Expression Of Porcine ACVR2B-Fc Fusion Protein In Pichia Pastoris And Activity Analysis

Muscle weight gain is the most important target of growth performance in pig production practice.At present,it is mainly through nutrition regulation technology,supplemented by injection of growth hormone or growth hormone-releasing hormone,feeding somatostatin exhausting agents or administration of somatostatin genetic engineering vaccines to promote the muscle growth of pigs.However,due to its transient effects,animal toxicity and biological safety,they have not yet been applied in livestock farms.Myostatin（MSTN）is an important negative regulatory gene for muscle growth,which inhibits the activation of skeletal muscle satellite cells before birth and the hypertrophy of muscle fibers after birth.It is found that activin receptor ⅡB（ACVR2B）has the highest affinity with MSTN.Therefore,ACVR2B can be used as an important candidate protein for MSTN neutralizing antibody design.The porcine fusion protein ACVR2B-Fc can be expressed in secretory type of Pichia Pastoris to increase protein yield and reduce animal immune response.1 Construction and expression of porcine ACVR2B-Fc fusion protein plasmidBioinformatics analysis was shown that porcine ACVR2B protein（NP₀01005350.1）has a total of 512 amino acids,among that,the signal peptide is from 1-22 amino acid,the binding regions of activin I and II receptor is from 39-117 amino acid,and serine/threonine kinase catalytic region is from 194-484 amino acid,the dynein light chain 1 interaction region is from 491-512 amino acid.In addition,the porcine IgG precursor protein（NP₉98993.1）has a total of 474 amino acids,among that,the signal peptide is 1-19 amino acid,the variable region of IgG is 23-145 amino acid,the constant region of IgG is 151-244 amino acid,and the Fc region of IgG is 265-474 amino acid.The primers were designed according to the sequences of the extracellular receptor binding region of pig ACVR2B and IgG Fc region,respectively.Then,the cloned gene fragments were inserted into the T vector plasmids and sequenced.The correctly sequenced T vector plasmids were used as a template,the 5’-SnaB I-ACVR2B-Fc-Not I-3’ fusion gene was obtained by overlapping PCR.Finally,the resultant fragment was inserted into the expression vector pPIC9K through double digestion.After linearization of the plasmid,the DNA were electroporated into Pichia Pastoris strain GS115.After the antibiotic G418 selection,the positive recombinant yeast strains were induced expression through methanol supplementation.After SDS-PAGE electrophoresis of yeast supernatant,the gel was stained with Coomassie brilliant blue.It was shown that the strain（#52）superlatively expressed the target protein in the supernatant for 72 h.The purified fusion protein ACVR2B-Fc can be obtained by using Protein A affinity chromatography column.2 Functional verification of porcine ACVR2B-Fc fusion protein in vitroConsidering of the activity of ACVR2B-Fc fusion protein,two requirements should be meet with:ACVR2B-Fc fusion protein could recognize MSTN protein,and macrophages could recognize ACVR2B-Fc fusion protein,then be endocytosed.First,the Rhodamine B-labeled ACVR2B-Fc fusion protein was with or without co-culture with the porcine macrophage cell line 3D4/21 for 2 h for immunofluorescence microscopy observation.It was shown that red fluorescent signals were visible in the cell membrane and cytoplasm after 2 h co-culture.For without co-culture,only the red fluorescent signal could be observed on the cell membrane surface.It was indicated that the ACVR2B-Fc fusion protein can be recognized by the receptors on the surface of macrophages and be endocytosed into cells.In addition,different dosages of the Rhodamine B-labeled ACVR2B-Fc fusion protein（0pmol,32pmol,48 pmol,96 pmol,128 pmol,192 pmol,382 pmol,510 pmol,1020 pmol）were incubated with 3D4/21 cells,and then analyzed by flow cytometry.It was shown that the binding curve of ACVR2B-Fc fusion protein to 3D4/21 cells is S-type,which may be due to multiple Fc receptors on the surface of 3D4/21 cells.About 1020 pmol of ACVR2B-Fc fusion protein is required for the 3×106 3D4/21 cells to reach saturation.In addition,the expressed MSTN precursor protein and maturation protein were analyzed by Dot-ELISA with ACVR2B-Fc fusion protein.It was shown that ACVR2B-Fc fusion protein can recognize MSTN precursor protein and maturation protein with increasing dosages.Then,flow cytometry was performed though FITC-labeled MSTN as a direct primary antibody binding with ACVR2B-Fc-3D4/21 cells.It was shown that ACVR2B-Fc fusion protein can bind to MSTN precursor protein and maturation protein,and the binding rate was S-type.In summary,the ACVR2B-Fc fusion protein expressed by Pichia Pastoris has the biological activity of antagonizing MSTN and being recognized by macrophages.