| Objective:Malaria is one of the most prevalent infectious diseases in the World.In 2017,there were about 219 million cases worldwide,and about 435,000 patients died of malaria(World malaria report,2018).Although considerable progress has been made,many factors have prevented malaria eradication.In recent years,studies suggest that after invading the body,the parasite have evolved a relatively perfect mechanism,in order to evade the host immune attack,which can escape the host immune system to attack them in a variety of ways,including the simulation of the host immune molecules?resistance of plasmodium falciparum parasite expression gene polymorphism?high variability and malaria disease infected host immune system suppression,etc.Among them,the molecular simulation provides a new immune escape mechanism for plasmodium.During its long evolution,parasites have always retained homologous molecules with similar characteristics to the host,which may be due to the fact that such molecules can escape attacks from the host and thus facilitate their survival,reproduction and transmission.T cell immunomodulatory protein(TIP)is a newly discovered immunomodulatory molecule found in humans and various animals.It is a transmembrane protein consisting of 612 amino acids.Bioinformatics analysis has found that proteins similar to TIP structure exist in various plasmodium parasites.However,it is still unclear whether the structure and function of TIP-like protein is similar with TIP,and whether it can inhibit the host's immune attack ability against plasmodium.This research mainly studies the function of Plasmodium berghei T cell immune regulatory proteins(PbTIP)in the process of host immune in parasite infection.The previous research and analysis found that PbTIP is a highly conservative kind of specific membrane proteins in parasite,it exists in the Plasmodium life cycle,and have already verify its function of the host immune adjustment in vivo.However,the effect of PbTIP on immune cells such as dendritic cells is unclear.Dendritic cells(DC)are the most functional antigen-presenting cells(APC)found so far,which are distributed in lymphatic and non-dendritic tissues of the body and participate in initial and adaptive immune responses.Although DC only accounts for about 1%of the mononuclear leukocytes in peripheral blood,but they are widely distributed in peripheral tissues,and they act as immune sentries to continuously patrol and extract environmental antigens.DC can initiate immune response to control and eliminate pathogens.DC play an important role in initiating acquired immunity and inducing self-tolerance by stimulating natural T cells,this indicating that DC participates in immune response as an important immune regulator in a wide range of immune processes.This experiment stimulated DC2.4 cell respectively in vitro with rPbTIP and trPbTIP,then detected the proliferation of DC2.4 cells and the changes in the levels of signaling molecules and cytokines.The purpose of this experiment is to explored whether PbTIP could play an immunomodulatory role on DC cells in vitro and stimulate DC cells to play an immunomodulatory role on malaria infection,to provide a new direction for controling cerebral malaria,reducing the mortality of severe malaria,researching and developing of new effective malaria vaccine and seeking new targets of the malaria parasite treatmentMethod:1.The source of the trPbTIPThe previous research and analysis used Group application of bioinformatics knowledge to carry on the detailed analysis and determine the segments of 204-335 aa to express the fragment protein of PbTIP(rPbTIP),the rPbTIP was synthesized in vitro.Its bioactivity and its role in the development of malaria was demonstrated2.The acquisition of purpose gene rPbTIP and construction of expression vectorThe bioinformatics prediction software was used to perform bioinformatics analysis on homology of plasmodium TIP like proteins and to obtain the whole PbTIP gene sequence information of mice?The whole PbTIP gene fragment was synthesized by Kingsry Biotechnology and cloned into the prokaryotic expression vectors pET32a(+)-rPbTIP and pET28a-MBP-rPbTIP3.Extracellular recombinant TIP expression and purificatiThe correctly sequenced prokaryotic expression vectors pET32a(+)-rPb TIP and pET28a-MBP-rPbTIP were transformed into E.coli BL-21,and rPbTIP was induced to express by IPTG.Select the vector with large amount and high purity to express rPbTIP.Collect and purify the labeled rPbTIP.Remove the MBP after digestion by TEV,rPbTIP was collected and purified after correct verification by SDS-PAGE and LC-MS/MS(sequence covering)4.CCK-8 was used to detect the optimal time and the concentration of PbTIPDC2.4 was cultured with 96 well plate,each well was 1×104/mL.After 24 hours,trPbTIP and rPbTIP were added and the volume of culture was adjusted to 100?L/well.The concentrations of trPbTIP and rPbTIP were 1.0ng/mL,10.0ng/mL and 100.0ng/mL respectively.Meanwhile,blank holes(1640 culture medium),non stimulated holes(DC alone)and LPS induced positive control holes(LPS concentration was 10 ?g/mL)were set and cultured for 24 hours,48 hours and 72 hours.4 hours before the end of culture,10 ?L CCK-8 reagent was added into each pore,and the culture continued for 4 hours.In the absence