弓形虫蛋白酶的鉴定及免疫学研究
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摘要
弓形虫是一种属于顶复门的专性细胞内寄生原虫,可引起弓形虫病。弓形虫病是广泛分布于温血动物包括人的一种人兽共患病,严重威胁着人类健康和畜牧业的发展,尤其对免疫功能受损的特殊人群,包括艾滋病患者、长期接受化学药物治疗的病人等往往有致命威胁。由于目前尚无针对弓形虫感染的有效治疗药物,所以加强对弓形虫病的预防工作显得尤为重要。本研究利用生物信息学的方法分析预测了弓形虫体内4种蛋白酶的基本理化性质、亚细胞定位、蛋白翻译后修饰以及B细胞和T细胞抗原表位；通过间接免疫荧光实验在激光共聚焦显微镜下观察了弓形虫天冬氨酸蛋白酶TgASP1和TgASP3在虫体内外的表达情况；通过构建相应的重组DNA疫苗来评价其免疫保护性。本研究填补了弓形虫天冬氨酸蛋白酶TgASP3的研究空白,丰富了有关弓形虫蛋白酶的基础理论知识,为进一步推进研制出安全有效的弓形虫疫苗奠定了基础。
目的：鉴定天冬氨酸蛋白酶TgASP1和TgASP3在弓形虫虫体内外的分布情况；构建重组基因疫苗,评价弓形虫半胱氨酸蛋白酶TgCPB、TgCPL及天冬氨酸蛋白酶TgASP1和TgASP3的免疫保护性。
方法：首先利用生物信息学方法来分析和预测弓形虫体内半胱氨酸蛋白酶和天冬氨酸蛋白酶的基本理化性质、亚细胞定位、蛋白翻译后修饰、B细胞抗原表位和T细胞抗原表位；然后构建重组原核表达载体转染大肠杆菌BL21(DE3)制备可溶性的重组蛋白,纯化后免疫小鼠获得特异性抗体,通过间接免疫荧光实验观察蛋白酶TgASP1和TgASP3在虫体内外的表达情况；最后通过构建重组真核表达载体接种于BALB/c小鼠评价其免疫保护性。
结果：利用生物信息学软件分析了弓形虫蛋白酶TgCPB、TgCPL、TgASP1和TgASP3的基本理化性质、亚细胞定位、蛋白翻译后的修饰等；利用多种方法预测了存在于弓形虫蛋白酶TgCPB、TgCPL、TgASP1和TgASP3上的B细胞和T细胞抗原表位,筛选出了大量亲水性好、可塑性强、可及性大和抗原指数高的潜在抗原表位区域,表明弓形虫蛋白酶TgCPB、TgCPL、TgASP1和TgASP3是良好的蛋白抗原；经PCR体外扩增获得了TgCPB、TgCPL、TgASP1和TgASP3的基因片段,大小分别为1710bp、1269bp、1863bp和1932bp；构建了重组原核表达质粒pET30a-TgCPB、pET30a-TgCPL、pET30a-TgASP1和pET30a-TgASP3及重组真核表达质粒pBudCE4.1-TgCPB、pBudCE4.1-TgCPL、pBudCE4.1-TgCPB-TgCPL、pEGFP-TgASP1和pEGFP-TgASP3,上述4种重组.原核表达质粒经转染大肠杆菌BL21(DE3)均获得高效表达的的重组蛋白,分离纯化后通过免疫BALB/c小鼠获得特异性较强的抗血清,进一步分离纯化后获得了抗上述蛋白酶的特异性抗体IgG。利用获得抗体IgG经间免疫荧光实验在激光聚焦荧光显微镜下观察了蛋白酶TgASP1和TgASP3在弓形虫体内外的表达位置及相对表达量;上述5种重组真核表达质粒通过免疫接种BALB/c小鼠,测定对照组与免疫组小鼠抗弓形虫总IgG、IgG1、IgG2a和细胞因子IL-4、IL-10、IFN-γ的水平评估了其刺激产生细胞免疫和体液免疫的能力：通过弓形虫速殖子攻击实验观测小鼠的生存时间评估了其免疫保护性。
结论：通过免疫荧光定位的方法观察了弓形虫蛋白酶TgASP1和FgASP3在弓形虫虫体内外的表达位置及相对表达量,其中TgASP1是一种膜蛋白,主要表达于弓形虫虫体顶部区域,表达区域高度集中,不呈细胞溶质状态,并且与弓形虫的主要分泌器官,包括棒状体、微线体和致密颗粒不在同一区域,可能存在介于高尔基体和内膜复合物之间的一个新的独立的区域；TgASP3蛋白几乎不在膜上分布,主要分布在细胞质中且呈现一定的细胞溶质状态,结合弓形虫的细胞结构,可以明显看出TgASP3主要分布在弓形虫虫体中部的高尔基体区域内,推测可能存在于高尔基体中。通过制备上述4种蛋白酶的重组DNA疫苗证实它们均可诱导较强的体液免疫和细胞免疫,并可显著增强小鼠抗弓形虫感染的能力,延长了生存时间,与生物信息学方法预测的结果一致。
Toxoplasma gondii is an apicomplexan and obligate intracellular parasite, it can cause toxoplasmosis which is a kind of widely distributed in warm-blood animals including human. This disease is a serious threat to human health and the development of stockbreeding; especially on people have impaired immune function, including AIDS patients and patients receiving long-term chemotherapy. Due to there are no drug treatments available to against T. gondii infection, so development of an effective vaccine is a vital strategy in the control of toxoplasmosis. In this study, we fistly analyze and predict the basic physical and chemical properties, subcellar localization, modification post translation, B-cell and T-cell antigen epitopes of four toxoplasma gondii protease. We have observed the expression of TgASPl and TgASP3in parasites by indirect immunofluorescence assay using laser scanning confocal microscope, among them, TgASP3was clearly indentified for the first time. Recombinant DNA vaccines were constructed to evaluate the immune protection. This study filled the research gap of TgASP3, enriched the basic theoretical knowledge about toxoplasma gondii proteases, and laid a foundation of the development of safe and effective vaccine against toxoplasma infections.
Objective:Identified the toxoplasma gondii proteases TgASPl and TgASP3, observed the distribution of them in toxoplasma gondii parasites, then constructed recombinant gene vaccines to evaluate the immune protection of cysteine proteases TgCPB, TgCPL and aspartic proteases TgASP1, TgASP3of toxoplasma gondii.
