Preparation Of Monoclonal Antibodies Against Goat IL-17A Using The Soluble Expressed Recombinant Protein And The Effect Of IL-17A On Goat Mammary Epithelial Cells

Interleukin-17(IL-17),the characteristic cytokine of Th17 cells,is considered to be a key inflammatory cytokine in host defense against extracellular bacteria,fungal infections,and also plays a pivotal role in various diseases such as inflammation,hypersensitivity reactions autoimmune diseases.Many inflammatory diseases such as mastitis have greatly damaged goat's health and hampered the healthy development of goat industry.However,there is few report about the relationship between IL-17 and inflammatory diseases such as mastitis in dairy goat,the lack of effective monoclonal antibodies against goat IL-17 may be an important reason of hindering the in-depth study of goat IL-17.In the present study,prokaryotic expression primers were designed according to the sequence of goat IL-17A CDS region(without the signal sequence)published on Gen Bank,and total RNA was extracted from Con A stimulated peripheral blood mononuclear cells(PBMCs)from dairy goats.The target sequence was amplified by reverse transcription PCR and ligated into the prokaryotic expression vector PET 32a.The recombinant plasmid was transformed into E.coli Tans B(DE3)competent cells for prokaryotic expression,and the prokaryotic expression conditions were optimized to obtain soluble expression of the target protein,and then purification conditions were optimized.Subsequently,the purified goat recombinant IL-17A(r IL-17A)was used as an antigen to immunize BABL/c mice,and mouse spleen cells were fused with SP2/0 myeloma cells to screen hybridoma cells which could stably secrete specific goat IL-17A monoclonal antibodies.The prepared hybridoma cells were injected into BABL/c mice to prepare ascites,and ascites was purified by the classical method of Octanoic acid-Ammonium sulfate precipitation,and then the m Ab isotype were identified.In order to verify the practical application of the prepared monoclonal antibody,the eukaryotic expression product of goat IL-17A was first prepared to replace the natural IL-17A protein.The eukaryotic expression primers were designed according to the complete CDS region of the goat IL-17A published on Gen Bank.The target fragment was amplified and ligated into the eukaryotic expression vector p EGFP-N1 and then the recombinant vector was transformed into E.coli Trans5?competent cells for storage,and the recombinant plasmid was named IL-17A-p EGFP-N1.The recombinant plasmid was transfected into HEK293T cells for transient expression.The prepared monoclonal antibody was used as the primary antibody,the eukaryotic expression supernatant was collected for western blot analysis and the IL-17A~+293T cells were detected by immunofluorescence analysis.The adult healthy dairy goats were selected and the clinical model of mastitis was established by injecting Staphylococcus aureus(S.aureus)through the milk duct.The purified ascites was used as the primary antibody,the mammary gland tissue of the infected goat was sampled for detection of natural IL-17A~+cells by immunofluorescence and immunohistochemistry analysis.Subsequently,another adult healthy dairy goat in the active lactation period were chosen to sample the mammary tissue and the primary goat mammary epithelial cells(GMECs)were prepared by the tissue block adherence method.The supernatant of the IL-17A-p EGFP-N1-293T cells was used to stimulate goat mammary epithelial cells,and the expression levels of various pro-inflammatory cytokines,chemokines and defensive proteins secreted by GMEC were detected by Real-Time PCR.The results obtained were as follows:1.The CDS region of goat IL-17A(without the signal sequence)was cloned and the prokaryotic expression system was established.The expression conditions were optimized and the IL-17A-PET 32a-Trans B(DE3)recombinant cell was found.The soluble recombinant fusion protein could be obviously obtained when IL-17A-PET 32a-Trans B(DE3)was induced by IPTG at a concentration of 0.3 mmol/L at 16°C for 42 h.The optimized purification conditions were as follows:80 mmol/L imidazole for wash of the unrelated proteins and 500 mmol/L imidazole for elution of the target protein;2.Two hybridoma cell lines stably secreting specific goat IL-17A antibodies were obtained,which were named B1 and H9,respectively.The chromosome numbers of B1 and H9 cell lines were about 82 and 96,respectively.The isotypes of B1 and H9 were both Ig G1subclass and?type;ELISA titers of the B1 and H9 cells culture supernatants were 1:2~8 and1:2~9respectively.ELISA titers of mouse ascites were 1:10~6 and 1:10~7 respectively before purification,and ELISA titers after purification were both 1:10~6.Only the antibodies secreted by H9 cell line could be used for further research;3.The eukaryotic expression system of goat IL-17A was established.The IL-17A protein secreted by IL-17A-p EGFP-N1-293T cells were detected by western blot analysis using the H9 monoclonal antibody.Moreover,the IL-17A~+293T cells were detected by immunofluorescence analysis using the H9 monoclonal antibody;4.S.aureus infected clinical dairy goat mastitis model was established.The natural IL-17A~+cells in the mammary gland of goat suffering clinical mastitis were detected by immunofluorescence and immunohistochemistry analysis using the H9 monoclonal antibody;5.Primary goat mammary epithelial cells(GMECs)were obtained,and after stimulated by the supernatant of IL-17A-p EGFP-N1-293T cells,a variety of pro-inflammatory cytokines,chemokines and defensive proteins were up-expressed.Conclusions were as follows:1.Established a soluble prokaryotic expression system of goat IL-17A and a purification system;2.Obtained 2 hybridoma cells named B1 and H9 which were able to stably secrete specific goat IL-17A monoclonal antibodies,and H9 antibody could be used for immunofluorescence,immunohistochemistry and Western blot analysis;3.The eukaryotic expression system of goat IL-17A was established,and the IL-17A protein in the supernatant and IL-17A~+cells were successfully detected by H9 antibody as the primary antibody;4.IL-17A can promote the secretion of various pro-inflammatory cytokines,chemokines and defensive proteins by goat mammary epithelial cells,which suggests IL-17A is an important cytokine that may mediate mammary defense.IL-17A has no significant effect on the secretion of apoptosis-related and proliferation-related factors.