Function Research Of Heparin Binding Protein ?PF3d7₁104400? Of Plasmodium Falciparum

Merozoites invasion into erythrocytes is the result of multiple receptor-ligand interactions in a series of successive events,which would multiply parasites heavily and lead to many pathological symptoms and changes.Many merozoite surface antigens have also shown strong potential as malaria vaccines,and sulfate heparin has been proved to be an important receptor mediating merozoites invasion and erythrocytes adhesion,so targeting merozoite surface proteins and sulfate heparin are believed to be effective methods for inhibiting growth and reproduction of Plasmodium,and also provide a new direction for the development of antimalarial vaccines and drugs.At present,proteins in the Plasmodium falciparum which have been confirmed to bind with heparin are in very limited number.Based on the important function of heparin for reproduction and pathogenicity of Plasmodium,researchers in our laboratory screened some proteins binding with heparin using affinity chromatography and mass spectrometry,including PF3D7₁104400 protein.The protein has multiple peptide fragments which can bind with heparin,so it is speculated to participate in invasion and adhesion of parasites.In the paper,we try to explore the sequence characters,conserved domain,transcription level,expression level,subcellular localization and function of the protein for revealing its role in merozoites invasion and erythrocyte adhesion and analyzing its availability as vaccine candidate antigen.Conserved Trx domain and GAG-binding motifs were found in PF3D7₁104400protein sequence by bioinformatics analysis.According to the location of Trx domain and GAG-binding motifs in the sequence,PF3D7₁104400 protein was divided into three fragments?PF3D7₁104400-N888,PF3D7₁104400-C522,PF3D7₁104400-1101?to express in Ecoli and purify recombinant proteins separately.Polyclonal antibodies were prepared by immunizing rabbits using His-tagged recombinant proteins,and then the specifity of purified antibodies were detected by Western blot.After obtaining antibodies and parasites samples,transcription and expression levels of PF3D7₁104400 gene and subcellular localization of its encoded protein wereinvestigated by RT-q PCR,Western blot,indirect immunofluorescence assay?IFA?and immune-electron microscopy?IEM?.Then erythrocyte-adhering and heparin-binding activities of the protein were analysed using GST-tagged recombinant proteins of three fragments,which were followed by invasion inhibition assay in vitro using specific Ig G of three fragments for exploring the role of PF3D7₁104400 protein in the merozoites invasion into erythrocytes.In the immunization protection assay,PBANKA₀942500 protein,orthologues of PF3D7₁104400 in the P.berhgei ANKA strain,was expressed in Ecoli and purified according to the manufacturer's instructions.Then we choosed His-tagged recombinant PBANKA₀942500 protein as immunogen to immunize mice for furher investigating the effect of immune protection of PBANKA₀942500-specific antibodies against parasite infection in mice and availability of PF3D7₁104400 as a potential vaccine candidate antigen.Via detections of SDS-PAGE and Western blot,purified recombinant proteins showed pretty high concentration and purity and prepared polyclonal antibodies were considerably specific,which could be applied for further research.RT-q PCR and Western blot showed that PF3D7₁104400 gene was transcribed and expressed in all stages after parasites invasion into erythrocytes.PF3D7₁104400 protein was localized on the surface of merozoites by analyzing the results of IFA and IEM.Furthermore,PF3D7₁104400 protein exhibited erythrocyte-adhering and heparin-binding activities,and PF3D7₁104400-N888 peptide fragment with Trx domain was necessary for PF3D7₁104400 protein to bind with erythrocytes.In addition,anti-PF3D7₁104400 specific antibody could inhibit merozoites invading into erythrocytes in vitro.The result of immunization protection assay indicated PBANKA₀942500-specific antibodies in mice can reduce parasitaemia and delay death dates of mice,and protect mice against parasite infection.In summary,PF3D7₁104400 protein is one of important ligands on the surface of merozoites participating in invasion of erythrocytes,and the protein is more likely to be a potential malaria vaccine candidate antigen.