Methods:Firstly, bioinformatics approaches were used to analyze and predict the physical and chemical properties, subcellar localization, modification post translation, B-cell and T-cell antigen epitopes of TgCPB, TgCPL, TgASP1, and TgASP3. The recombinant prokaryotic expression vectors were constructed and were transfected into E.coli BL21(DE3) to prepare soluble recombinant proteins. Mice were immunized subcutaneously with purified rTgCPB, rTgCPL, rTgASP1and rTgASP3proteins to prepare anti sera. Through immunolocalization experiments, TgASP1and TgASP3were observed in both extracellular parasites and intracellular parasites grown overnight in HeLa cells under a laser scanning confoal microscope. Finally, recombinant eukaryotic expression vectors were inoculated into BALB/c mice to evaluate the immune protection.
Results:The physical and chemical properties, subcellar localization, modification post translation of TgCPB. TgCPL, TgASP1, and TgASP3were analyzed using bioinformatics softwares, B-cell and T-cell antigen epitopes of TgCPB. TgCPL. TgASP1, and TgASP3were predicted by many bioinformatics approaches and filtered out a lot of potential antigen epitopes areas with good hydrophilic, strong plasticity, strong accessibility and high antigen index. The results showed that toxoplasma gondii proteases TgCPB, TgCPL, TgASP1and TgASP3are all good protein antigens. TgCPB, TgCPL, TgASP1and TgASP3gene fragments were amplified by PCR, molecular weight size are respectively1710bp,1269bp,1863bp and1932bp. and then they were cloned into pET30a prokaryotic expression vector in order to construct cloned plasmids pET30a-TgCPB. pET30a-TgCPL. pET30a-TgASP1and pBT30a-TgASP3, meanwhile, TgCPB and TgCPL gene fragments were cloned into pBudCE4.1eukaryotic expression vector in order to construct cloned plasmids pBudCE4.1-TgCPB. pBudCE4.1-TgCPL and pBudCH4.1-TgCPB-TgCPL. TgASP1and TgASP3gene fragments were cloned into pEGFP eukaryotic expression vector in order to construct cloned plasmids pEGF'P-TgASP1and pEGFP-TgASP3. E, coli BL21(DE3) cells were transformed by pET30a-TgCPB, pET30a-TgCPL, pET30a-Tg ASP1and pET30a-TgASP3. Recombinant proteins TgCPB, TgCPL, TgASP1and TgASP3were induced by IPTG and purified by NiNTA resin. Mice were immunized subcutaneously with purified rTgCPB, rTgCPL, rTgASP1and rTgASP3proteins to prepare anti sera, specific antibody IgG was obtained by further separation and purification. The expression area and expression level of TgASPl and TgASP3were observed by indirect immunofluorescence assay under a flurescence microscope. Through immunolocalization experiments, both extracellular parasites and intracellular parasites grown overnight in HeLa cells were observed under a laser scanning confocal microscope. BALB/c mice were immunized with eukaryotic expression plasmids and the determination of anti-toxoplasma total IgG, IgGl, IgG2a and cytokines IL-4, IL-10, IFN-y levels between experimental and control groups were used to assess its ability to stimulate cellular and humoral immunity. To evaluate the immunoprotection induced by the DNA vaccines, all the mice were challenged intraperitoneally (i.p.) with T.gondii RH strain.
