The Transportation Mechanism Analysis Of Plasmodium Falciparum SURFIN_1.1 Protein In Blood Stage Parasites

Objective:Malaria is a parasitic infectious disease which is transmitted by Anopheles mosquitoes.There are about 270 million malaria cases every year in the world,and600,000 people died of malaria infection.In human body,the development of malaria parasites can be divided into two stages:liver stage and asexual blood stage.Of which,blood stage is the one cause serious clinical symptoms during malaria infection.The parasites escape the elimination of host immune system and pathogenicity is due to parasite-induced modifications of the infected red blood cells.A series of modifications of the infected red blood cells were caused by different parasite-encoded proteins that are exported into the RBC cytoplasm.The secretion of parasite-encoded protein depends on the unique network protein transport system established by the parasite.SURFIN is a protein family of Plasmodium found in recent years,which belongs to the PNEPs?PEXEL negative exported proteins,PNEPs?protein family whose transport mechanism has not been elucidated.SURFIN protein plays an important role in the adhesion of erythrocytes infected with Plasmodium falciparum to host endothelial cells and merozoite invasion.Therefore,this study intends to select the SURFIN_1.1 protein of Plasmodium falciparum and clarify the related sequences that mediate the transport of SURFIN_1.1 protein through molecular biology and immunological techniques,so as to provide a theoretical basis for the research and development of new anti-malarial drugs.Methods:The domain and transmembrane region of SURFIN_1.1.1 were predicted by bioinformatics software.Construction C-terminal fusion GFP tagged strain of SURFIN_1.1 protein by single homologous arm recombination technique.At the same time,the expression vectors containing different domains of SURFIN_1.1 were constructed by multi-site recombination technique?MultiSite Gateway Technology?and transfected into Plasmodium falciparum 3D7 strain and transfection strain was obtained by drug screening.Western blotting?WB?and indirect immunofluorescence?IFA?were used to detect the expression and localization of SURFIN_1.1 transfected strains.Results:1.The domain region of SURFIN_1.1 were predicted by bioinformatics software.SURFIN_1.1 contains N-terminal,cysteine-rich region?Cysteine-rich domain,CRD?,variable region?Variable domain,Var?,transmembrane region?Transmembrane domain,TM?and three intracellular tryptophan-rich regions?Tryptophan-rich domains,WRDs?.2.The pSLI-2×FKBP-SURFIN_1.1-GFP vector was successfully constructed,and the SURFIN_1.1^full-GFP transfection strain was selected.IFA detection showed that the green fluorescence of SURFIN_1.1^full-GFP was punctate in the cytoplasm of infected red blood cells?infected red blood cell,iRBC?and co-located with Maurer's clefts localization protein SBP1.3.The recombinant protein of SURFIN_1.1^NCVT-Cyt?N:N-terminal;C:cysteine rich domain,CRD;V:variable domain,Var;T:transmembranedomain,TM;Cyt:cytosolicregion,Cyt?and SURFIN_1.1^N-T-Cyt were successfully expressed in Plasmodium.IFA detection showed that the recombinant proteins of SURFIN_1.1^NCVT-Cyt and SURFIN_1.1^N-T-Cyt were punctately distributed in the cytoplasm of iRBC and co-located with Maurer's clefts localization protein SBP1.4.Successful expression of SURFIN_1.1^N-T,SURFIN_1.1^N-Cyt,SURFIN_1.1^T-Cyt recombinant protein in Plasmodium.IFA detection showed that the recombinant SURFIN_1.1^N-T protein was punctately distributed in the cytoplasm of iRBC and co-located with Maurer's clefts localization protein SBP1.The recombinant SURFIN_1.1^N-Cyt,SURFIN_1.1^T-Cyt protein was localized in the cytoplasm of parasite.5.A series of vector recombinant proteins truncated at the N-terminal of SURFIN:SURFIN_1.1^N1-T-cyt?N1:1-20aa?,SURFIN_1.1^N2-T-cyt?N2:1-40aa?,SURFIN_1.1^N3-T-cyt?N3:40-50aa?,SURFIN_1.1^N4-T-cyt?N4:20-40aa?,SURFIN_1.1^N5-T-cyt?N5:10-30 aa?were successfully expressed.IFA showed that the recombinant proteins of SURFIN_1.1^N1-T-cyt and SURFIN_1.1^N3-T-cyt were located in the cytoplasmofPlasmodiumparasite.Therecombinant SURFIN_1.1^N2-T-cyt,SURFIN_1.1^N4-T-cyt,SURFIN_1.1^N5-T-cyt protein was punctately distributed in the cytoplasm of iRBC and co-located with Maurer's clefts localization protein SBP1.Conclusions:1.SURFIN_1.1 is export protein in blood stage parasites and Located at the Maurer's clefts in the cytoplasm of iRBC.2.SURFIN_1.1 N-terminal 50 amino terminal plus transmembrane region and Cytosolic tail contain protein motif to mediate protein transport to iRBC cytoplasm.3.The TM of SURFIN_1.1 protein mediates that the recombinant protein enters the classical secretion pathway through endoplasmic reticulum Golgi.4.The N-terminal sequence"RSGPFEIWRK"?21-30aa?is an important motif in the N-terminal transport of SURFIN_1.1^N-T-Cyt-T-Cyt recombinant protein.