3)The pET28a-Sumo-MSTN expression vector was successfully constructed,and with the induction of IPTG,the Sumo-His-MSTN fusion protein was expressed in a soluble manner in Escherichia coli;(4)The Sumo-His-MSTN fusion protein obtained by affinity chromatography purification can specifically bind to His-tagged antibodies,proving that it is immunogenic;(5)Derived from white rabbits immunized by Sumo-His-MSTN fusion protein,the rabbit anti-chicken MSTN polyclonal antibody could specifically bind to both Sumo-His-MSTN fusion protein and MSTN protein in chicken embryonic muscle tissue,and the antibody titer was higher than 1: 256 000;(6)Western blot detection proved that the minimum detectable amount of Sumo-His-MSTN fusion protein by anti-chicken MSTN polyclonal antibody was 2.5 μg;(7)The content of MSTN protein in the pectoralis major and leg muscles on ED17 were higher than that on ED13 and D1;(8)The expression of MSTN protein in leg muscle and pectoralis muscle on ED17 was significantly higher than that in other tissues,which was consistent with the expression profile of MSTN m RNA;(9)Immunohistochemical staining demonstrates that endogenous MSTN expressed mainly in the cytoplasm.In a word,rabbit anti-chicken polyclonal antibody was obtained in this experiment,and the expression of endogenous MSTN protein in chicken embryonic tissue was detected by Western blot,which further proved the effectiveness of chicken MSTN polyclonal antibody.This will lead to studying the relevant molecular mechanism of chicken MSTN on regulation of muscle development.