Conclusions:The results of immunolocalization experiments show that TgASP1is a membrane protein and has a punctuate apical localization rather than being cytosolic. In addition, it seems like that TgASP1does not localize in typical regions where the secretory organs, including rhoptries, micronemes or dense granules localized in. Previous studies suggested that TgASP1resides in a novel compartment of the secretory system that potentially serves a link between the Golgi and the inner membrane complex (IMC). The results confirmed that recombinant DNA vaccines can induce strong cellular and humoral immunity, significantly enhance the ability of mice against toxoplasma gondii infection, prolong the survival times and verify the feasibility of prediction of protein antigen epitopes using bioinformatics approaches.
目的：鉴定天冬氨酸蛋白酶TgASP1和TgASP3在弓形虫虫体内外的分布情况；构建重组基因疫苗,评价弓形虫半胱氨酸蛋白酶TgCPB、TgCPL及天冬氨酸蛋白酶TgASP1和TgASP3的免疫保护性。
方法：首先利用生物信息学方法来分析和预测弓形虫体内半胱氨酸蛋白酶和天冬氨酸蛋白酶的基本理化性质、亚细胞定位、蛋白翻译后修饰、B细胞抗原表位和T细胞抗原表位；然后构建重组原核表达载体转染大肠杆菌BL21(DE3)制备可溶性的重组蛋白,纯化后免疫小鼠获得特异性抗体,通过间接免疫荧光实验观察蛋白酶TgASP1和TgASP3在虫体内外的表达情况；最后通过构建重组真核表达载体接种于BALB/c小鼠评价其免疫保护性。
结果：利用生物信息学软件分析了弓形虫蛋白酶TgCPB、TgCPL、TgASP1和TgASP3的基本理化性质、亚细胞定位、蛋白翻译后的修饰等；利用多种方法预测了存在于弓形虫蛋白酶TgCPB、TgCPL、TgASP1和TgASP3上的B细胞和T细胞抗原表位,筛选出了大量亲水性好、可塑性强、可及性大和抗原指数高的潜在抗原表位区域,表明弓形虫蛋白酶TgCPB、TgCPL、TgASP1和TgASP3是良好的蛋白抗原；经PCR体外扩增获得了TgCPB、TgCPL、TgASP1和TgASP3的基因片段,大小分别为1710bp、1269bp、1863bp和1932bp；构建了重组原核表达质粒pET30a-TgCPB、pET30a-TgCPL、pET30a-TgASP1和pET30a-TgASP3及重组真核表达质粒pBudCE4.1-TgCPB、pBudCE4.1-TgCPL、pBudCE4.1-TgCPB-TgCPL、pEGFP-TgASP1和pEGFP-TgASP3,上述4种重组.原核表达质粒经转染大肠杆菌BL21(DE3)均获得高效表达的的重组蛋白,分离纯化后通过免疫BALB/c小鼠获得特异性较强的抗血清,进一步分离纯化后获得了抗上述蛋白酶的特异性抗体IgG。利用获得抗体IgG经间免疫荧光实验在激光聚焦荧光显微镜下观察了蛋白酶TgASP1和TgASP3在弓形虫体内外的表达位置及相对表达量;上述5种重组真核表达质粒通过免疫接种BALB/c小鼠,测定对照组与免疫组小鼠抗弓形虫总IgG、IgG1、IgG2a和细胞因子IL-4、IL-10、IFN-γ的水平评估了其刺激产生细胞免疫和体液免疫的能力：通过弓形虫速殖子攻击实验观测小鼠的生存时间评估了其免疫保护性。
结论：通过免疫荧光定位的方法观察了弓形虫蛋白酶TgASP1和FgASP3在弓形虫虫体内外的表达位置及相对表达量,其中TgASP1是一种膜蛋白,主要表达于弓形虫虫体顶部区域,表达区域高度集中,不呈细胞溶质状态,并且与弓形虫的主要分泌器官,包括棒状体、微线体和致密颗粒不在同一区域,可能存在介于高尔基体和内膜复合物之间的一个新的独立的区域；TgASP3蛋白几乎不在膜上分布,主要分布在细胞质中且呈现一定的细胞溶质状态,结合弓形虫的细胞结构,可以明显看出TgASP3主要分布在弓形虫虫体中部的高尔基体区域内,推测可能存在于高尔基体中。通过制备上述4种蛋白酶的重组DNA疫苗证实它们均可诱导较强的体液免疫和细胞免疫,并可显著增强小鼠抗弓形虫感染的能力,延长了生存时间,与生物信息学方法预测的结果一致。
