| Objective:Malaria,a global parasitic disease,is a serious threat to human health caused by the transmission of Plasmodium through the bite of Anopheles mosquitoes.The world health organization?WHO?has identified it as one of the top three human public health issues of the 21st century,along with tuberculosis?TB?and HIV/AIDS.Malaria accounts for a large proportion of the global burden of disease,with about 200 million cases and about 430,000 deaths each year.Although medical research over the past 15 years have made considerable progress in the eradication of malaria,but along with Anopheles of insecticide resistance enhancement,a parasite on commonly used antimalarial drug artemisinin,the development of drug resistance such as chloroquine,and at this stage is still lack of effective malaria vaccine,makes the treatment and prevention and control of malaria still is facing a difficult task.Cerebral malaria?CM?:one of the clinical types of acute malaria,which is the most common and severe.It is caused mainly by Plasmodium falciparum infection and is the leading cause of death in children under five years of age.It is widely believed that the occurrence of cerebral malaria is mainly associated with excessive inflammatory immune response in patients.Excessive production of inflammatory factors prompted the endothelial cell adhesion molecules,such as CD36,CSA and ICAM-1,which expression level was rised,increased the malaria parasite infection of red blood cells?pRBC?,lymphocyte in microcirculation of platelet aggregation and blocking microcirculation,which stimulate the endothelial cells to produce too much other material such as endothelin-1,then caused the cerebrovascular contraction,brains lack of oxygen.Histopathology also confirmed that the pathological basis of cerebral malaria was that pRBC adhered to vascular endothelial cells and remained in deep cerebral blood vessels,causing microcirculation disorders.The mechanisms that cause death are not well understood,and there are many hypotheses for the occurrence and development of cerebral malaria,but no definitive conclusions have been reached.At present,the mortality rate of CM is as high as 10-20%,accounting for 80%of the total number of malaria deaths.Even if the survivors are lucky enough to survive,there are still10%20%survivors with neurological sequelae.Therefore,it is an urgent and important issue to better study and clarify the occurrence mechanism of CM and formulate effective and reasonable prevention and treatment strategies.Research on the role of Plasmodium parasites in the process of malaria has made some new discoveries in recent years.Previous studies have shown that certain proteins expressed or secreted by Plasmodium can be involved in regulating the host's immune response.Fiscella et al.found a T cell immunomodulatory protein?TIP?in mammalian.Their study confirmed that TIP can significantly inhibit the immune rejection induced by grafts and has a similar protective effect in the rat model of host anti-acute transplantation immunity.Nono et al.found in echinococcus multiloculata that there is a TIP homologue-EmTIP protein,which can be secreted into early echinococcus multiloculata in vitro and is involved in the regulation of host Th1 immune effect.Through bioinformatics analysis and comparison,Kaczanowski et al.found that there was a protein homologue,with high homology of TIP in mammalian genome,which was marked as"Q813H7".The presence,role and function of the Plasmodium TIP homologue?PbTIP?in Plasmodium have not been reported.To this end,we identified the TIP of Plasmodium parasite by its distribution,location and whether has the same or similar functions of mammal TIP in the development process of cerebral malaria,which could have similar protective immune regulation effect.Aims to provide a basis for effective prevention and control of CM pathological damage,then develop more effective treatment strategies for cerebral malaria.Methods:1.The PbTIP was analyzed by bioinformaticsPlasmodium berghei TIP?PbTIP?gene was screened in PlasmoDB database.The characteristics of PbTIP protein were predicted and analyzed using SMART.Alignment alignment between BLAST and ClustalW alignment alignment was performed.2.Culture and isolation of P.berghei ANKA Schizont/Gametocyte/Ookinete.Schizonts:Mice were intraperitoneally injected with pRBC,and the infection rate reached about 5%10%.Hearts were collected and mixed with fission body culture medium,and 55%Nycodenz separation liquid was separated by gradient density.Gametocytes:Mice were intraperitoneally injected with pRBC,and mice were given sulfadiazine drinking water on the fourth day after infection.On the sixth day,heart blood samples were collected and placed in 48%?v/v?Nycodenz solution for gradient density separation.Ookinetes:Mice were intraperitoneally injected with pRBC,and on the third day