of light,the absorbance(OD value)was measured at 450 nm on the enzyme reader5.DC2.4 signal changes after PbTIP were detected by RT-PCRDC cells were inoculated into the 6-well plate at a concentration of 105/mL.according to the CCK-8 experiment results,trPbTIP and rPbTIP at a concentration of 1.0ng/mL and 100.0ng/mL were selected to stimulate DC2.4 cells,and LPS at a concentration of 10 ?g/mL was used to stimulate DC2.4 cells as the positive control group.The culture time was 48h.Then the supernatant of each group was collected for cytokine detection,and the cells were collected according to the tradition RNA was extracted by Trizol method,and the concentration and OD value of RNA were determined.Each group of cDNA was obtained by reverse transcription.RT-PCR was used to compare the mRNA content of MyD88,NF-?B,TLR9 and TLR4 in each group.6.Detection of cytokinesThe collected DC2.4 culture supernatant from different experimental groups was used to detect several cytokines including IL-10?TNF-??TGF-? and IFN-?,respectively,according to the operation procedure of R&D Systems ELISA Kit.OD value of each indicator was detected at 450nm with an enzyme marker,and the concentration of cytokines in each experimental group was calculated according to the standard curve drawn by the Standard Kit7.Statistical analysisData were expressed as mean ± standard deviation(X±SD).SPSS 21.0 statistical software was used to process the experimental data.The inter-group differences were analyzed by independent sample t-test or one-way analysis of variance,P<0.05 was considered statistically significant,and P<0.001 was considered statistically significant.Results:1.The acquisition of purpose gene and construction of expression vectorAccording to the whole gene sequence information of PbTIP in mice newly published by Plasmodb,we commissioned Kingsry Biotechnology to synthesize the whole PbTIP extracellular outland gene fragment,and then cloned it into the prokaryotic expression vectors pET32a(+)and pET28a-MBP.The sequencing results were consistent with those published in Genebank,and the results of enzyme digestion showed that the size of the target fragment was about 120bp,which was consistent with the expectation.So the protein expression vector of rPbTIP was successfully constructed.2.The expression of recombinant proteinThe prokaryotic expression vectors were transformed into E.coli BL-21,after the inducible expression,we found that rPbTIP was expressed more by pET28a-MBP than by pET32a(+).The optimal expression condition was induced at 15? for 16 hour,and the content of rPbTIP in the supernatant of cell lysis was the highest.3.The purification of recombinant proteinAfter two steps of purification,the labeled rPbTIP was obtained.The ratio of TEV enzyme to rPbTIP was 1:20,which was digested at room temperature for 2 hours.The protein was collected after the tag protein MBP was removed,SDS-PAGE and LC-MS/MS(sequence covering)were used to confirm that the molecular weight of the protein was correct,the purity of protein was about 85%.The collected rPbTIP were detected with the BSA Kit,and the Quantized-BSA standard curve was drawn.The concentration of rPbTIP was calculated to be 0.15mg/mL.4.CCK-8 was used to detect the optimal time and concentration of PbTIPThe optimal time for PbTIP to act on DC2.4 cells was 48h.At 24h,the cell growth was not vigorous and there was no difference between groups,while at 72h,there was no significant difference between groups due to excessive cell growth.rPbTIP with a concentration of 1ng/mL may have a certain promoting effect on DC2.4 cells,P<0.05,the difference was statistically significant.rPbTIP at a concentration of 100ng/mL may have inhibitory effects on DC2.4 cells,P<0.05,the difference was statistically significant.The results of trPbTIP were basically the same as those of rPbTIP?5.DC2.4 signal changes after PbTIP were detected by RT-PCRAfter inhibiting DC2.4 cells,the mRNA expression levels of MyD88,NF-?B and TLR9 in DC2.4 cells were increased by 100ng/mL trPbTIP,P<0.001,the difference was statistically significant,but TLR4 mRNA expression was not significant.After inhibiting DC2.4 cells,the 100ng/mL rPbTIP increased the mRNA expression level of NF-?B on the surface of DC2.4 cells,P<0.001,the difference was statistically significant,while the mRNA levels of MyD88,TLR4 and TLR9 were not significantly expressed.6.Detection of cytokinesHowever,the concentration of 100ng/mL rPbTIP had a significant effect on the secretion of TNF-?,P<0.001,and the difference was statistically significant.The amount of TNF-? secreted by DC2.4 was increased by 1ng/mL and 100ng/mL trPbTIP compared with the negative control group,P<0.001,the difference was statistically significant,and the concentration of 1ng/mL trPbTIP was more significant.Conclusion:1.Low concentration of rPbTIP and trPbTIP can promote the proliferation of DC,while high concentration of rPbTIP and trPbTIP can inhibit the proliferation of DC.2.trPbTIP regulated the RNA level of NF-?B,MyD88 and TLR9 of DC signal molecules,while rPbTIP regulated the RNA level of NF-?B of DC DC signal molecules.3.High concentration of rPbTIP can stimulate the secretion of TNF-? by DC;trPbTIP can stimulate the secretion of TNF-? by DC. |
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