| Malaria,which is caused by Plasmodium spp,remains one of the most serious parasitic diseases.According to WHO World Malaria Report 2018,an estimated 219 million cases of malaria occurred worldwide in 2017 and estimated deaths due to malaria declined to 435000 cases.There was a slight upward trend in the number of malaria cases between 2015 and 2017,indicating that no significant progress was made in the prevention and control of malaria at this stage.Currently,insecticide-treated mosquito nets,rapid diagnostic tests,artemisinin-based combination therapy and other control measures have achieved remarkable results.However,the emergence of insecticide-resistant Anopheles mosquitoe and artemisinin-based resistant Plasmodium spp.pose a great challenge to the elimination of malaria worldwide.Five plasmodial species present a significant health threat for humans.In terms of complicated pathogenic mechanism,wide transmission range,high fatality rate and serious harm.Plasmodium falciparum is usually considered the most important.The life cycle of P.falciparum is characterized by alternation between female Anopheles mosquitoes and vertebrate hosts.The intraerythrocytic development cycle represents all of the stages in the development of P.falciparum responsible for all clinical symptoms of malaria,and the replication of parasites can not take place outside the host cells.Therefore,the intraerythrocytic development cycle is a critical stage for the survival and pathogenesis of Plasmodium spp.,and erythrocyte invasion by merozoites is most important step.A better understanding of the cellular and molecular mechanisms and the host immune responses during this stage is indispensable to the study of pathogenesis and vaccines of malaria parasites.The P.falciparum 3D7 nuclear genome is composed of 22.8 megabases distributed among 14 chromosomes.Of the 5,268 predicted proteins,about 60%did not have the sufficient similarity with proteins in other organisms to provide functional annotations.Since routine genetic tools for studying Plasmodium parasites are time-consuming,inefficient and technically demanding,many high throughput methods are used to collect gene functional informations.And gene expression profile analysis has become an important method to study gene functions.The analysis for expression profiling of the.falciparum intraerythrocvtic development cycle is generated by measuring relative mRNA abundance levels in samples collected from a highly synchronized in vitro culture of parasites.The common methods include oligonucleotide microarray,RNA sequencing and real-time quantitative PCR.To provide more accurate results,real time PCR is usually used to detect the expression level of a single gene,and validate microarray or sequencing results.This study undertakes parts of the research task of "Research on the basis and control technology of important parasitic diseases",which belongs to the medical and health science technology innovation project of the Chinese Academy of Medical Sciences.In this study,we screen and perform functional study to find novel ligands for host erythrocytes invasion by P.falciparum.Highly synchronized parasites were collected at six different time points in the intraerthrocytic development cycle.Real time PCR was used to analyze the transcription levels of 243 protein-coding genes on chromosome 3 and 246 protein-coding genes on chromosome 4 of P.falciparum.After gene cluster analysis,the genes with high specific expression level at Oh after red blood cell invasion were selected for subsequent functional analysis.Among the genes in chrosome 3 with high specific expression level at Oh,four genes were selected after literature reading and bioinformatics analysis and all soluble recombinant proteins were obtained.In erythrocyte binding assays.two recombinant proteins were eluted from red blood cells which indicate they have the ability to bind to the surface of erythrocytes.To determine the properties of receptors on the erythrocyte membrane,we tested the ability of the two recombinant proteins that if they could bind to enzyme-treated red blood cells.The results show that the binding of the two recombinant proteins to erythrocyte is sialic acid independent and has no relation with the outside regions of glvcophorin A and C.Then the two parasite proteins were cloned and expressed in CHO cells and RBCs could bind to CHO cells expressing two parasites proteins separately.According to the results of heparin binding and heparin binding inhibition assays,the two parasite proteins can bind to heparin-like receptors on erythrocyte surface.Then parasite proteins were detected by Western blot using polyclonal antibody.And the cellular locations of target proteins were obtained by immunofluorescence assays.The serum of patients infected with Plasmodium falciparum.other fever patients and healthy volunteers were collected to investigated the antigenicity of the proteins by enzyme-linked immunosorbent assays.Finally,combined with in vitro invasion inhibition assays,the potential cell functions and potential antimalarial infection values were further evaluated.For the first time,the transcriptome analysis of protein-coding genes on chromosome 3 and 4 of P.falciparum throughout the intraerythrocytic development was carried out by real-time PCR.These results improved the transcriptomics data of.falciparum obtained by microarray and sequencing,and play a positive role in the in-depth understanding of intraerythrocytic development cycle of parasites.Subsequent functional studies by screening genes which are highly expressed when merozoites invade RBCs provide clues for the identification of new invasion-related parasite antigens and potential chemotherapeutic or vaccine candidates. |
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