| Objective: Malaria is a disease caused by Plasmodium parasites in humans and is one of the most serious infectious diseases.2022 World Malaria Report released by WHO shows that malaria cases are still on the rise from 2020 to 2021,with 619,000 malaria deaths worldwide in 2021.Current malaria control relies heavily on the use of vaccines,and the life history of Plasmodium is so complex that malaria vaccines should ideally target multiple developmental stages and multiple malaria parasite species.Based on the life cycle of Plasmodium,malaria vaccines are divided into three main categories:pre-erythrocytic vaccines(PEVs),intraerythrocytic vaccines(BSVs),and transmission-blocking vaccines(TBVs)for the mosquito stage,of which the most widely sought-after is the malaria transmission-blocking vaccine.Significant efforts have been put worldwide to research malaria vaccines,with children in areas of moderate transmission receiving some degree of protection after RTS,S vaccination,and a marked increase in rebound of clinical malaria infection after 3 to 6 years.The development of more novel and effective transmission-blocking vaccine candidate antigens is also needed to achieve the goal of malaria elimination.In this study,a conserved gene PBANKA＿1452300,which is expressed in the sexual stage of Plasmodium berghei,was identified through a search of the Plasmo DB database.The immunogenicity of the protein and its localization in the developmental stages of Plasmodium berghei were investigated by indirect immunofluorescence(IFA),respectively,and the effect of anti-rPb25 antibody on the development of Plasmodium berghei in mice was evaluated,and the transmission-blocking activity of the above antibody was verified by direct mosquito feeding assay.Methods: 1.Candidate gene screening.Search qualification was set by database Plasmo DB;homology was analyzed by applying Clustal W;transmembrane region and structural domain of target protein were analyzed by Tm Hmm2.0;antigenic determinant cluster was analyzed by Kolaskar and Tongaokar.2.Expression and purification of recombinant protein.We selected a 100 amino acid fragment(92-192aa)containing only one transmembrane region for protein expression,ligated the fragment into the linearized vector p ET32a(+)(which contains a His tag),transformed into recipient cells DE3 for expression and purified the protein.3.Immunization of experimental animals and serum collection.The recombinant protein and PBS were emulsified separately with adjuvant,and mice were injected subcutaneously with booster immunization at 14 and 28 days after the initial immunization.Blood of mice was collected from tail vein and serum was obtained.4.Detection of immunogenicity of recombinant protein.ELISA was performed to detect rPb25 antibody titers.lysosomes,gametophytes and kinetochores were obtained and purified by in vitro culture to detect the reaction of worm antigen with antiserum.5.Mouse survival rate evaluation.The infection rate and survival rate of malaria parasites in mice immunized with protein were tested.6.purified lysosomes,male and female gametophytes,male gametophytes and kinetochores were labeled with anti-rPb25 mouse serum.Indirect immunofluorescence assay(IFA)was used for localization.7.In vitro kinetochore formation assay was performed to observe whether the antiserum had an inhibitory effect on male gametophyte and kinetochore formation.8.direct mosquito feeding assay was performed to observe the effect of antiserum on the number of oocysts in mosquitoes.Results: 1.rPb25 is highly homologous among different Plasmodium species,with eight antigenic determinants.analysis revealed that it is expressed in both asexual and sexual stages.transcriptomic data suggest that Pb25 is associated with the growth and development of Plasmodium,and the protein product encoded by this gene has a molecular weight of 25 k Da.2.the recombinant protein was detected by SDS-PAGE to reveal a molecular weight of about 31.4 k Da Protein bands with a molecular weight of about 31.4 k Da were seen by SDS-PAGE.3.The antibody titers of all three immunizations collected were significantly higher than those of the control group.4.The antibody titer of the last collected serum reached 1:204800,indicating that the rPb25 protein is highly immunogenic.5.The antiserum had an effect on the infection rate,gametophyte rate and survival rate of mice in vivo after protein immunization,and the antiserum produced a protective effect on mice.6.IFA showed that Pb25 fluorescence signal was weak in female gametophyte and female gametophyte stages and strong fluorescence signal on the surface of kinetochore.In in vitro culture experiments,the addition of anti-rPb25 serum at 1:5 and 1:10 dilutions to the culture medium reduced male gametophyte emergence by 66% and 58%,respectively,and reduced kinetochores by 61% and 45%,respectively.8.In direct mosquito feeding experiments,the mosquito infection rate was reduced by 5% in the anti-rPb25 serum group compared with the control group.The infection rate was reduced by 5% and the density of intestinal oocysts in mosquitoes was reduced by 90.15% compared to the control group.Conclusion: 1.The recombinant protein rPb25 was successfully expressed by prokaryotic expression system,which has good immunogenicity.2.Pb25 is expressed in both gametophytes and zygotes,and the expression level is high in the zygote stage.3.The anti-rPb25 serum showed good transmission blocking function. |
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