Preliminary Study On The Protein Expression And Function Of Plasmodium Falciparum PF3D7?1372300

Plasmodium falciparum extensively remodels host cells by translocating numerous proteins into the cytoplasm of red blood cells after invasion.Among these exported proteins,Plasmodium helical interspersed subtelomeric(PHIST)family are crucial for host cell remodelling and host-parasite interactions,and thereby contribute to malaria pathogenesis.In this experiment,we explored the function of PF3D7?1372300 in the PHISTa-like/PHIST subfamily.Bioinformatics analysis found that the PF3D7?1372300 protein has a signal peptide and without transmembrane region,indicating that the protein is a secreted protein,which contains a typical PHIST domain.Sequence alignment with homologs from other species revealed ?93% sequence identity with different Plasmodium falciparum strains and Plasmodium spp.that infects gorillas or chimpanzees.Plasmodium falciparum 3D7 were cultured in vitro,and collected at 8 h,16 h,24 h,32 h,40 h and 48 h after two consecutive synchronizations,then extracted RNA from the parasites and quantitatively reverse transcription into c DNA.The transcription level was detected by q PCR and the results showed that the gene was fully transcribed at blood stage,reached the peak of transcription at the late stage,and had a higher transcription level at the early stage.Then the PF3D7?1372300 gene fragments were amplified,and ligated to p ET-28 a and p GEX-4T-1 expression vectors,respectively,then PF3D7?1372300-His and PF3D7?1372300-GST recombinant proteins were purified.The His-tagged recombinant protein was used to immunize rabbits and rats to obtain specific serum and purify Ig G.The indirect immunofluorescence assay was used to detect the localization of PF3D7?1372300protein,and the results showed that it was spotted on red blood cells at the early stage,and during the trophozoite and schizont stages,there was fluorescence in the entire red blood cells and a large amount of fluorescence accumulation in the erythrocyte membrane,which results were consistent with the predicted result of signal peptide,indicating that the protein can be secreted into red blood cells.Meanwhile,8 h,16 h,24 h,32 h,40 h and 48 hours of parasites by percoll were collected and prepared into whole protein lysate samples,then the changes in expression levels at 6 time points at blood stage were detected by Western blot.The results showed that PF3D7?1372300 protein was expressed throughout the blood stage and the expression level at the late stage was slightly higher than that at the early and trophozoite stages.The specific interaction between the PF3D7?1372300 protein and the ATS protein of the intracellular domain of Plasmodium falciparum erythrocyte membrane protein(PfEMP1)were verified by in vitro molecular interaction and Dot blot assays,and it was found that the fluorescence of the two proteins at the trophozoite and schizont stages of the blood stage overlapped in the red blood cell membrane through the colocalization of the two proteins.Finally,the interaction sites between PF3D7?1372300 protein and ATS protein were identified through molecular dynamics simulation,and the results showed that 34 amino acids of PF3D7?1372300 protein participated in the interaction and 36 amino acids of the ATS protein were also involved in the interaction.Further PF3D7?1372300 protein was divided into two segments according to the binding sites and two recombinant proteins PF3D7?1372300-(27-129)-GST and PF3D7?1372300-(130-206)-GST were expressed,respectively.Molecular interaction instrument was used to detect the affinity between two recombinant proteins and ATS protein.The results showed that PF3D7?1372300-(27-129)-GST protein has stronger affinity with ATS protein than PF3D7?1372300-(130-206)-GST,which is consistent with the results of molecular dynamics simulations,because the protein of PF3D7?1372300-(27-129)-GST contains more interacting amino acids.This study investigated the transcription level,expression level and localization of the PF3D7?1372300 protein in the PHISTa-like/PHIST subfamily of Plasmodium falciparum.The interaction between PF3D7?1372300 and PfEMP1 ATS was verified and the interaction sites were further determined.The findings deepen our understanding of host-parasite interactions and malaria pathogenesis,and provide a basis for exploring the functions of this important protein family.