弓形虫蛋白质二硫键异构酶的免疫保护作用
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摘要
为了探讨皮下免疫弓形虫蛋白质二硫键异构酶(TgPDI)对弓形虫急性感染的保护作用,本试验通过原核表达获取了重组TgPDI,将纯化并去除内毒素的重组蛋白皮下免疫昆明小鼠,利用ELISA方法监控抗体水平变化,通过实时荧光定量PCR检测感染小鼠的组织载虫量并记录小鼠存活时间,评价TgPDI的免疫保护作用。结果显示,免疫重组TgPDI可以有效刺激小鼠产生抗体(1∶16 000);免疫该蛋白可以极显著抑制弓形虫感染小鼠血液、肝脏、脾脏和脑组织中的弓形虫增殖(P<0.01),并能延长小鼠感染后的存活时间。结果表明,皮下免疫重组TgPDI可以刺激小鼠产生保护性免疫应答,TgPDI是预防弓形虫感染的免疫候选分子。
To study the protective effect of Toxoplasma gondii protein disulfide isomerase(TgPDI),the recombinant TgPDI was obtained by prokaryotic expression in this study.After being purified and removed endotoxin,recombinant TgPDI was immunized to Kunming mice.Antibody levels were monitored by ELISA method.Fluorescence quantitative PCR was used to detect the tissue tachyzoites burden.The survival time of mice inoculated with tachyzoites was recorded to evaluate the immune protection of TgPDI.The results showed that immunization of recombinant TgPDI could effectively stimulate the production of antibody(1∶16 000),which could significantly inhibit the proliferation of tachyzoites in blood,liver,spleen and brain of mice infected with T.gondii(P<0.01).Immunization of TgPDI could prolong the survival time of mice infected with the parasites.The study indicates that subcutaneous immunization with recombinant TgPDI can stimulate mice to produce protective immune responses.TgPDI is an immunological candidate to prevent T.gondii infection.
To study the protective effect of Toxoplasma gondii protein disulfide isomerase(TgPDI),the recombinant TgPDI was obtained by prokaryotic expression in this study.After being purified and removed endotoxin,recombinant TgPDI was immunized to Kunming mice.Antibody levels were monitored by ELISA method.Fluorescence quantitative PCR was used to detect the tissue tachyzoites burden.The survival time of mice inoculated with tachyzoites was recorded to evaluate the immune protection of TgPDI.The results showed that immunization of recombinant TgPDI could effectively stimulate the production of antibody(1∶16 000),which could significantly inhibit the proliferation of tachyzoites in blood,liver,spleen and brain of mice infected with T.gondii(P<0.01).Immunization of TgPDI could prolong the survival time of mice infected with the parasites.The study indicates that subcutaneous immunization with recombinant TgPDI can stimulate mice to produce protective immune responses.TgPDI is an immunological candidate to prevent T.gondii infection.
引文
[1] DUBEY J P.Advances in the life cycle of Toxoplasma gondii[J].Int J Parasitol,1998,28(7):1019-1024.
[2] BAHIA-OLIVEIRA L M,JONES J L,AZEVEDO-SILVA J,et al.Highly endemic,waterborne toxoplasmosis in north Rio de Janeiro state,Brazil[J].Emerg Infect Dis,2003,9(1):55-62.
[3] XUE,J X,JIANG W,CHEN Y J,et al.Twenty-six circulating antigens and two novel diagnostic candidate molecules identified in the serum of canines with experimental acute toxoplasmosis[J].Parasit Vectors,2016,9(1):374.
[4] CARRUTHERS V B.Host cell invasion by the opportunistic pathogen Toxoplasma gondii[J].Acta Trop,2002,81:111-122.
[5] ZHOU X W,KAFSACK B F C,COLE R N,et al.The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins[J].J Biol Chem,2005,280:34233-34244.
[6] TURANO C,COPPARI S,ALTIERI F,et al.Proteins of the PDI family:unpredicted non-ER locations and functions[J].J Cell Physiol,2002,193:154-163.
[7] XIAO R,WILKINSON B,SOLOVYOV A,et al.The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum[J].J Biol Chem,2004,279:49780-49786.
[8] FLORENT I,MOURAY E,DALI ALI F,et al.Cloning of Plasmodium falciparum protein disulfide isomerase homologue by affinity purification using the antiplasmodial inhibitor 1,4-bis[3-[N-(cyclohexyl methyl) amino]propyl]piperazine[J].FEBS Lett,2000,484:246-252.
