| Purpose:Malaria is an infectious parasitic disease caused by the transmission of malaria parasites through Anopheles mosquitoes.It is one of the most serious public health problems in the world.Traditional measures to eliminate malaria have their limitations,and the incidence and mortality of malaria in some areas are still increasing.Establishing new malaria prevention and control strategies and speeding up the elimination of malaria are important issues that need to be resolved urgently in the world today.Transmission-blocking vaccines(TBVs)are considered to be one of the effective methods.The basic principle of transmission blocking vaccine TBVs is to immunize individuals with surface antigens in the sexual reproduction stage of Plasmodium(including the two stages before and after the fertilization of the male and female gametes)to induce the body to produce antibodies.The mosquitoes suck human blood and inhale at the same time.The specific antibodies produced by the human body then undergo an antigen-antibody reaction in the mosquito,which affects the sexual reproduction and spore proliferation of the malaria parasite,thereby preventing the subsequent development of the malaria parasite in the mosquito and blocking the transmission of the malaria parasite.In the study of TBVs,many antigens were found to be expressed on the surface of male and female gametes,zygote,and zygote,but only a few proteins have clear transmission blocking activity.The insufficient candidate antigens for effective malaria vaccines hindered the development of vaccines.Therefore,screening new candidate antigens for the sexual stage of Plasmodium,determining its molecular structure and immunological characteristics,and developing efficient and safe TBV vaccines have become a key strategy for malaria prevention and control.Based on the importance of TBVs in the elimination of malaria,in this study,we used bioinformatics,molecular biology and immunology and other related experimental techniques to screen out the Pb33 membrane surface protein of Plasmodium berghei,and clarified its expression characteristics and transmission interruption.active.This research provides necessary theoretical and experimental basis for Pb33 as a new candidate antigen for malaria transmission blocking vaccine.Research methods:1.Materials.Prokaryotic expression vector p ET-32a(+);Escherichia coli DE3 strain;Plasmodium berghei ANKA strain(P.berghei ANKA)is a gift from****university;BALB/c mouse was purchased from Beijing Weitong Lihua Experimental Animal Technology Co.,Ltd.;Anopheles stephensi.Animal experiments were approved by the***animal ethics committee.2.Bioinformatics analysis.Plasmo DB(http://www.plasmodb.org/plasmo/)was used to screen out the PBANKA＿081330(P.berghei protein 33)gene sequence and its homologous and upstream and downstream genes,and BLAST was used for gene comparison.Predict the signal peptide and domain of the protein online in SMART(http://smart.embl-heidelberg.de/).primer-premier 5.0 software design primer.Use software to draw a plasmid map.DOG 2.0 requires software to draw protein pattern diagrams.3.Construct Pb33-HA.In order to clarify the expression stage of the target gene,we added tags through homologous recombination technology to construct a Pb33-HA strain.4.Use IFA to detect the expression stage of Pb33.The plasmodium was washed with PBS and fixed with 4%paraformaldehyde.The cells were permeabilized with 0.1%Triton X-100,and then blocked with PBS at 37°C after rinsing.Add diluted rabbit anti-HA tag and incubate with mouse Pbs21 m Ab.Add Alexa Fluor555/anti-rabbit Ig G and incubate with FITC-labeled anti-mouse Ig G.After DAPI staining,fluorescence microscope observation,Adobe Photoshop analysis results.5.Acquisition and identification of the knockoutΔPb33 Plasmodium strain.The 5’UTR fragment was amplified by PCR and cloned into the PL0034 vector to construct the PL0034-Pb33-5U plasmid.Then amplify the 3’UTR fragment and clone it into the previously successfully constructed vector,and use double enzyme digestion to identify the successfully constructed PL0034-Pb33-5U-3U targeting vector.6.Observe the infectivity ofΔPb33Plasmodium in the blood of mice.Five groups of female BALB/c mice,of which 4groups were injected intraperitoneally with 1×10~6ΔPb33 P.berghei Plasmodium,and the1 group was injected with WT P.berghei.Starting from the third day after infection,the Giemsa method was used for staining.,Observe the red blood cell infection in the peripheral blood of mice under the microscope.And take blood to observe the gametophyte rate and the ratio of male and female gametophytes between groups.7.Observe the influence ofΔPb33 Plasmodium on the development of sexual stage.