Toxoplasma gondii is an apicomplexan and obligate intracellular parasite, it can cause toxoplasmosis which is a kind of widely distributed in warm-blood animals including human. This disease is a serious threat to human health and the development of stockbreeding; especially on people have impaired immune function, including AIDS patients and patients receiving long-term chemotherapy. Due to there are no drug treatments available to against T. gondii infection, so development of an effective vaccine is a vital strategy in the control of toxoplasmosis. In this study, we fistly analyze and predict the basic physical and chemical properties, subcellar localization, modification post translation, B-cell and T-cell antigen epitopes of four toxoplasma gondii protease. We have observed the expression of TgASPl and TgASP3in parasites by indirect immunofluorescence assay using laser scanning confocal microscope, among them, TgASP3was clearly indentified for the first time. Recombinant DNA vaccines were constructed to evaluate the immune protection. This study filled the research gap of TgASP3, enriched the basic theoretical knowledge about toxoplasma gondii proteases, and laid a foundation of the development of safe and effective vaccine against toxoplasma infections.
Objective:Identified the toxoplasma gondii proteases TgASPl and TgASP3, observed the distribution of them in toxoplasma gondii parasites, then constructed recombinant gene vaccines to evaluate the immune protection of cysteine proteases TgCPB, TgCPL and aspartic proteases TgASP1, TgASP3of toxoplasma gondii.
Methods:Firstly, bioinformatics approaches were used to analyze and predict the physical and chemical properties, subcellar localization, modification post translation, B-cell and T-cell antigen epitopes of TgCPB, TgCPL, TgASP1, and TgASP3. The recombinant prokaryotic expression vectors were constructed and were transfected into E.coli BL21(DE3) to prepare soluble recombinant proteins. Mice were immunized subcutaneously with purified rTgCPB, rTgCPL, rTgASP1and rTgASP3proteins to prepare anti sera. Through immunolocalization experiments, TgASP1and TgASP3were observed in both extracellular parasites and intracellular parasites grown overnight in HeLa cells under a laser scanning confoal microscope. Finally, recombinant eukaryotic expression vectors were inoculated into BALB/c mice to evaluate the immune protection.
