| Objective:Plasmodium is the causative agent of malaria and can infect a wide range of vertebrates worldwide,including birds,reptiles,amphibians and humans.A pathogen that spreads so widely poses a great threat to human health.There is currently no widely available malaria vaccine and the main obstacles to the development of an effective vaccine are the complex life history of the malaria parasite and the inadequate understanding of the complex immune response to malaria infection.Malaria vaccine candidates are classified according to different target antigens of the plasmodium lifecycle.Malaria vaccines can be divided into three categories according to the stage of parasite development:(1)Pre-Erythrocytic Stage Vaccines(PEVs),(2)Blood Stage Vaccines(BSVs)and(3)Transmission Blocking Vaccines(TBVs).TBVs not only effectively controls malaria,but also prevents the emergence and spread of drug-resistant or vaccine-resistant parasites.Discovering new TBVs candidate gene is one of the most critical tasks at present.TBVs blocks malaria transmission by affecting the mosquito stage of infection.Candidate antigens with good transmission blocking effect include gametocyte and gamete surface antigens P48/45 and P230,and zygote and ookinete surface antigens P25and P28.P48/45 and P230 knockout results in reduced zygote formation.P25 and P28affect numbers of ookinete and oocyst development.Studies have shown that the loss of both P25 and P28 results in almost no oocyst formation in the midgut of mosquitoes.The knockout of HAP2 affects the membrane fusion after the combination of male and female gametes.Antigens expressed in the post-fertilization stage of male and female gametes can produce longer immunity with less variation because of the absence of human immune selection pressure.Since ookinete stage is important after fertilization due to its invasion ability,the discovery of new antigens from the surface of ookinete may be an important direction for the development of TBVs.The ookinetes cultured by in vitro culture system and separated and purified by gradient density centrifugation method can be used to isolate ookinete from other stages and RBC to a great extent.The purity of ookinetes obtained is high and can be used for further research.Transcriptome refers to the transcriptional level of all genes in a cell at a given time.Transcriptomics is a subject that studies the transcription and regulation of genes in cells on an overall level.In short,transcriptomics is the study of gene expression at the RNA level.The study and comparison of the transcriptional level of plasmodium from zygote to ookinete can reveal the large amount of expression of certain genes at a certain stage,or provide information about the expression of certain genes under certain conditions,and reveal the mechanism of action of certain genes.Therefore,this study intends to identify the transcription characteristics of the ookinete by analyzing the transcriptomics of the ookinete cultured for 2h,6h,12h,24h after the combination of male and female gametes,and find the potential TBVs candidate genes.Methods:1).Culture and purification of ookinete of wild-type Plasmodium berghei.Female KM mice aged 6-8 weeks were intraperitoneally injected with 0.06mg/ml phenylhydride for 3 days and then injected 5×10~6 wild-type Plasmodium berghei infected iRBC through tail vein.When parasitemia reached about 10%,eye blood was collected and cultured for 2h,6h,12h and 24h,respectively.The culture was stained by Gimesa to identify the effect of culture.The plasmodium was purified with 62%Nycodenz solution.The purified products lysed with 0.2%saponin and were washed twice with PBS.2).Transcriptomics test.Total RNA samples were obtained,and their secondary structures were opened by thermo-suitable denaturation.mRNA was enriched with Oligo(d T)magnetic beads.Interrupting reagent was added,and the mRNA fragments were fragmented.A strand of cDNA was synthesized on the PCR instrument,and the second strand cDNA was synthesized after a certain temperature reaction time.The double-stranded cDNA ends were repaired,and A base was added to the 3’ends to prepare the joint connection reaction system,so that the joint could be connected with cDNA.After denaturing the PCR product into a single strand,the cyclization reaction system was prepared,and the single stranded ring product was obtained by sufficient mixing and proper temperature reaction for a certain period of time.After digesting the linear DNA molecules that had not been cyclized,the final library was obtained.The Agilent 2100 Bio Analyzer was used to detect the fragment size and concentration of the library.Sequencing reads of 50bp/100bp/150bp were obtained by combined probe anchoring polymerization.3).Bioinformatics analysis of transcriptomics.Screening of cultured 2h,6h,12h and24h on the base of the differentially expressed genes.According to the expression quantity do log2 conversion,the MEV was used for cluster analysis,and a group of clusters with continuously up-regulated 2h,6h,12h and 24h expression was selected to show the expression difference by heat map,GO analysis was performed on this cluster,one gene whose expression level was continuously up-regulated and expressed on the membrane during 2h,6h,12h and 24h was selected for comprehensive analysis.4).Transcriptomic data were verified by qRT-PCR.RNA from 2h,6h,12h and 24h ookinetes of wild-type P.berghei was extracted and reverse-transcripted into cDNA.The gene PBANKA＿1241500 was selected to design the qRT-PCR primer,andβ-tubulin was used as the control for the qRT-PCR experiment.The products were visualized by agarose gel electrophoresis and Tanon gel scanner.5).Construction of HA labeled strain.The HA labeled strains were constructed by double cross homologous recombination.The plasmids were electrically transfected into the mature wild-type P.berghei schizonts,and the transfected schizonts was injected into mice through the tail vein.Tagged strains with drug-resistant genes were screened by mice drinking pyrimidine to extract plasmodium genome,and PCR was used to identify the success of electrotransfer.Monoclonal experiments were carried out by limit dilution method after successful electrolysis,and PCR was used to identify the success of the monoclonal experiments.6).Protein localization of candidate genes.Indirect immunofluorescence assay(IFA)was used to detect the expression stages and locations of the proteins,and Western blotting was used to verify the expression of the proteins.Results:1).GO functional classification of transcriptome data.Bioinformatics analysis showed that after the combination of both male and female gametes develop 2h,6h,12h,24h,expression of genes can be detected 193 continues to increase,the GO function classification is displayed in a biological process(BP)the categories include organic nitrogen compounds in metabolic process,the process of biosynthesis,cell biological synthesis process,such as metabolic process of organic nitrogen compounds accounted for the highest,accounted for 8.8%;The cellular component(CC)can be divided into membrane segment,intrinsic component and cytoplasmic part,among which the membrane segment has the highest proportion(27.8%);molecular function(MF)can be divided into binding,heterocyclic compound binding and catalytic activity,among which the binding function has the highest catalytic activity,accounting for 10.0%,respectively,followed by heterocyclic compound binding function,accounting for 8%.2).Verification of transcriptome data by qRT-PCR.The qRT-PCR primers were designed according to the PBANKA＿1241500 sequence,and the qRT-PCR results showed that the expression level of PBANKA＿1241500 was continuously up-regulated during 2h,6h,12h,24h,which was consistent with the transcriptomic results.3).Prediction of protein expression stages of candidate genes.IFA and WB results showed that PBANKA＿1241500 was expressed at 2h,6h,12h and 24h and was expressed on the membrane,in the ookinete stage,it was highly expressed in the basal end.Conclusion:1).PBANKA＿1241500 is conserved in plasmodium species,and is highly expressed in the ookinete basal end;2).The ookiente transcriptome data in combination with the malaria parasite database can provide a basis for the discovery of candidate genes for transmission-blocking vaccines. |
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