[9] BEN ACHOUR Y,CHENIK M,LOUZIR H,et al.Identification of a disulfide isomerase protein of Leishmania major as a putative virulence factor[J].Infect Immun,2002,70:3576-3585.
[10] PADILLA A,NOIVA R,LEE N,et al.An atypical protein disulfide isomerase from the protozoan parasite Leishmania containing a single thioredoxin-like domain[J].J Biol Chem,2003,278:1872-1878.
[11] 薛俊欣.弓形虫急性感染犬血清中标志蛋白的筛选及其功能与应用研究[D].江苏南京:南京农业大学,2016.
[12] CONG H,MUI E J,WITOLA W H,et al.Towards an immunosense vaccine to prevent toxoplasmosis:protective Toxoplasma gondii epitopes restricted by HLA-A*0201[J].Vaccine,2011,29:754-762.
[13] YIN H,ZHAO L,WANG T,et al.A Toxoplasma gondii vaccine encoding multistage antigens in conjunction with ubiquitin confers protective immunity to BALB/c mice against parasite infection[J].Parasit Vectors,2015(8):498.
[14] CHEN J,LI Z Y,HUANG S Y,et al.Protective efficacy of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) adjuvated with recombinant IL-15 and IL-21 against experimental toxoplasmosis in mice[J].BMC Infect Dis,2014,14:487.
[15] HUANG X,LI J,ZHANG G,et al.Toxoplasma gondii:protective immunity against toxoplasmosis with recombinant actin depolymerizing factor protein in BALB/c mice[J].Exp Parasitol,2012,130:218-222.
[16] YU Q,HUANG X,GONG P,et al.Protective immunity induced by a recombinant BCG vaccine encoding the cyclophilin gene of Toxoplasma gondii[J].Vaccine,2013,31:6065-6071.
[17] COSTA-SILVA T A,MEIRA C S,FERREIRA I M,et al.Evaluation of immunization with tachyzoite excreted-secreted proteins in a novel susceptible mouse model (A/Sn) for Toxoplasma gondii[J].Exp Parasitol,2008,120:227-234.
[18] KHOSROSHAHI K H,GHAFFARIFAR F,SHARIFI Z,et al.Comparing the effect of IL-12 genetic adjuvant and alum non-genetic adjuvant on the efficiency of the cocktail DNA vaccine containing plasmids encoding SAG-1 and ROP-2 of Toxoplasma gondii[J].Parasitol Res,2012,111:403-411.
[19] MACHADO A V,CAETANO B C,BARBOSA R P,et al.Prime and boost immunization with influenza and adenovirus encoding the Toxoplasma gondii surface antigen 2 (SAG2) induces strong protective immunity[J].Vaccine,2010,28:3247-3256.
[20] WU X N,LIN J,LIN X,et al.Multicomponent DNA vaccine-encoding Toxoplasma gondii GRA1 and SAG1 primes:anti-Toxoplasma immune response in mice[J].Parasitol Res,2012,111:2001-2009.
[21] ISMAEL A B,DIMIER-POISSON I,LEBRUN M,et al.Mic1-3 knockout of Toxoplasma gondii is a successful vaccine against chronic and congenital toxoplasmosis in mice[J].J Infect Dis,2006,194:1176-1183.
[22] MEVELEC M N,DUCOURNAU C,BASSUNY ISMAEL A,et al.Mic1-3 knockout Toxoplasma gondii is a good candidate for a vaccine against T.gondii-induced abortion in sheep[J].Vet Res,2010,41:49.
[23] XUE J X,JIANG W,CHEN Y J,et al.Thioredoxin reductasefrom Toxoplasma gondii:an essential virulence effector with antioxidant function[J].FASEB J,2017,31:4447-4457.
[24] KWOK L Y,SCHLUTER D,CHRISTINE C,et al.The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury[J].Mol Microbiol,2004,51:47-61.
[2] BAHIA-OLIVEIRA L M,JONES J L,AZEVEDO-SILVA J,et al.Highly endemic,waterborne toxoplasmosis in north Rio de Janeiro state,Brazil[J].Emerg Infect Dis,2003,9(1):55-62.
[3] XUE,J X,JIANG W,CHEN Y J,et al.Twenty-six circulating antigens and two novel diagnostic candidate molecules identified in the serum of canines with experimental acute toxoplasmosis[J].Parasit Vectors,2016,9(1):374.
[4] CARRUTHERS V B.Host cell invasion by the opportunistic pathogen Toxoplasma gondii[J].Acta Trop,2002,81:111-122.
[5] ZHOU X W,KAFSACK B F C,COLE R N,et al.The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins[J].J Biol Chem,2005,280:34233-34244.
