| Background: Malaria is a mosquito-borne disease caused by Plasmodium.Together with tuberculosis and AIDS,malaria is one of the three major infectious diseases in the world.Plasmodium falciparum and P.vivax are the predominant species that infect human.The increasing resistance of the vector Anopheles to insecticides,as well as the parasite to antimalarial drugs have hampered malaria prevention and control.With no effective vaccine available,there is an urgent need to find new treatments.During the period of malaria infection,the activation of host immune cells eliminates plasmodium,but plasmodium can also cause systemic inflammation by acting on host cells to induce dysregulation of immune response.Among them,Macrophages are the important source of pro-inflammatory factors in the inflammatory response,and Plasmodium related proteins could modulate inflammation by binding to macrophage membrane receptors.But the specific molecular mechanism needs to be further elucidated.Reducing host inflammatory response and screening out Plasmodium molecules which modulate host inflammatory response could provide a new strategy to malaria vaccine.Plasmodium surface related antigen(SRA)is a novel antigen that could be exported to infected erythrocyte membrane,and is observed on the surface of Plasmodium merozoites.The N-terminal of PfSRA is highly conserved,and the antibody restricted Plasmodium invasion into red blood cells.PvSRA participated in the immune evasion of Plasmodium vivax by binding to human splenic fibroblasts.These studies confirmed that the export protein SRA participates in the invasion of plasmodium and regulates the host immune response by interacting with host cells.In a word,SRA is a potential vaccine candidate antigen.Objective: In this study,PySRA was the object of this study,aiming to clarify the mechanism of macrophage-mediated inflammatory response which was modulated by PySRA.So as to provide a new strategy for the development of malaria vaccine.Methods: The pysra-F1,pysra-F2 and pysra-F3 were amplified according to the structural characteristics of pysra and the homology comparison with the hydrolyzed fragments of PfSRA.After constructing prokaryotic expression plasmids,the recombinant proteins were expressed by Escherichia coli system.Balb/c mice were immunized with the purified recombinant protein and were infected with P.yoelii 17 XL strain.The phenotype of the mice were analyzed.The obtained mice spleen tissue were stained with HE to analyze the protective effect of anti-PySRA antibody on mouse spleen.The serum of mice were analyzed by ELISA to detect the level of inflammation in mice.After culture medium containing PySRA-F1,PySRA-F2 and PySRA-F3 recombinant proteins were co-incubated with RAW264.7 macrophages,flow cytometry was used to detect the activation effect of PySRA on RAW264.7 macrophages.At the same time,qRT-PCR and Western blot were used to detect the regulatory effects of PySRA recombinant protein on pro-inflammatory factors and related signaling pathways in RAW264.7 macrophages.The effect of PySRA on macrophage activity was analyzed by CCK-8 kit and flow cytometry.In addition,flow cytometry and Western blot were used to detect the interaction between PySRA recombinant protein and RAW264.7 macrophages.Macrophage membrane receptor molecules bound to PySRA were screened by tandem affinity chromatography and mass spectrometry.The gene of receptor was amplified from macrophage cDNA and was cloned into pGEX-6P-1 vector to construct prokaryotic expression plasmid.The recombinant receptor protein was obtained through the expression system of Escherichia coli,and the recombinant protein was immunized rabbits to prepare polyclonal antibodies.After the polyclonal rabbit antibody was co-incubated with macrophages to block the surface receptor,Western blot and ELISA were used to detect the regulatory effect of PySRA recombinant protein on RAW264.7macrophages.The full-length pysra and pysra-F2 gene knockout plasmid were constructed by CRISPR/Cas9 gene editing technique.The plasmids were transfected into purified Plasmodium yoelii by electric shock.The infected ICR mice were treated with pyrimethamine to screen monoclonal knockout strains.Results: The soluble recombinant proteins of PySRA-F1,PySRA-F2 and PySRA-F3 were obtained by Escherichia coli system.The sera obtained from mice was verified after immunizing mice with the recombinant protein,Western blot results showed that PySRA-F1,PySRA-F2 and PySRA-F3 immunized mice sera could recognize the corresponding recombinant proteins.ELISA results showed that the antibody titers in the three groups were from 1:640 000 to 1:2 560 000.Anti-PySRA-F2 antibody showed protective effect on P.yoelii17 XL mice,and the parasitemia remained at 20% low level,the survival rate was 66.67%,and the spleen structure of mice was intact.ELISA results showed that the levels of IL-1β,TNF-αand IL-6 pro-inflammatory factor protein in PySRA-F2 immunized mice were significantly lower than those infected with P.yoelii 17 XL.Western blot results indicated that PySRA-F2 activated NF-κB and MAPK signaling pathways in RAW264.7 macrophages.PySRA-F2 inhibited the activity of RAW264.7 macrophages and induced apoptosis of macrophages after24 h treatment.Flow cytometry and Western blot showed that PySRA-F2 protein effectively bound RAW264.7 macrophages in a dose-dependent manner.Pull down results indicated that CD68 is the macrophage cell membrane receptor molecule that specifically binds to PySRA.After using CD68 polyclonal rabbit to block RAW264.7 macrophage surface receptor,Western blot results showed that the binding ability of PySRA to macrophages attenuated,and phosphorylation levels of p65,p38 and ERK proteins in MAPK and NF-κB signaling pathway were attenuated.ELISA results showed that after CD68 antibody blocked the surface receptor on RAW264.7 macrophages,the level of PySRA-F2 protein regulating inflammatory response and the secretion levels of IL-1β and IL-6 factors were attenuated.The full length of pysra and pysra-F2 gene could not be knocked out in Plasmodium.Conclusion: PySRA-F2 activated downstream MAPK and NF-κB signaling pathway by binding to CD68 molecule on the surface of macrophage membrane,and regulated inflammatory response.When the mice obtained anti-PySRA-F2 antibody,the antibody was protective against P.yoelii 17 XL mice,and the survival rate was improved and the proinflammatory response was attenuated. |
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