Preliminary Study On Screening Candidates Proteins Of Novel Plasmodium Vivax Blood Stage Vaccine By Wheat Germ Cell-free System

Objective: Plasmodium falciparum and Plasmodium vivax account for more than 95%infections among the five species of human malaria which cause a serious public health challenge.Although the mortality of P.falciparum is the highest,studies in recent years have found that the morbidity and mortality of P.falciparum and P.vivax are consistent.And because of some unique biological characteristics and geographical distribution of P.vivax,it has gradually become one of the biggest obstacles to eliminate malaria.Therefore,the development of vaccines for P.vivax is imminent.Since the clinical symptoms of malaria occur in the intra-erythrocyte stage,and the invasion of red blood cells by merozoites is critical to the development of parasites in the erythrocyte stage,the development of vaccines against merozoite antigens is particularly important.Due to transcriptomics and proteomics data of P.vivax merozoites are not available,so this study mainly focuses on the screening of mature merozoite proteins of P.vivax.The recombinant protein expressed in wheat germ cell-free expression system was preliminarily screened with the patient sera of P.vivax,so as to provide theoretical support for the future research.Methods: 1)Screening of target gene.Screening was performed using omics data on the Plasmodium Database website and MATCH function.Firstly,mature merozoite genes were screened by transcriptomics data provided on Plasmo DB website.Then,the genes co-expressed in P.vivax,P.falciparum and P.berghei and those genes containing signal peptides and/or transmembrane regions were screened by MATCH function.Finally,based on the transcription level and gene description of these genes,candidate antigens that can be used as vaccines in the erythrocyte stage are selected.2)The selection of Open Reading Frame(ORF).Based on the relevant data provided by the Plasmodium database website,the protein domain of the target gene was predicted by SMART website,the protein transmembrane domain was predicted by TMHMM Server V.2.0 website,and the B cell epitope was predicted by Immunomedicine Group website.Finally,the appropriate ORF was selected according to the above biological information analysis.3)The amplification of target gene.Firstly,the amplification templates needed for this study were prepared,which were divided into genomic DNA(g DNA)and c DNA.Genomic DNA was extracted from the blood of patients with P.vivax on the border between China and Myanmar through the blood genomic DNA extraction kit,while c DNA was mainly produced by extracting RNA from the blood of patients with P.vivax and reverse transcription with reverse transcription kit.According to the requirements of In-Fusion cloning technology,specific primers for ORF were designed,that is,the 5’end of the primer should have 15 bases homologous to the end of the vector,and the3’ end of the primer should have a specific primer sequence of the target gene.Finally,the extracted genome DNA was used as a template,the target gene fragment was obtained by PCR using Prime STAR GXL DNA polymerase,d NTP mixture,Nuclease free water and specific primers.4)Construction of protein expression vector.The target fragment was ligated with the linearized p EU vector by In-Fusion cloning technique,and the ligated product was transformed into DH5α competent cells.The successful construction of the recombinant plasmid was analyzed by PCR identification,plasmid restriction enzyme digestion identification and Huada gene sequencing.5)Expression and identification of recombinant protein.Firstly,the wheat germ cell-free expression system(WGCFS)was used to express the recombinant protein,which was divided into in vitro transcription and translation.Then,the recombinant protein was verified by Western Blot(WB).Finally,the successful expression of the recombinant protein was verified by ECL luminescence imaging.6)Reactivity of the recombinant protein with vivax malaria patients’ sera.Firstly,the blood of the P.vivax patients is centrifuged to obtain sera.Then,the protein was separated and transferred to PVDF membrane(the process is the same as the above 5).The the PVDF membrane was incubated with P.vivax patients’ sera(1:200)and rabbit anti-human Ig G H&L(HRP)secondary antibody(1:5000),respectively.Finally,image analysis was performed by chemiluminescence.Results: 1)492 target genes were successfully screened.Firstly,according to the transcriptome information provided on the Plasmo DB website,a total of 5180 mature merozoite genes expressed in P.v were screened.Then,the homologous genes expressed only in mature merozoites and containing SP and/or TMD were screened by MATCH function.Finally,492 target genes that expressed on the membrane surface were screened according to the description of gene information.2)102 target fragments were obtained successfully.In this study,100 target genes were analyzed by bioinformatics related websites,and the fragments contains more B cell epitopes and/or domains without SP and TMD were selected as open reading frames.In order to successfully use the cell-free expression system to express proteins,we selected a total of 120 ORF with no more than 500 amino acids per ORF.A total of 120 pairs of specific primers were designed for ORF.In this study,102 target fragments with single and clear bands were successfully obtained.3)82 expression vectors were successfully constructed.In this study,91 plasmids were screened by colony PCR and double restriction endonuclease digestion,and 82 expression vectors were successfully constructed by comparing with the standard sequence.4)67recombinant proteins were successfully expressed.82 successfully constructed expression vectors were expressed by WGCFS.After WB verification and ECL photoluminescence imaging,67 proteins were correctly expressed.5)The recombinant protein reacted with vivax malaria patients’ sera.The successfully expressed 67 recombinant proteins were tested with vivax malaria patients sera by WB,and seven proteins were recognized by sera from vivax malaria patients.Conclusion: 1)492 target genes screened in this study were conserved mature schizont proteins of Plasmodium vivax,which could be used as potential targets for blood stage vaccine research.2)In-Fusion cloning technique and WGCFS can effectively express the recombinant protein of the Plasmodium vivax’ mature schizont protein.3)The recombinant proteins expressed by WGCFS has antigenicity,which can be used for screening of serological reaction.