after infection,blood was collected from the heart and mixed in the culture medium of Ookinetes.Gradient density separation of 62%Nycodenz separation solution.3.Preliminary detection of PbTIP--mRNA expression phase.pRBC infected BALB/c mice with celiac infection.Female anopheles mosquitoes fed on hungry mice fed on blood were extracted from the midgut?oocyst stage?and salivary glands?sporozoic stage?of anopheles mosquitoes on day 15 and 21 and stored in Trizol.Liver tissues of mice infected with plasmodium for 24h and 48h were collected and stored in Trizol.The Schizonts,Gametocytes and Ookinetes cultured and isolated by P.berghei ANKA were also preserved in Trizol.RNA of each sample was extracted by Trizol method.Application TaKaRa PrimeScript TMRT reagent Kit with gDNA Eraser retroviruses Kit for each sample cDNA,specific primers RT-PCR method to detect PbTIP expression in different stages of the situation.4.Cloning and identification of Plasmodium PbTIP HA-tagAccording to the experimental procedure provided by PlasmoGEM,PlasmoGEM was used to electrocute the linear PbTIP HA-tag and perform homologous recombination with the mature mitosome.The genome integrates hDHFR as a drug stress selective gene,and hdhfr-resistant Plasmodium parasites are screened by pyrimidine drugs.The resistant plasmodium was identified by PCR.5.Amplification of PbTIP gene fragment?rPbTIP?and construction of pET32a-rPbTIP expression vectorMice were intraperitoneally injected with pRBC,and the infection rate reached about 30%.Using DNA as template,specific primers were used to amplify the target gene fragment.The T4 ligase was linked to the digested prokaryotic expression vector pET32a?+?,and the linked vector was transferred to the host bacterium E.coli BL-21 for fragment protein expression.6.Expression and purification of rPbTIP fragment proteinWhen the colony was placed in LB liquid medium,OD value of the bacterial solution reached between 0.4 and 0.6,and IPTG with a final concentration of1.0mM was added for induction.After the supernatant was filtered,the protein was purified by niaminotriacetic acid agarose resin.The purified and concentrated rPbTIP was identified by 10%polyacrylamide gel electrophoresis.7.Preparation of mice polyclonal antiserumBALA/c mice were randomly divided into two groups.The experimental group was the recombinant fragment protein rPbTIP immunization group,and the control group was PBS group.Purified and concentrated rPbTIP was mixed with complete freund's adjuvant and subcutaneously injected into immune mice.Then at week 3and week 5,the mice were mixed with incomplete freund's adjuvant and given enhanced immunity twice.ELISA was used to detect the titer of polyclonal antibody.8.The rPbTIP immuno-antiserum was used to detect the nucleoprotein,cytoplasmic and membrane protein of P.berghei ANKA of Schizont/Gametocyte/Ookinete phase by Western Blot.Mice intraperitoneal injection of pRBC,infection rate of about 30%,heart blood collection.Blood cells were placed in 0.17m NH4Cl to lyse red blood cells on ice.Nycodenz separation liquid was used to conduct gradient density separation for insect bodies of each stage.The obtained insect bodies of each stage were separated and purified according to the steps of the Invent Biotechologies protein separation and extraction kit to extract different component proteins of each stage for Western Blot detection9.IFA method was used to localize the expression of rPbTIPPlasmodium was extracted from blood and fixed with 4%paraformaldehyde.The first antibody was incubated with diluted anti-rpbtip immune serum,the second antibody was added with FITC-labeled goat anti-mouse IgG,DAPI nuclear staining,observed with OLYMPUS BX53 microscope imager,and images were processed with Adobe Photoshop10.Construction and identification of a PbTIP gene knockout Plasmodium bergeni strainThe aim gene knockout plasmodium strain was constructed by double cross homologous gene recombination technique.The HDHFR expression structure was used to replace and knock out the PbTIP target gene sequence.Using P.berghei genomic DNA as template,3UTR and 5UTR were amplified by PCR.The target vector was completely linearized and electrotransfected,and the plasmodium with hDHFR knockout was screened by pyrimidine,and the peripheral blood gene was extracted for train PbTIP identification.11.Establishment and experimental grouping of mice cerebral malaria modelIn addition to the normal control group,C57BL/6 mice were randomly divided into two groups after peritoneal infection with pRBC:one group was treated with wild PbA infection+PBS tail vein injection?PBS group?,and the other group was treated with wild PbA infection+rPbTIP tail vein injection?rPbTIP group?.In the rPbTIP group,2mg/kg of rPbTIP was injected into the tail vein of mice once a day on day 0,1,3,5 and 7,respectively.12.Blood brain barrier