| Objective:Malaria is the most serious parasitic infectious disease transmitted by the malaria parasite through the vector insect mosquito.Hundreds of thousands of people diefrom malaria.The development of Plasmodium is through three stages:intrahepatic,erythrocyte and mosquito,as well as vertebrate and insect mosquito hosts.Thegametocyte is the only bridge between the host of vertebrate and the host of mosquito,and is the key between human and mosquito transmission.During the development of Plasmodium,there is a phenomenon of natural transmission blocking,that is,with the proliferation of Plasmodium,the number of gametophytes increases but the infectivity of gametocytes decreases.It shows that gametocyte is an important link in thedevelopment of Plasmodium.Exploring its mechanism of blocking naturaltransmission will undoubtedly provide new scientific ideas and solutions for blocking human mosquito transmission and eradicating malaria.In this study,by comparing the differences in the expression proteins of Plasmodium gametocytes at different timepoints after infection,a preliminary screening of related protein genes affecting the viability of Plasmodium gametocytes was performed,and biological functions were predicted for candidate genes.In this study,proteomics methods were used tocompare the differences in the expression proteins of Plasmodium gametocytes atdifferent times after infection.The relevant protein genes that affected the viability of Plasmodium gametocytes were initially selected,and candidate genes were predicted by biological recombination technology.Methods:1.Establishment of mouse malaria model and detection of gametocyte activity.At6-8 weeks,female BALB/c mice were intraperitoneally injected with 1×10~6wild-type Plasmodium yoelii and Plasmodium berghei to infect red blood cells,and the red blood cell infection of the mice was monitored dynamically on thesecond day after infection Rate and gametocyte rate;male gamete exflagellation;number of zygote cultured in vitro.2.Screen for protein genes with differential expression of Plasmodium gametocyte.Density gradient centrifugation purification of Plasmodium mature schizonts andgametocyte proteins on days 3 and 5 after infection,trypsinization,Tandem MassTag?(TMT)labeling,high performance liquid chromatography fractionation,liquid chromatography-mass spectrometry Tandem analysis,using 1.5 as thecoefficient of difference,genes with differential expression of gametocyteproteins at different times were obtained.Proteomics analysis and networkinformation resource query are used to predict bioinformatics and evaluateprotein function of differential proteins.3.Establishment of HA tag gene recombinant strains.The HA tag strain wasconstructed by the gene double-crossover homologous recombination technology,the plasmid was transfected into mature Plasmodium schizonts,and the micewere injected intravenously.Extract the genome for PCR identification.4.Basic biological analysis of candidate genes.Indirect immunofluorescence assay(IFA)was used to determine the localization and expression period of HA-taggedprotein.Western blotting(WB)was used to verify the molecular weight ofHA-tagged protein.Results:1.The murine malaria model simulates the phenomenon of natural transmissionblocking.The gametocyte rate of wild-type P.yoelii and P.berghei increased withthe increase of the infection rate,and the number of exflagellation and zygoteswas the highest on the third day after infection.There was a peak of vitality onthe 3rd day and then a downward trend,which did not increase with the increaseof the infection rate.2.Proteomics results suggest that as the plasmodium infection progresses,genesthat down-regulate protein expression mainly occur during the gametocyte phase.There were 167 protein expressions upregulated and 106 protein expressionsdownregulated on the 5th day compared with the 3rd day after infection.ThePlasmoDB database predicts that among the 167 up-regulated genes,101 genesare transcribed in the erythrocyte period,29 genes are transcribed in thegametocyte phase,and 37 genes are found in both periods.Erythrocyte intraphasetranscription genes accounted for 82.63%of the total up-regulated genes;9 of the106 down-regulated genes were erythrocyte intraphase transcription genes,81gametocyte phase transcription genes,and 16 genes were found in both periods.The gametocyte phase transcribed genes accounted for 91.51%of the totaldown-regulated genes.3.Functional analysis of the down-regulated protein revealed that its functions aremainly concentrated in the following categories:triphosphate hydrolase domain,Dynein domain,Kinesin domain,6-Cys and leucine Domain.These structuresplay an important role in gametocyte development and male-female gametocytebinding.4.Network information resources screening of genes that affect gametocytefunction.Through network information resources,81 gametocyte phasedown-regulated genes with signal peptide,transmembrane protein or GPIstructure in structure are predicted.Genes with domain function are predicted ascandidate candidates Genes,preliminary screening of 20 genes that meet theabove conditions.Among them,genes that meet the above conditions,such asP48 and P230,have proven to be important genes in the development ofPlasmodium gametocytes.5.Functional prediction of candidate genes.Five HA tag P.berghei strains weresuccessfully constructed by genetic recombination technology;the molecularweight identification and protein location of Pb120990 gene was completed.Conclusions:1.Proteomics experiments confirmed that the protein expression of Plasmodiumgametocytes with important biological functions was down-regulated as theinfection progressed.These down-regulated proteins weakened the viability ofthe Plasmodium gametocyte;2.Proteomics results suggest that natural transmission is blocked during theinfection of Plasmodium,which occurs during the infection period of thevertebrate host;3.Proteomics experiments combined with network information resource screeningand genetic recombination experiments can provide laboratory evidence forrevealing the mechanism of natural transmission blocking. |
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