| Toxoplasma gondii(T.gondii)is an intracellular Apicomplexan parasite that infects a wide range of warm blooded animals,and causes a zoonotic parasitosis called toxoplasmosis.Toxoplasmosis is opportunistic infections,leading to congenital infection impaired immune defense of individual serious illness and infants.This infection causes significant morbidity,costs for care and loss of productivity and suffering.The most effective measures to minimize this parasite’s harm to patients are prompt diagnosis and treatment and preventing infection.While researchers have found that some specific antigen,but there is still no effective and safe for toxoplasmosis vaccine.Some major proteins or enzymes of Toxoplasma gondii are involved in invasion by Toxoplasma.These proteins or enzymes can be used as potential vaccine antigens.This paper chooses three enzymes of Toxoplasma gondii involved in glycolysis and the energy generation process,phosphoglycerate mutase(PGAM),citrate synthase 1(CS1)and Malate Dehydrogenase(MDH).In this thesis we describe their cloning and sequence analysis,prokaryotic expression and the function of macrophages in vitro.The studies were carried out as follows:1.Cloning and sequence analysis of TgPGAM1,TgPGAM2,and TgCSlWe cloned the full-length cDNA encoding Toxoplasma gondii phosphoglycerate mutase 1(TgPGAM1)and phosphoglycerate mutase 2(TgPGAM2)and citrate synthase 1(TgCS1)from the cDNA of RH tachyzoites,respectively.The amplified product was recovered and purified,followed by ligation with pMD-19T cloning vector.Resultant ligation product was used to transform E.coli competent bacteria(DH5a).Restriction enzymes were used to digest and identify everyone positive clones with the right inserted DNA fragment,which was detected by resolving the digestion product by electrophoresis on 1%agarose gel,this step was confirmed by submitting the recombinant product for further analysis by sequencing(GENEWIZ Inc,Suzhou)and bioinformatics analysis(DNASTAR Inc.).Bioinformatics analysis revealed the following:As for TgPGAM1(798bp),the ORF of this gene encodes a protein of 29.26kDa which contains 266 amino acids,while TgPGAM2(795bp)was translated into 265 amino acids with a 29.15kDa protein deduced protein.On the other hand,TgCS1(1665bp)deduced a protein of size 61.05kDa containing 555amino acids.2.Prokaryotic expression of TgPGAMl,TgPGAM2,TgCSl and TgMDH and development of antiseraTo obtain the recombinant proteins of TgPGAM1,TgPGAM2,TgCS1 and TgMDH,the cloned DNA fragments were digested with the proper enzymes,recovered and sub-cloned into pET28a/pET32a vector,the recombinant plasmid was,after that,used to transform E.coli(BL21)and expression of the recombinant protein was induced with IPTG,and all the proteins were found to be expressed.The expressed recombinant protein was checked by running the bacterial extracts on SDS-PAGE gel,revealing band of bands of-29kDa(PGAM1),-29kDa(PGAM2),～61kDa(CS1)and～34kDa(MDH).The recombinant proteins were passed through a scheme of dialysis and refolding processes.Antisera against TgPGAM1,TgPGAM2,TgCS1 and rTgMDH,were raised after rats were experimentally immunized with purified and re-natured recombinant proteins.Animals at the end of immunization session were bled and the sera were searated and western blot technique was used for verification by probing the recombinant proteins immobilized on a proper western blot membrane with the developed anti-sera.western blot revealed a specific strong band of the expected size,That indicated recombinant protein having immunogenic.3.TgPGAM1,TgPGAM2,TgCS1 and TgMDH binding to mouse macrophages and effect on the proliferation of Ana-1 cellsThe purpose of this study was to observed four recombinant proteins binding to mouse macrophages(Ana-1 cell)and cell proliferation after incubation with recombinant protein.Mouse macrophage Ana-1 cells were incubated with different concentrations of recombinant proteinsPGAM 1,PGAM2,CS1 and MDH.The cell viability was determined by an CCK-8 assay.The binding of the recombinant proteins to Ana-1 cell was observed by immunofluorescence antibody technique.The result showed that all the four recombinant proteins can bind to Ana-1 cell,and decreased the proliferation of Ana-1 cells.4.Effects of four recombinant proteins of T.gondii on the cell apoptosis and phagocytotic of murine macrophages in vitroIn this study,we evaluated the effects of four recombinant proteins of T.gondii on murine macrophage cell line Ana-1.Mouse macrophage Ana-1 cells were incubated with recombinant proteins of different concentrations.The phagocytotic assay and cell apoptosis were analyzed by flow cytometric analysis.The results showed that rTgPGAM1,rTgPGAM2,rTgMDH and rTgCS1 of low doses increased the phagocytosis of macrophages.Moreover,rTgPGAMl,rTgPGAM2 of high doses and rTgCSl,rTgMDH of low doses induced early-stage apoptosis,rTgPGAM1,rTgPGAM 2,rTgMDH and rTgCSl of low doses induced late-stage apoptosis.5.Effects of four recombinant proteins of T.gondii on the cytokines and NO secretion of murine macrophages in vitroIn this study,we evaluated the effects of four recombinant proteins of T.gondii on murine macrophage cell line Ana-1.Mouse macrophage Ana-1 cells were incubated with recombinant proteins of different concentrations.The ELISA assay was used to examne the expression of interleukin-1β(IL-1β),interleukin-10(IL-10),tumor necrosis factor-α(TNF-α),transforming growth factor-β1(TGF-β1)of macrophages Ana-1 after incubation with recombinant proteins.The results showed that rTgPGAM1,rTgPGAM2(Except 20 and 40 μg/mL),rTgMDH and rTgCS1 of high doses upregulated the expression of anti-inflammatory cytokines interleukin-10(IL-10)and transforming growth factor-β1(TGF-β1),and pro-inflammatory cytokines tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the culture supernatant of Ana-1 cells.Moreover,Incubation with rTgPGAM1、rTgPGAM2 inhibited the production of Nitric oxide(NO),rTgMDH and rTgCS1 of high doses boosted the production of Nitric oxide(NO). |
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