(1)Sexual stage in vitro:a.BALB/c female mice were injected intraperitoneally withΔPb33or WT P.berghei Plasmodium 1×10~6P.berghei,on the 3rd day of infection,blood was collected from the tail vein to detect gametophyte silkiness and female sex Gamete activation.The plasmodium is cultured at 19 to 20°C for 24 hours.The cultures were fixed and labeled,and the zygote formation rate was observed under a fluorescence microscope.b.Five minutes before activation or incubation,WT andΔPb33 gametophyte samples were treated with different concentrations of saponin to lyse the red blood cell membrane,and the in vitro gametophyte silking experiment and the motility formation rate experiment were repeated.It can be distinguished that the gametophytes of theΔPb33 group cannot synthesize flagella and participate in the formation of motility zygote,or that flagella can be formed in the cytoplasm but cannot penetrate the envelope,resulting in the inability to form motility.c.Previous studies have confirmed thatΔPb48/45 can block male gametophytes from producing silk,andΔPb47 can block the activation of female gametes.Combine samples of WT,ΔPb48/45,ΔPb47,andΔPb33gametophytes with the same total number of gametophytes for fertilization test.(2)Oocyst formation rate and mosquito infection rate:mice were injected intraperitoneally with 1×10~6ΔPb33 and P.berghei in the WT group,and fed at least 50 female Anopheles mosquitoes for 30 minutes after 3 days.For 10 days,check the number of oocysts on the stomach wall of mosquitoes and the infection rate of mosquitoes.8.The expression and purification of r Pb33.IPTG induces the p ET32a-Pb33 expression vector,after ultrasonically disrupting the bacteria,the recombinant protein is purified,and the eluted and dialyzed samples are detected by SDS-PAGE electrophoresis.9.r Pb33 immune serum specific antibody detection.On the 13th day after the initial immunization,ELISA was used to detect r Pb33 immune serum specific antibodies.Boost immunization was performed on the 28th and 42nd day after the initial immunization,and antibodies were detected by ELISA.10.Determine the expression position of Pb33.The purified P.berghei schizont(Sch),gametophyte(Gam)and motif(Ook)reacted with r Pb33 immune sera,respectively,for Western Blot detection.11.Determination of the ability of anti-Pb33 serum to block transmission in vivo.(1)In vitro sexual phase:1×10~6P.berghei infected mice,blood was collected from the tail vein on the 3rd day,r Pb33 full-length protein/truncated protein/control group immune serum was co-cultured,and the male gametophyte was observed to produce silk.Another blood was added to the motility culture medium,and recombinant full-length protein/truncated protein/control group immune serum was added,and the number of motility formed was observed by fluorescence microscope after culture and labeling.(2)Ozygote formation rate and mosquito infection rate in vivo:r Pb33 full-length protein/truncated protein/control immunized mice,infected mice after three immunizations,and fed female Anopheles mosquitoes with mice on the 3rd day.For 10 days,check the number of zygotes on the stomach wall of mosquitoes and the infection rate of mosquitoes.12.Statistical analysis.Results:1.Pb33 is a highly conserved protein,expressed in the sexual stage of Plasmodium.In Plasmo DB,we choose PBANKA＿081330(Pb33)for detailed functional research.The gene encodes 331 amino acids.SMART analysis predicts that the Pb33protein contains a signal peptide,a low-density complexity region and multiple transmembrane regions.The BLAST multiple sequence alignment showed that the PBANKA＿081330 gene is highly conserved in the Plasmodium species and has no homology with the biological population outside the Plasmodium.2.The Pb33 gene has no effect on the development of the blood stage of Plasmodium.The results showed that on the 3rd day after the mouse infection,the red blood cells infected by Plasmodium could be found in the peripheral blood,and then the protozoaemia of theΔPb33 and WT groups increased rapidly,but the red blood cell infection rate of theΔPb33 group was not significantly different from that of the WT group..Similarly,there was no significant difference in survival curves between theΔPb33 group and the WT group after infection with Plasmodium.In addition,on the 3rd day of the mouse infection,there was no significant difference between theΔPb33 and WT groups in the ratio of gametophytes and the ratio of male to female gametophytes.The above results indicate that the Pb33gene has no effect on the development of the blood stage of Plasmodium.3.The effect ofΔPb33 Plasmodium on the development of sexual stage.To observe the effect of PlasmodiumΔPb33 on the development of sexual stages,after infection with P.bergheiΔPb33 or WT Plasmodium,the tail vein blood was collected for in vitro gametophyte silking,activation and motor zygote formation experiments.The results showed that compared with the normal silk production of male gametophytes in the WT group,the male gametophytes of theΔPb33 group could not produce silk at all,and the female gametes of theΔPb33 group were not activated.After the male and female gametophytes were cultured for 24 hours in vitro,most of the WT group could develop into mature motility zygote,while theΔPb33 group did not see the formation of motility zygote.Oocyst formation