| Malaria,tuberculosis,and AIDS are known as the three major infectious diseases in the world.Among the five species of Plasmodium that can infect humans,Plasmodium falciparum infection causes the most severe clinical symptoms and the highest fatality rate.Plasmodium falciparum surface-related antigen(Pf SRA)can be exported to the surface of the red blood cell(RBC)membrane and participate in the process of Plasmodium falciparum invading RBCs.Previous studies have shown that the serum of patients with falciparum malaria can recognize Pf SRA synthetic peptides,but whether Pf SRA can be used as a falciparum malaria vaccine candidate still needs further experimental data to support.The intra-erythrocytic phase of the malaria parasite’s life cycle is the only period in which the parasite causes clinical symptoms.The research on the pathogenic mechanism of the intra-erythrocyte phase is undoubtedly the most important in the development of vaccines and drugs for malaria.Because infected red blood cells(i RBCs)combine with normal RBCs to form rosettes and adhere to the blood vessel wall,thereby blocking the blood vessels and leading to a series of pathological manifestations.Studies have shown that endothelial cells undergo activation and apoptosis after contacting i RBC with vascular endothelial cells,but the molecular mechanism has not yet been clarified.Human umbilical vein endothelial cells(HUVECs)as stem cells can pass indefinitely and are a good cell model for vascular endothelial cell research.Therefore,this study mainly explores the feasibility of Pf SRA as a falciparum malaria vaccine candidate,and studies whether Pf SRA is involved in endothelial cell apoptosis and the specific molecular mechanism by using HUVEC as the object.The main results are as follows:Part Ⅰ.Genetic polymorphism analysis of pfsra.In this study,a total of 74 serum samples from patients with falciparum malaria were collected,the pfsra gene was divided into four segments,sequencing primers were designed,and genomic DNA was extracted from serum samples,PCR amplification and gene sequencing were performed.Using Mega7,Dna SP,and other software to analyze the gene polymorphisms of pfsra,the results showed that the amino terminus(N terminus)of Pf SRA is a highly conserved,and the carboxyl terminus(C terminus)is relatively polymorphic.Part Ⅱ.Immunogenicity and antibody protection analysis of Pf SRA.The recombinant Pf SRA protein(r Pf SRA)was expressed by a prokaryotic expression system,and the purified recombinant protein was used to immunize mice.The results of enzyme-linked immunosorbent assay(ELISA)showed that r Pf SRA could induce mice to produce high levels of Ig G antibodies,and the affinity of antibodies in the F1 a group could reach more than 97%.The CCK8 cell proliferation assay results showed that Pf SRA could promote the proliferation of mouse-specific lymphocytes.In addition,flow cytometry showed that the immune serum of mice could inhibit the invasion of RBCs by Plasmodium falciparum.Part Ⅲ.Effects of Pf SRA on HUVEC phenotype.The results of flow cytometry showed that only r Pf SRA-F3 a could bind to HUVEC and promote HUVEC apoptosis.The results of the CCK8 cell proliferation assay and scratch assay indicated that r Pf SRA-F3 a inhibited the proliferation and migration of HUVEC.Western blot detected the down-regulation of the anti-apoptotic protein B cell lymphoma-2(Bcl-2)after apoptosis,as well as the up-regulation of cleaved aspartate specific proteinase-3(Cysteinyl aspartate specific proteinase-3,Caspase-3),cleaved poly ADP-ribose polymerase(PARP)and apoptosis-promoting Bcl-2associated x protein(Bax).Part Ⅳ.To initially explore the molecular mechanism of Pf SRA regulating HUVEC apoptosis.First,the subcellular localization of Pf SRA in HUVECs was clarified.The enriched in vitro cultured Plasmodium falciparum was co-cultured with HUVEC.After staining,it was observed that the Plasmodium-related substances in HUVEC gradually increased with the increase of incubation time.Subsequently,the eukaryotic plasmid of pfsra was constructed,and the expression of the fusion protein of the F3 segment was found in the nucleus of the cell by transfecting human embryonic kidney cell 293T(HEK293T).The same results were obtained in the nucleocytoplasmic separation experiment,and it was predicted that the F3 gene had four nuclear localization signals(NLS).Through Co-immunoprecipitation(Co-IP)and protein in vitro binding experiments,it was found that F3 was associated with The NLS receptor protein,Karyopherin alpha(KPNA)1-6,which has different degrees of binding.After co-incubation of i RBC and HUVEC,immunofluorescence assay showed that Pf SRA entered the cells and co-localized with nucleolin(NCL,C23),and Co-IP assay found that F3 a protein bound to C23.During the incubation of HUVEC,the expression of C23 by F3 a protein decreased with time.And C23 has the function of binding and stabilizing Bcl-2m RNA.Flow cytometry showed that the pro-apoptotic effect of purified r Pf SRA-F3aΔNLS3on HUVEC was significantly lower than that of Pf SRA-F3 a,indicating that the effect of nuclear entry was related to apoptosis.After HUVEC overexpressed C23,the proportion of apoptosis decreased.Western blot detection showed that F3 a could activate the NF-κB and MAPK signaling pathways that regulate cell proliferation and apoptosis.In conclusion,this study found that the N-terminus of Pf SRA is highly conserved and can significantly induce the adaptive immune response in mice.Pf SRA-F1 a antibody can inhibit the invasion of RBCs by Plasmodium falciparum.After the r Pf SRA-F3 a protein was introduced into the nucleus through KPNA,it combined with C23 in the nucleus,resulting in a decrease in the binding and stabilizing effect of C23 on Bcl-2 m RNA,and the activation of NF-κB and MAPK signaling pathways,thereby regulating the apoptosis of HUVECs.This study provides new research evidence for elucidating the mechanism of vascular endothelial cell apoptosis caused by Plasmodium falciparum and provides a theoretical basis for the prevention and control of falciparum malaria. |
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