Study On The Regulation And Mechanism Of Plasmodium Yoelii Tryptophan-rich Antigen 7 On Macrophage Inflammation

Malaria is still a major public health problem,and Plasmodium yoelii is an important model for studying the pathogenesis of malaria.During malaria infection,the early response of the innate immune system is to stimulate macrophages to produce the immune response.If the immune response is inappropriately regulated to cause inflammation,it will be harmful to the host.The main cause of P.yoelii 17 XL(P.y17XL)strain causing death in mice is inflammation,but the specific mechanism is still unclear.The tryptophan rich region is a tandem repeat sequence,and the Plasmodium tryptophan-rich antigen is an export protein that participates in host cell invasion and Plasmodium-host interaction.This study mainly describes the pathological characteristics of Balb/c mice infected with P.y 17 XL and the regulation and mechanism of P.yoelii tryptophan-rich antigens(PyTRAgs)on the inflammatory response of macrophages,it provides a theoretical basis for the Plasmodium-host interaction and the pathogenic mechanism of malaria.This research mainly includes four parts:Part Ⅰ,pathological characteristics and spleen proteomics analysis of mice infected with P.y 17 XL.Infection of Balb/c mice by intraperitoneal injection of P.y 17 XL,the parasitemia of mice increased rapidly after P.y 17 XL infection,reaching 80-90%,and the mice all died on the 6th and 7th days.The spleen of the mice infected with P.y17 XL was also significantly enlarged and darkened,the spleen tissue had pathological changes,and a large amount of malarial pigment deposition was found.Further analysis of the spleens of P.y 17XL-infected mice by proteomics methods revealed that compared with uninfected mice,immune-related proteins were significantly upregulated,and the levels of serum inflammatory factors were also significantly higher than those of normal mice.The above results indicate that mice infected with P.y 17 XL have a significant inflammatory response and lead to death of the mice.Part Ⅱ,construction of PyTRAg prokaryotic expression plasmid and expression and purification of recombinant protein.The pytrag1,pytrag2,pytrag7,pytrag8 and pytrag11 genes were successfully amplified by PCR,and the prokaryotic expression plasmid pET30a-PyTRAgs was constructed using In-fusion cloning technology.Transform the successfully expressed plasmid into BL21 competent cell,and use IPTG to induce expression and purify the PyTRAgs soluble proteins for use.The subcellular localization of PyTRAgs proteins was detected by immunofluorescence method(Immunological Fluorescence Assay,IFA).The results showed that PyTRAgs is expressed on the surface of P.y 17 XL i RBC and is an export protein.Part Ⅲ,the effect of PyTRAgs on the immune regulation of macrophages.Through flow cytometry,it is found that PyTRAg7 in PyTRAgs can interact with RAW264.7 cells and promote the proliferation of RAW264.7 cells.Through inverted microscope and flow cytometry,it was found that PyTRAg7 could change the morphology of RAW264.7 cells and polarize to M1.Furthermore,RTq-PCR(Real Time quantitative PCR,RTq-PCR)and enzyme-linked immunosorbent assay(Enzymelinked immunosorbent assay,ELISA)were used to detect the transcription and secretion of inflammatory cytokines in RAW264.7.The results showed that PyTRAg7 increased RAW264.7 The transcription and secretion levels of i NOs,IL-1β,IL-6 and TNF-α in cells.Western blotting was used to detect the changes in MAPK and NF-κB signaling pathway related proteins in RAW264.7 cells.The results showed that PyTRAg7 can activate the MAPK and NF-κB signaling pathways in RAW264.7 cells,and through IFA,it was found that PyTRAg7 promoted the p65 nucleus of RAW264.7cells.Transposition.The above results indicate that PyTRAg7 polarizes RAW264.7cells towards M1,activates the MAPK and NF-κB signaling pathways of RAW264.7cells,and stimulates RAW264.7 cells to secrete NO,inflammation cytokines such as IL-1β,IL-6,and TNF-α.Part Ⅳ,the preliminary study of PyTRAg7 on RAW264.7 cell inflammation regulation mechanism.The RAW264.7 cell membrane protein bound to PyTRAg7 was screened by nickel column and Flag beads tandem affinity chromatography.The eluted protein complex was analyzed by silver staining,and the difference bands were cut to identify the membrane protein bound to PyTRAg7 by mass spectrometry.Through Pulldown to further verify the binding of PyTRAg7 to the membrane protein identified by mass spectrometry,CD71 is the specific receptor for PyTRAg7 to interact with RAW264.7 cells.After blocking the receptor CD71 of RAW264.7 cells with monoclonal antibodies,flow cytometry showed that blocking the receptor CD71 can attenuate the interaction between PyTRAg7 and RAW264.7 cells.Blocking the CD71 receptor of RAW264.7 cells reduced the protein levels of PyTRAg7 on the NF-κB signaling pathway p-p65 and p-IKKα/β in RAW264.7 cells,and also reduced the effect of PyTRAg7 on RAW264.7 cells NO and inflammatory cytokines IL-1β,IL-6,TNF-αsecretion.The above results indicate that CD71 is the specific receptor that PyTRAg7 interacts with RAW264.7 cells.PyTRAg7 binds to the receptor CD71 on the surface of RAW264.7 to activate the NF-κB signaling pathway and stimulate RAW264.7 to secrete NO,inflammation cytokines such as IL-1β,IL-6,and TNF-α.In summary,this study found that mice infected with P.y 17 XL produced a significant inflammatory response and eventually led to the death of the mice.The export protein PyTRAg7 can bind to the RAW264.7 macrophage receptor CD71,thereby activating the NF-κB signaling pathway,and stimulating RAW264.7macrophages to secrete NO,inflammatory cytokines such as IL-1β,IL-6 and TNF-α.The inflammatory regulation effect of PyTRAg7 on RAW264.7 macrophages and its mechanism are described,which provides a theoretical basis for the Plasmodium-host interaction and the pathogenic mechanism of malaria.