The Immune Regulation Research On Cyptosporidium CypA Protein To Monocyte-macrophage Cell Line Of THP-1Cells

Objective:The cyclophilin A (CypA) of Cryptosporidium parvum is a secreted protein. It is not yet clear whether the CypA could regulate the function of immune cells or not.To know the functional role of CypA of Cryptosporidium parvum on macrophages, we designed and synthesized polypeptides based on CypA homology domain which might have regulatory effects on macrophages, and revealed their mechanisms in signaling pathway. The innate immune is the first line of defense after the human body is infected by cryptosporidium, and macrophages are very important cells in innate immune. So it is very significant for us to do research about the function effects of secreted CypA of Cryptosporidium parvum on macrophages, and it also important for us to understand the immune escape and opportunities pathogenicity of Cryptosporidium.Methods:1. cyclophilin A protein of Cryptosporidium was identified as a secreted proteinWe used pGEX-4T-1-CypA and pGEX-4T-1-CypA-30plasmid which contain CypA and CypA-30target gene constructed previously in our laboratory. Then we designed the primers of eukaryotic expression vector pcDNA3.1+. After the CypA and CypA-30gene was amplified by PCR, we subcloned the CypA-pcDNA3.1+and CypA-30-pcDNA3.1+eukaryotic expression vector. The correct recombinant vector were evaluated by double digestion, DNA electrophoretic and sequencing. The above plasmids were transformed into E.coli DH5a, and a large number of target genes were amplified by colony PCR, then we extracted plasmids by plasmid extraction kit, transfected the above three correct plasmids into Hela cells by LipofectamineTM2000, collected supernatant and cell, and detected the CypA secretion by Western blot.2. The functional regulation of protein and polypeptides of Cryptosporidium CypA on THP-1cellswe transfected the above eukaryotic recombinant plasmids into THP-1cells by LipofectamineTM2000, and the effects of CypA on proliferation of THP-1cells was assayed by cck-8method. According to the bioinformatics analysis, α,β,γ three polypeptides were designed and synthesized, and we used different concentrations of polypeptides to stimulate the THP-1cells, filtering out the right concentration of each polypeptide which could regulate the THP-1cells. Cell cycle and apoptosis were detected by Flow cytometry and cell cytokines IL-8and TNF-α in THP-1were assayed by ELISA, and the signaling pathway moleculars effected by polypeptides were detected by Western blot.Results:1. Cryptosporidium cyclophilin A protein identified as a secreted proteinCypA and CypA-30gene was amplified successfully, the recombinant plasmid were confirmed correctly by double digestion, DNA electrophoresis, and sequencing. The CypA-pcDNA3.1+,CypA-30-pcDNA3.1+and pcDNA3.1+were transfected into Hela cells with LipofectamineTM2000, and we collected supernatant and cell. The CypA protein was detected both in supernatant and cell by Western blot. We could detect in cell but in supernatant in CypA-30-pcDNA3.1+group. We detected this protein neither in supernatant nor in cell in pcDNA3.1+group. This result proved that CypA from Cryptosporidium was a secretory protein. 2. The functional mechanisms of CypA protein and polypeptides of Cryptosporidium on THP-1cellsWe transfected the above three correct plasmids into THP-1cells by LipofectamineTM2000. THP-1cells proliferation was not effected by CypA protein.We used different concentrations of α,β,γ three polypeptides to stimulate the THP-1cells and found out that β polypeptide promoted proliferation of THP-1cells. There was no significant differences of THP-1Cell cycle and apoptosis detected by Flow cytometry. The β polypeptide of CypA had influence on the expression and phosphorylation of ERK1/2and JNK MAPK cell signaling channel in THP-1cells. There were no significant difference of cell cytokines IL-8and TNF-a in THP-1cells.Conclusion:CypA-pcDNA3.1+and CypA-30-pcDNA3.1+recombinant plasmids were constructed successfully, and CypA was identified as a secreted protein, which laid the foundation for our research of CypA on the regulation of cell function. P polypeptide come from CypA promoted THP-1cells proliferation, and had influence on the expression and phosphorylation of ERK1/2and JNK MAPK cell signaling channel. So we conclude that β polypeptide of CypA might regulate THP-1cells proliferation by the ERK1/2and JNK MAPK cell signaling pathway.