Study On Antibacterial Activity Of Porcine S100A8 And S100A9 Proteins And Production And Application Of Their Monoclonal Antibodies

In the S100 protein family,the proteins S100A8 and S100A9 act primarily as congenital immunomodulators.S100A8 and S100A9 chelate the essential metal nutrients for pathogens to invade the host through the way of "nutritional immunity",and inhibit the growth and reproduction of pathogens in the host.On the other hand,these two proteins can also bind to cell surface receptors such as RAGE and TLR4 to initiate inflammatory signal transduction and induce the expression of pro-inflammatory cytokines,thus participating in the inflammatory response and immune regulation.Clinical studies have found significant increases in the expression levels of S100A8 and S100A9 proteins in various types of tumors,and inflammatory diseases.Therefore,these two proteins can be used as biomarkers of the dysregulation of inflammatory response and the tumor microenvironment in vivo for screening and detection of related diseases.Since the biological activity of S100A8 and S100A9 proteins is affected by many factors,the correlation between the expression levels of these two proteins and their biological activity is still unclear.Therefore,exploring the expression of the S100A8 and S100A9 proteins and their antibacterial activity in vitro will help to further understand the function of the above two proteins.In addition,the proteins S100A8 and S100A9 are only expressed in vertebrates,and the current reports on them have focused on humans.There have been few reports on the use of porcine S100A8 and S100A9 in the surveillance of porcine disease and other potential role in porcine disease.However,S100A8 and S100A9 proteins have the property and advantage of being well-stabilized and not easily degraded and have the potential to be used as reliable biomarkers in the detection of the health status of pigs.In this study,the porcine S100A8 and S100A9 proteins were expressed and purified by the Escherichia coli prokaryotic expression system.The antibacterial activity of S100A8 and S100A9 proteins was investigated by in vitro experiments.At the same time,multiple monoclonal antibodies against S100A8 and S100A9 from pigs were prepared and obtained,and the characteristics of the monoclonal antibodies were identified.Tests on serum samples of piglets showed that the above monoclonal antibodies specifically identified the proteins S100A8 and S100A9 in the serum and were able to measure the content of the above proteins in these serum samples.The purpose of this study is to provide preliminary data for further understanding of the antibacterial activity range of S100A8 and S100A9 proteins,to develop antibacterial drugs,and to lay a foundation for the development of porcine disease detection reagents based on the monoclonal antibodies against porcine S100A8 and S100A9 proteins.The main contents are as follows:1.Expression and antibacterial activity of porcine S100A8 and S100A9 proteinsThe strains pET28a(+)-S100A8-BL21(DE3)and pET28a(+)-S100A9-BL21(DE3)stored in our laboratory were resuscitated,and the expression conditions of their proteins were optimized.After IPTG induction,porcine S100A8 and S100A9 proteins,which are mainly expressed in soluble form,were obtained.The purification effect of S100A8 and S100A9 was detected by SDS-PAGE after the proteins were purified by nickel column.The results show that the protein sizes of S100A8 and S100A9 have high purity and no impurity bands.The protein sizes of S100A8 and S100A9 are 11.2 kDa and 17.0 kDa,respectively,which are consistent with the expected sizes.On this basis,the purified S100A8 and S100A9 protein concentrations were determined by the BCA assay,and S100A8 and S100A9 proteins with concentrations of 25,50,100,200,and 300 μg/mL were selected for in vitro bacteriostatic experiments.To explore the in vitro antibacterial activities of different concentrations of proteins against enterotoxigenic Escherichia coli(ETEC)F4ac,Staphylococcus aureus ATCC 29213.The bacteriostatic results showed that 100,200,and 300 μg/mL S100A8 and S100A9 had a certain bacteriostatic effect on the ETEC F4ac strain,and the bacteriostatic effects were dose-dependent in a certain range.When the concentration of S100A9 protein reached 200μg/mL,it also had a certain bacteriostatic effect on Staphylococcus aureus.These results indicate that the S100A8 and S100A9 proteins expressed by the prokaryotic expression system in this experiment can perform an antimicrobial function in vitro and have good biological activity.2.Preparation of monoclonal antibodies against the porcine S100A8 and S100A9 proteinsBALB/c mice were immunized with purified porcine S100A8 and S100A9 proteins.After four immunizations,spleen cells were obtained and SP2/0 fusion was performed.Positive hybridoma cells were screened by indirect ELISA,7 hybridoma cell lines that stably secretion monoclonal antibodies against porcine S100A8 protein,and 6 hybridoma cell lines that stably secretion monoclonal antibodies against porcine S100A9 protein were obtained.Subsequently,the preparation of mouse ascites was performed and purified with the protein A+G Agarose.The purification effects of S100A8 and S100A9 were determined by SDS-PAGE.It was shown that the purified monoclonal antibodies have high purity and their molecular weights are consistent with the characteristics of monoclonal antibodies.The concentrations of purified S100A8 and S100A9 monoclonal antibodies were determined using the BCA method.The successful preparation of the monoclonal antibody provided the basic material for the characterization and application of the S100A8 and S100A9 monoclonal antibodies.3.Characterization and application of monoclonal antibodies against the porcine S100A8 and S100A9 proteins7 strains of monoclonal antibodies against porcine S100A8 protein and 6 strains of monoclonal antibodies against porcine S100A9 protein successfully prepared were tested for subclasses,antibody titer,specificity,and relative affinity.The results showed that the subclasses of the 7 strains of S100A8 monoclonal antibody were IgG1,Kappa(κ);the 6 strains of S100A9 monoclonal antibody were IgG2b,Kappa(κ);the titer of S100A8 monoclonal antibody could reach 1:1.6×105,and that of S100A9 monoclonal antibody could reach 1:3.2×105.According to the above results,S100A8-F8F9 and S100A9-C92H monoclonal antibodies were selected for a comprehensive follow-up trial.Western-Blot and indirect immunofluorescence assay results demonstrate that the S100A8-F8F9 and S100A9-C92H monoclonal antibodies can specifically recognize their corresponding recombinant proteins and cell expressed proteins.The S100A8-F8F9 monoclonal antibody does not cross-react with the S100A9 and S100A12 proteins of the S100 protein family,nor does the S100A9-C92H monoclonal antibody cross-react with the S100A8 and S100A12 proteins of the S100 protein family.These results indicate that the S100A8-F8F9 and S100A9-C92H monoclonal antibodies have excellent specificity.ELISA was used to detect the relative affinity of the S100A8-F8F9 and S100A9-C92H monoclonal antibodies.According to the results,the above monoclonal antibodies have a strong affinity.At the same time,the monoclonal antibodies S100A8-F8F9 and S100A9-C92H have been successfully applied for the serum sample detection in piglets,the levels of S100A8 and S100A9 proteins were significantly increased in the serum samples of piglets challenged with pathogens compared to the healthy piglets.These results establish a foundation and platform for the subsequent application of S100A8 and S100A9 monoclonal antibodies in swine disease surveillance and the study of the function of S100A8 and S100A9 proteins.