Effect Of Proteomics-based Cysticercus Cellulosae Excretory Secretory Antigen LRRC15 Protein On T-cell Immune Response In Piglets

Objective:In this study,we screened LRRC15(Leucine-Rich Repeat Containing 15)protein of cysticercus cellulosae ESA,which was validated by parallel reaction monitoring(PRM),for eukaryotic expression based on proteomics,and observed the effect of LRRC15 protein on the porcine T cell immune response.Methods:1.Cysticercus cellulosae and its ESA were extracted for protein and then enzymatically digested by trypsin and liquid chromatography-tandem mass spectrometry(LC-MS/MS)identification,combined with GO functional annotation,subcellular localization analysis and validated using parallel reaction monitoring to find the highly expression differential protein.2.Bioinformatics online software was used to predict the LRRC15 protein physicochemical properties,hydrophilicity,transmembrane region,signal peptide prediction,subcellular localization,secondary structure,tertiary structure and antigen epitope.LRRC15 was synthesized by PAS(PCR-based accurate synthesis)method,cloned into eukaryotic expression vector pc DNA3.4 and transferred into Top10 competent cells.The positive clones were sequenced and digested to identify the recombinant plasmid pc DNA3.4-LRRC15.The recombinant plasmid pc DNA3.4-LRRC15 was transfected into human embryonic kidney HEK293 cells and purified by Ni column.The purified LRRC15 protein was identified by SDS-PAGE and Western blot.3.The peripheral blood PBMC and spleen CD4~+T cell of normal piglets were stimulated with LRRC15 protein,respectively the T cell immune response was analyzed by the following analyses:(1)Under PHA treatment,PBMC were stimulated with LRRC15,ESA and ConA were used as positive control and the number of CD4~+and CD8~+T lymphocytes were detected by flow cytometry;(2)LRRC15 stimulated PBMC,the expression levels of CD4~+CD25~+Foxp3~+and CD8~+CD25~+Foxp3~+Treg cells were detected by flow cytometry;(3)In the presence or absence of LPS,PBMC were stimulated with LRRC15,and the secretion levels of IL-10 in culture supernatants were detected by ELISA.(4)Analysis of the effect of LRRC15 protein on initial CD4~+Th cell differentiation at different time periods at 24 h,48h and 72 h.Firstly,the induced piglet medullary-derived DCs were isolated and stimulated by adding LRRC15,respectively,and the expression of DC surface markers CD80,CD86and MHCⅡwere detected by flow cytometry,and the secretion levels of IL-6,IL-10,IL-12and TNF-αin the cell culture supernatant were detected by ELISA;(5)Subsequently,piglet spleen CD4~+T cells were isolated and co-cultured with immature medullary-derived DC,after LRRC15 stimulation,ELISA was performed to detect the secretion levels of IL-4,IL-5,IL-10,IL-17,IFN-γin the co-culture supernatants at 24 h,48 h and 72 h.Results:1.The proteomic analysis of cysticercus cellulosae and its ESA identified 475 proteins highly expressed in cysticercus and 331 in ESA.LRRC15 proteins were screened according to GO functional annotation and protein expression differences and PRM validation.2.According to the analysis of biological information,LRRC15 protein belongs to hydrophilic protein,which consists of 644 amino acids,contains 1 signal peptide,1 transmembrane region and multiple antigen epitopes,hasαhelix,β-rotation,extended chain and irregular crimp secretory protein,and contains leucine-rich domain.The recombinant plasmid pc DNA3.4-LRRC15 was successfully constructed and the target protein LRRC15 with molecular weight of about 70 KDa was expressed in human embryonic kidney HEK293 cells.3.PBMC was stimulated by LRRC15 protein,the number of CD4~+T lymphocytes increased and CD4~+CD25~+Foxp3~+and CD8~+CD25~+Foxp3~+Treg were induced,but IL-10 secretion was not induced.4.LRRC15 could induce the expression of CD80,CD86 and MHC Ⅱ in DC with or without LPS,but inhibit the production of cytokines IL-6,IL-10,IL-12 and TNF-αsecreted by DC.The secretion levels of IL-4 and IL-17 were decreased by LRRC15 protein at 24 h,48 h and72 h,respectively,and the secretion level of IL-5 was increased at 24 h compared with 48 h after induction with LRRC15 protein,but there was no significant difference between 72 h and 48 h(P<0.05).LRRC15 protein induced the increase of IFN-γsecretion,but there was no significant difference at 24 h,48 h and 72 h,but LRRC15 could not induce IL-10 secretion at all three time periods.Conclusion:1.LRRC15 protein can induce the immune imbalance of CD4~+/CD8~+T cells in piglet PBMC and induce the expression of Treg cells which may play an immunosuppressive role through independent IL-10 pathway,It is assumed that it may be related to the secretion of TGF-βsecretion..2.LRRC15 protein can induce Th1 immune response in the early stage and mixed immunity with Th1/Th2 in the later stage.