permeability?BBB?test in miceOn the 6th day of infection,the mice showed symptoms of cerebral malaria,such as shallow breathing,weak body,paralysis of limbs,unsteady gait and paroxysmal tremor.Each group was randomly selected and injected with Evans blue dye into the tail vein.The mice were anesthetized for 1h,and the brain tissue of the mice was extracted after normal saline heart perfusion and immersed in formamide liquid.The content of Evens blus in the supernatant was detected by spectrophotometer at 630nm13.Immunohistochemistry and HE staining The mice developed symptoms of cerebral malaria on day 6 of infection.Each group was randomly selected and brain tissue was taken for histological examination.Brain tissue sections were used for hematoxylin-eosin?HE?staining and immunohistochemical staining of adhesion factors ICAM-1,VCAM-1 and CD36.To observe microvascular occlusion and leakage of cerebral tissue in rat model of cerebral malaria14.RNA extraction and real-time PCR were used to compare the cytokine levels of the two groups.Mice in each group were randomly selected on the 0,3,5 and 7 days after infection.Spleen and brain tissues of the mice were taken out under aseptic conditions,and RNA was extracted by traditional Trizol method.Application TaKaRa PrimeScriptTMRT reagent Kit with gDNA Eraser ldpe-g-nvp cDNA reverse transcription kits for each sample.cDNA was used as the template,and real-time PCR was performed for each specific primer.The RNA expressions of VCAM-1,ICAM-1 and CD36 in brain tissues and CXCL9,CXCL10 and CXCR3in the spleen of PBS group and rPbTIP group were compared.15.SplenocytosisAt the 0,3,5 and 7 days after infection,each group of mice were randomly taken out,and their spleens were taken out under aseptic conditions.Spleen cells were obtained from cell suspension prepared by grinding mouse spleen tissue in a sieve,and the final concentration was adjusted to 107/mL,which was cultured in a24-well cell culture plate for later use.16.The expression levels of cytokines in serum and supernatant of spleen cell culture were detected by ELISAAt the 0,3,5 and 7 days after infection,each group of mice were randomly selected.Blood samples were collected from the heart under sterile conditions,and serum was centrifuged.R&D Systems ELISA kit was used to detect the expression levels of IFN-??TNF-??IL-1?IL-12?IL-10 and TGF-?in the supernatant medium and serum of collected spleen cells.17.Flow cytometryOn the 0,3,5 and 7 days after infection,mice in each group were randomly selected and their spleenes were taken out under aseptic conditions.The spleen tissues of the mice were screened and ground,and the spleen cells were suspended.The cell concentration was counted,and the final concentration of the spleen cells was adjusted to 107/mL.The cells to be tested were stained with specific antibodies and fixed with 4%paraformaldehyde in each flow tube.18.Statistical methodsThe data results were analyzed and processed using SPSS 17.0 statistical software,and the mean and standard error of each group was calculated.Independent sample t test method or single factor analysis of variance were used in the intergroup differences.Kaplan-Meier long-rank test was used to analyze the survival difference of each group,and P<0.05 was considered statistically significant.Results:1.Bioinformatics analysis of PbTIP.According to PlasmoDB database,Plasmodium TIP?PbTIP?,ID:PBANKA124360,is a conserved Plasmodium protein with 703 amino acid coding proteins and molecular weight of 80.4kDa on chromosome 12.Its function is unknown at present.Bioinformatics was used to analyze that PbTIP had a small segment of signal peptide at the N end,and there were transmembrane regions at both ends of N and C.PbTIP belongs to the protein family PF13517?family:VCBS?.According to the analysis of the Pfam database.The PbTIP 204-335aa fragment domain also exists in other species.The area about100 residues,exists in multiple vibrio,slow Colwellia,rhizobia Shewanella and several other bacterial proteins,synthetic protein size,duplicate copy number of other species distribution and activity of protein hint.PbTIP aa 204-335 regions may have similar some adhesion effect,bioinformatics analysis for late PbTIP authentication and protein fragment screening2.PbTIP--detection of mRNA expression level.The liver tissues of the mice infected with pRBCs for 24h and 48h were extracted.The RNA was extracted from the Schizont/Gametocyte/Ookinete and oocyst stage and salivary gland of infected anopheles on day 15 and day 21.The cDNA was amplified by PbTIP specific primers.The results showed that PbTIP mRNA was expressed in all the above stages,indicating that PbTIP can be expressed in both the infrared and red phases of the host as well as in the body of the vector anopheles mosquitoes.So PbTIP can exist throughout the entire life cycle of the malaria parasite.3.rPbTIP fragment vector construction and fragment protein expression purification.The rPbTIP fragment gene was successfully amplified and compared with the published sequence in Genebank.Construct pET32a?