Results:The physical and chemical properties, subcellar localization, modification post translation of TgCPB. TgCPL, TgASP1, and TgASP3were analyzed using bioinformatics softwares, B-cell and T-cell antigen epitopes of TgCPB. TgCPL. TgASP1, and TgASP3were predicted by many bioinformatics approaches and filtered out a lot of potential antigen epitopes areas with good hydrophilic, strong plasticity, strong accessibility and high antigen index. The results showed that toxoplasma gondii proteases TgCPB, TgCPL, TgASP1and TgASP3are all good protein antigens. TgCPB, TgCPL, TgASP1and TgASP3gene fragments were amplified by PCR, molecular weight size are respectively1710bp,1269bp,1863bp and1932bp. and then they were cloned into pET30a prokaryotic expression vector in order to construct cloned plasmids pET30a-TgCPB. pET30a-TgCPL. pET30a-TgASP1and pBT30a-TgASP3, meanwhile, TgCPB and TgCPL gene fragments were cloned into pBudCE4.1eukaryotic expression vector in order to construct cloned plasmids pBudCE4.1-TgCPB. pBudCE4.1-TgCPL and pBudCH4.1-TgCPB-TgCPL. TgASP1and TgASP3gene fragments were cloned into pEGFP eukaryotic expression vector in order to construct cloned plasmids pEGF'P-TgASP1and pEGFP-TgASP3. E, coli BL21(DE3) cells were transformed by pET30a-TgCPB, pET30a-TgCPL, pET30a-Tg ASP1and pET30a-TgASP3. Recombinant proteins TgCPB, TgCPL, TgASP1and TgASP3were induced by IPTG and purified by NiNTA resin. Mice were immunized subcutaneously with purified rTgCPB, rTgCPL, rTgASP1and rTgASP3proteins to prepare anti sera, specific antibody IgG was obtained by further separation and purification. The expression area and expression level of TgASPl and TgASP3were observed by indirect immunofluorescence assay under a flurescence microscope. Through immunolocalization experiments, both extracellular parasites and intracellular parasites grown overnight in HeLa cells were observed under a laser scanning confocal microscope. BALB/c mice were immunized with eukaryotic expression plasmids and the determination of anti-toxoplasma total IgG, IgGl, IgG2a and cytokines IL-4, IL-10, IFN-y levels between experimental and control groups were used to assess its ability to stimulate cellular and humoral immunity. To evaluate the immunoprotection induced by the DNA vaccines, all the mice were challenged intraperitoneally (i.p.) with T.gondii RH strain.
Conclusions:The results of immunolocalization experiments show that TgASP1is a membrane protein and has a punctuate apical localization rather than being cytosolic. In addition, it seems like that TgASP1does not localize in typical regions where the secretory organs, including rhoptries, micronemes or dense granules localized in. Previous studies suggested that TgASP1resides in a novel compartment of the secretory system that potentially serves a link between the Golgi and the inner membrane complex (IMC). The results confirmed that recombinant DNA vaccines can induce strong cellular and humoral immunity, significantly enhance the ability of mice against toxoplasma gondii infection, prolong the survival times and verify the feasibility of prediction of protein antigen epitopes using bioinformatics approaches.
引文
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[35]Martin DM, Berriman M, Barton GJ. GOtcha:a new method for prediction of protein function assessed by the annotation of seven genomes. BMC Bioinformatics. 2004,5:178.
[36]吴忠道,黄静,徐劲.生物信息学及在寄生虫学上的应用[J].热带医学杂志2001,1(1)：80-83.
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[39]沈雅婧,柳建发.生物信息学技术对寄生虫的影响.地方病通报,2009, 24(2)：66-68.
[40]Bhasin M, Lata S, Raghava GP:Searching and mapping of T-cell epitopes, MHC binders, and TAP binders. Methods Mol Biol 2007,409:95-112.
[41]Vider-Shalit T, Louzoun Y:MHC-I prediction using a combination of T cell epitopes and MHC-I binding peptides. J Immunol Methods 2011,374(1-2):43-46.
[42]Constant S, Pfeiffer C, Woodard A, Pasqualini T, Bottomly K:Extent of T cell receptor ligation can determine the functional differentiation of naive CD4+T cells. J Exp Med 1995,182(5):1591-1596.
[43]Duffy MM, Ritter T, Ceredig R, Griffin MD:Mesenchymal stem cell effects on T-cell effector pathways. Stem Cell Res Ther 2011,2(4):34.
[44]Takacs AC, Swierzy IJ, Luder CG:Interferon-γ restricts Toxoplasma gondii dev-elopment in murine skeletal muscle cells via nitric oxide production and immunity-related GTPases. PLoS One.2012, e45440 p.
[45]Martin DM, Berriman M, Barton GJ. GOtcha:a new method for prediction of protein function assessed by the annotation of seven genomes. BMC Bioinformatics. 2004,5:178.