[6] TURANO C,COPPARI S,ALTIERI F,et al.Proteins of the PDI family:unpredicted non-ER locations and functions[J].J Cell Physiol,2002,193:154-163.
[7] XIAO R,WILKINSON B,SOLOVYOV A,et al.The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum[J].J Biol Chem,2004,279:49780-49786.
[8] FLORENT I,MOURAY E,DALI ALI F,et al.Cloning of Plasmodium falciparum protein disulfide isomerase homologue by affinity purification using the antiplasmodial inhibitor 1,4-bis[3-[N-(cyclohexyl methyl) amino]propyl]piperazine[J].FEBS Lett,2000,484:246-252.
[9] BEN ACHOUR Y,CHENIK M,LOUZIR H,et al.Identification of a disulfide isomerase protein of Leishmania major as a putative virulence factor[J].Infect Immun,2002,70:3576-3585.
[10] PADILLA A,NOIVA R,LEE N,et al.An atypical protein disulfide isomerase from the protozoan parasite Leishmania containing a single thioredoxin-like domain[J].J Biol Chem,2003,278:1872-1878.
[11] 薛俊欣.弓形虫急性感染犬血清中标志蛋白的筛选及其功能与应用研究[D].江苏南京:南京农业大学,2016.
[12] CONG H,MUI E J,WITOLA W H,et al.Towards an immunosense vaccine to prevent toxoplasmosis:protective Toxoplasma gondii epitopes restricted by HLA-A*0201[J].Vaccine,2011,29:754-762.
[13] YIN H,ZHAO L,WANG T,et al.A Toxoplasma gondii vaccine encoding multistage antigens in conjunction with ubiquitin confers protective immunity to BALB/c mice against parasite infection[J].Parasit Vectors,2015(8):498.
[14] CHEN J,LI Z Y,HUANG S Y,et al.Protective efficacy of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) adjuvated with recombinant IL-15 and IL-21 against experimental toxoplasmosis in mice[J].BMC Infect Dis,2014,14:487.
[15] HUANG X,LI J,ZHANG G,et al.Toxoplasma gondii:protective immunity against toxoplasmosis with recombinant actin depolymerizing factor protein in BALB/c mice[J].Exp Parasitol,2012,130:218-222.
[16] YU Q,HUANG X,GONG P,et al.Protective immunity induced by a recombinant BCG vaccine encoding the cyclophilin gene of Toxoplasma gondii[J].Vaccine,2013,31:6065-6071.
[17] COSTA-SILVA T A,MEIRA C S,FERREIRA I M,et al.Evaluation of immunization with tachyzoite excreted-secreted proteins in a novel susceptible mouse model (A/Sn) for Toxoplasma gondii[J].Exp Parasitol,2008,120:227-234.
[18] KHOSROSHAHI K H,GHAFFARIFAR F,SHARIFI Z,et al.Comparing the effect of IL-12 genetic adjuvant and alum non-genetic adjuvant on the efficiency of the cocktail DNA vaccine containing plasmids encoding SAG-1 and ROP-2 of Toxoplasma gondii[J].Parasitol Res,2012,111:403-411.
[19] MACHADO A V,CAETANO B C,BARBOSA R P,et al.Prime and boost immunization with influenza and adenovirus encoding the Toxoplasma gondii surface antigen 2 (SAG2) induces strong protective immunity[J].Vaccine,2010,28:3247-3256.
[20] WU X N,LIN J,LIN X,et al.Multicomponent DNA vaccine-encoding Toxoplasma gondii GRA1 and SAG1 primes:anti-Toxoplasma immune response in mice[J].Parasitol Res,2012,111:2001-2009.
[21] ISMAEL A B,DIMIER-POISSON I,LEBRUN M,et al.Mic1-3 knockout of Toxoplasma gondii is a successful vaccine against chronic and congenital toxoplasmosis in mice[J].J Infect Dis,2006,194:1176-1183.
[22] MEVELEC M N,DUCOURNAU C,BASSUNY ISMAEL A,et al.Mic1-3 knockout Toxoplasma gondii is a good candidate for a vaccine against T.gondii-induced abortion in sheep[J].Vet Res,2010,41:49.
[23] XUE J X,JIANG W,CHEN Y J,et al.Thioredoxin reductasefrom Toxoplasma gondii:an essential virulence effector with antioxidant function[J].FASEB J,2017,31:4447-4457.
[24] KWOK L Y,SCHLUTER D,CHRISTINE C,et al.The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury[J].Mol Microbiol,2004,51:47-61.