rate and mosquito infection rate in vivo:Mice were injected intraperitoneally with 1×10~6ΔPb33 and P.berghei in the WT group,and fed at least 50female Anopheles mosquitoes for 30 minutes after 3 days.On the 10th day after the female Anopheles sucked blood,Check the number of oocysts on the stomach wall of mosquitoes and the infection rate of mosquitoes.It was found that the infection rate and the density of oocysts in theΔPb33 group were significantly reduced.Samples of WT andΔPb33 gametophytes,lysed the red blood cell membrane,and repeated the in vitro gametophyte silking experiment and the motility formation rate experiment.The experimental results showed that the gametophytes of the WT group could produce silk,but the gametophytes of theΔPb33 group did not produce silk at all.The WT group can develop mature motility zygotes,but theΔPb33 group does not.It proves that theΔPb33group gametophyte cannot synthesize flagella and participate in the formation of motifs,while the non-cytoplasmic flagella cannot penetrate the capsule and thus cannot form motifs.Combine samples of WT,ΔPb48/45,ΔPb47,andΔPb33 gametophytes with the same total number of gametophytes,and conduct a fertilization test,which proves thatΔPb33 can simultaneously affect male gametophyte production and female gametophyte activation.Therefore,the Pb33 gene plays a vital role in the production of male gametophytes,the activation of female gametes,the development of zygote and the formation of oocysts.4.Expression and purification of r Pb33.The p ET32a-Pb33expression vector was induced by IPTG,after the bacterial cells were ultrasonically disrupted,the resulting recombinant protein was purified,and the protein eluted and dialyzed samples were collected and detected by SDS-PAGE electrophoresis.Obvious target bands can be observed.5.r Pb33 immune serum specific antibody detection.On the13th day after the initial immunization,the ELISA method showed that the serum antibody level of the mice immunized with r Pb33 protein increased significantly.The booster immunization was performed on the 28th and 42nd day after the initial immunization.Compared with the control group,the specific antibody has been maintained at a higher level.6.Determination of Pb33 expression characteristics.The purified P.berghei schizont(Sch),gametophyte(Gam)and motif(Ook)reacted with r Pb33 immune sera,respectively,for Western Blot detection.Western blotting bands appeared in the schizont,gametophyte and motozygote stages,indicating that the anti-Pb33 immune serum can react with the Plasmodium antigen.IFA tests were performed on Plasmodium and Pb33 immune sera at different stages,and it was shown that Pb33 was mainly expressed in schizonts,gametophytes and motifs,and mainly expressed on the cell surface.7.Anti-Pb33 antibody can prevent the formation of some male gametophytes and zygote.In order to further determine the transmission blocking activity of the protein Pb33 expressed in the sexual stage of Plasmodium,in vitro motility formation and direct mosquito feeding experiments were used respectively.The co-cultivation of anti-Pb33 immune serum and Plasmodium can reduce the silking of male gametophytes,and the formation rate of motility zygote is significantly reduced.When the antiserum is diluted 1:10,compared with truncated protein immune serum,the full-length protein immune serum has more obvious blocking ability on male gametophyte silk and zygote formation,which also shows that anti-Pb33 antibody can prevent male gametophyte.The formation of the zygote and the zygote.Compared with the control protein immunization group,the mosquito infection rate of the r Pb33truncated protein immunization group was reduced by 6.66%,the oocyst density was reduced by 55.32%,the r Pb33 full-length protein immunization group was reduced by14.66%,and the oocyst density was reduced by 49.23%.The results of in vivo experiments showed that compared with the control group,the mosquito infection rate and the density of oocysts in the anti-Pb33 immune group were reduced.Antiserum can affect the silking of male gametophytes,interfere with the formation of zygote and oocysts,reduce the infection rate of mosquitoes,and have obvious transmission blocking effect.Conclusion:1.Pb33 is a conservative Plasmodium berghei protein with a molecular weight of 331aa,a signal peptide,a low-density complex region and multiple transmembrane domains.2.Pb33 is mainly expressed on the surface of the gametophytes and motor zygotes in the sexual reproduction stage of Plasmodium,and has no effect on the development of the blood stage of Plasmodium.3.Anti-Pb33 immune serum has a significant blocking effect on the formation of male gametophytes,zygote and oocyst formation.4.Compared with the wild-type Plasmodium,after the Pb33 gene knockout,the male gametophyte production,zygote and oocyst formation completely inhibit the mosquito infection rate by 100%.This study provides a new theoretical and experimental basis for the follow-up study of Pb33 as a candidate antigen for TBVs. |