+?-rPbTIP carrier after BamH?,Xho?agarose electrophoresis identification of double after enzyme digestion.As expected,that rPbTIP fragment protein carrier build is successful.E.coil BL-21 strain containing pET32a?+?-rPbTIP vector was used to express the rPbTIP recombinant protein.The protein was purified by His-tag agarose,and the sample showed clear target bands at the 35kDa position by SDS-PAGE electrophoresis.The protein purity was greater than 85%,and the concentration was about 1mg/mL.The fragment protein was detected by SDS-PAGE electrophoresis and anti-His tag antibody Western blot,and showed clear Western blotting bands at the position of 35kDa.The results confirmed the successful expression of rPbTIP protein.4.Detection of rPbTIP polyantiserum and its specificity.The recombinant protein rPbTIP immunized SPF Grade 6-8 weeks female BALB/c mice to obtain the polyclonal immune antiserum.ELISA results showed that serum specific antibodies were significantly increased in mice at week 2 after initial immunization.In the subsequent two rounds of immunization,the antibody peaked and remained at a high level.5.Western Blot-PbTIP protein expression identification.The nucleoprotein,cytoplasmic protein and membrane protein 30?g/pore were obtained from Schizont/Gametocyte/Ookinete phases.The rPbTIP multi-antibody was used to react with the PVDF membrane transfected with proteins of different components in each stage of plasmodium malaria by Western Blot.The results showed that only the membrane protein had a clear western blotting band at 80kDa.6.IFA-PbTIP detection.The obtained rPbTIP immune serum was used to locate the PbTIP plasmodium at different stages in wild P.berghei.FITC fluorescence antibody labeling and DAPI staining showed that all stages of P.berghei presented green fluorescence on the membrane surface,which was consistent with the results of Western Blot,indicating that PbTIP was a kind of specific membrane surface protein expressed in P.berghei.7.rPbTIP reduces immunopathologic damage of ECM brain tissue.In this study,the differences in the expression levels of PBS,rPbTIP,and the histopathological changes of the brain tissue were compared with that of the adhesion molecule VCAM-1,ICAM-1 and CD36.Compared with the PBS group,the ewencyan dye indicator,which was used as a blood-brain barrier in the rPbTIP group,exuded a low degree of exudation,suggesting that rPbTIP protects the integrity of the blood-brain barrier in mice.At the same time,the number of blood vessels deposited in the rPbTIP group was significantly lower than that in the PBS group.In addition,the expression of VCAM-1,ICAM-1 and CD36 in microvascular endothelial cells of rPbTIP group decreased.The mRNA expression levels of VCAM-1,ICAM-1and CD36 in the rPbTIP group were also correspondingly decreased with the spleen CXCL9,CXCL10 and CXCR3.8.Mice in PBS group died in the 6th to 12th day,but the level of plasmodium in rPbTIP group was relatively low,and less than 30%of mice died in ECM and the death occurred in the 12th day after infection.9.rPbTIP inhibits excessive expression of T cell mediated pro-inflammatory cytokines.In this study,the changes of the expression of cytokines mRNA in the serum of the two groups of IFN-??TNF-??IL-1and IL-12 in the serum of PBS and rPbTIP were compared.Compared with the PBS group,the serum concentration of I IFN-??TNF-??IL-1and IL-12 cytokines in the rPbTIP group decreased significantly,as well as the level of serum and mRNA expression levels in the culture of the spleen cells.10.rPbTIP promotes the expression of Tregs and anti-inflammatory cytokines IL-10/TGF-?.This study found that excessive rPbTIP could promote the expression level of Tregs and anti-inflammatory cytokines IL-10/TGF-?and effectively inhibit ECM.Compared with PBS group,the expression of IL-10/TGF-?increased in serum and spleen cells in rPbTIP group.It is known that the absolute value and percentage of CD4+CD25+Foxp3+Treg cells in the rPbTIP group were significantly higher than that in the PBS group.Conclution:1.PbTIP is a kind of specific membrane surface protein expressed by P.berghei.2.In the rPbTIP group of mice cerebral malaria model,immunopathological damage of brain tissue was reduced and the integrity of blood-brain barrier was enhanced.3.The high incidence of ECM was alleviated in the rPbTIP group of mice cerebral malaria model.4.In the rPbTIP group of mice cerebral malaria model excessive pro-inflammatory was decreased by Th-1 mediation.5.The expression levels of Tregs and the anti-inflammatory cytokine IL-10?TGF-?were increased in the rPbTIP group of mice cerebral malaria model. |
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