猪弓形虫体内诱导抗原的鉴定及功能分析
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摘要
弓形虫(Toxoplasma gondii)是能够感染真核细胞的顶器门寄生原虫,呈世界性分布,宿主范围广,传播途径多样,生活史复杂。可导致孕妇流产,新生儿先天性疾病,以及继发免疫抑制患者致死性疾病。弓形虫可致家畜流产和生长发育不良,给畜牧业带来巨大的经济损失。通过摄食被弓形虫污染的肉类可使人感染弓形虫病。猪肉是人类主要的肉食来源之一,猪的弓形虫感染严重威胁人类的生命健康。弄清弓形虫的致病机理和开发安全有效的疫苗是预防和控制人类和动物弓形虫病的基础和重要手段。
为此,本研究构建了RH株弓形虫速殖子cDNA表达文库,利用体内诱导抗原技术(IVIAT),鉴定出14个猪弓形虫体内诱导抗原；利用实时荧光定量PCR(Real-time PCR)对其中5个抗原基因进行了体内、外表达差异的验证和体外原核表达；对弓形虫钙调蛋白(CaM)基因进行了RNA干扰(RNAi),评价其表达下调对弓形虫速殖子滑行运动、细胞粘附、入侵和溢出及胞内繁殖等表型的影响；以弓形虫体内诱导抗原微线体蛋白11(MIC11)α链构建了DNA疫苗,并在BALB/c鼠模型中检测到保护性免疫应答的产生。(1)猪弓形虫体内诱导抗原基因的筛选与鉴定
大量收集并纯化RH株弓形虫速殖子,提取总RNA,利用商业化SMARTTM技术构建以噬菌体为载体的cDNA表达文库。构建的文库重组率达到98%以上,初始文库和扩增文库滴度分别为2.1x106pfu/mL和1.6x109pfu/mL,文库容量为3.2×1011个重组子。混合10份猪弓形虫阳性血清并通过逐级吸附弓形虫速殖子全细胞、超声破碎裂解液和热裂解物,去除血清中体外弓形虫抗体。运用吸附后血清对表达文库的3264个克隆进行免疫筛选,得到14个阳性克隆。对筛选得到的体内诱导抗原进行EST序列分析和功能预测,14个抗原中包括涉及离子/蛋白结合、信号转导、蛋白折叠、细胞入侵和生物合成与代谢等8个功能蛋白,同时包括6个未知功能的假定蛋白。
(2)体内诱导抗原体内外表达差异的验证及原核表达
选择其中5个体内诱导抗原基因,即CaM、致密颗粒蛋白5(GRA5)、MIC11、18KDa亲环蛋白(C-18)和丝氨酸蛋白酶抑制剂(PI),针对各自的ORF设计引物,real-time PCR检测感染BALB/c鼠72h和在BHK细胞中培养72h的速殖子中,5个基因mRNA相对表达水平的差别。结果显示,宿主体内速殖子中5个基因mRNA表达量均高于体外培养速殖子中表达量,统计学分析差异显著(p<0.05)。其中CaM基因体内、外mRNA相对表达量差异最大,可达15.04倍。利用5个基因全长CDS序列设计原核表达引物,分别克隆各自ORF片段,连接原核表达载体pGEX-6p-1,形成重组GST融合表达质粒。除pGEX-6p-1/GRA5表达失败以外,其余4个重组质粒均可在E. coli BL21系统中高效表达。分别利用弓形虫阳性猪血清和商业化GST一抗血清对表达产物进行Western-blot分析,4个体内诱导抗原均具有良好抗原性,且CaM和C-18抗原性较强。
(3)弓形虫CaM的下调表达对速殖子表型的影响
针对弓形虫CaM基因序列不同位点设计3对siRNA (siRNA707, siRNA865, siRNA900),对CaM的表达进行体外干扰。利用real-time PCR检测转录水平干扰效果,结果显示,3对siRNA对弓形虫CaM的表达均有一定程度的干扰,对其他与弓形虫CaM有同源性的基因无干扰作用,表明干扰特异性强,无脱靶效应。确定siRNA最优作用时间为24h,最佳工作浓度为200nmol/L。用鼠抗弓形虫CaM多克隆抗体对干扰前后速殖子进行Western-blot,检测蛋白质水平干扰效果。三对siRNA中,siRNA900在转录和蛋白表达水平上干扰效果最显著。在BHK细胞模型中,观察CaM被干扰后弓形虫速殖子表型的变化。结果显示,弓形虫CaM的表达下调显著降低速殖子的细胞粘附、入侵和溢出能力(p<0.05),但对其胞内繁殖无影响(p>0.05)。
(4)体内诱导抗原MIC11免疫原性的研究
将弓形虫MIC11-a链基因片段与真核表达载体pcDNA3.1连接,构建DNA疫苗pcDNA3.1/MIC11。间接免疫荧光(IFA)证明重组质粒在BHK细胞中可高效表达,并能被弓形虫多克隆抗血清特异性识别。重组质粒100μg免疫BALB/c小鼠,同时,设相同剂量pcDNA3.1空载体和100μL PBS阳性对照组。首次免疫后两周,利用TLA-ELISA可在pcDNA3.1/MIC11免疫鼠血清中检测到特异性TLA抗体,随免疫时间延长,抗体水平持续升高,对照组无抗体产生。细胞因子ELISA结果证明,构建的DNA疫苗可有效刺激机体产生高水平的IL-2、IL-12及IFN-y,但对IL-4无刺激作用。淋巴细胞增殖实验显示,pcDNA3.1/MIC11免疫鼠淋巴细胞增殖水平显著高于空载体组(p<0.05)和PBS对照组(p<0.05)。免疫后第8周,小鼠腹腔接种RH株弓形虫速殖子,对照组小鼠在10天内全部死亡,而疫苗组小鼠在攻虫后15天仍有17%的存活率。以上结果表明,DNA疫苗pcDNA3.1/MIC11可有效诱导机体产生特异性弓形虫抗体和Thl型细胞免疫反应,同时产生一定保护力。
本研究通过利用IVIAT对弓形虫体内诱导抗原的鉴定及功能分析,深化了人们对弓形虫病致病机制的理解,并为弓形虫疫苗的研究提供了理论基础。
Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite infecting all kinds of eukaryocyte. The parasite has a complex life cycle and distributes worldwide due to the extensive host rang and the various routs of transmission. T. gondii usually causes abortion in pregnant women and congenital diseases in neonates, it also causes life-threatening diseases in immune-compromised individuals. Toxoplasmosis in livestock always results in abortion and mal-development which brings serious economic losses. People become infection with T. gondii by ingesting the undercooked meat contaminated with cysts. Pork is one of main meat sources for human, thus, toxoplasmosis in pigs is thought to be a threat for human health. Understanding the pathogenic mechanism and development of vaccine are important for preventing and controlling T. gondii.
In the present study, a cDNA expression library of T. gondii RH strain tachyzoites was constructed. Then in vivo induced antigen technology (TVIAT) was used for screening the constructed library by anti-T. gondii convalescence sera from pigs and yielded14in vivo induced antigens of T. gondii in pigs. The differential expressions of five of the identified antigens between in vivo and in vitro were confirmed using real-time PCR. Prokaryotic expression of the five antigens was also performed. RNA interference (RNAi) was carried out on T. gondii calmodulin (CaM) to evaluate the function in cell events of tachyzoites such as gliding movement, cell attachment, invasion, egress and intracellular replication. Finally, a DNA vaccine against T. gondii encoding micromene protein11(MIC11) was constructed and the protective immunity was detected in BALB/c mice model.(1) Screening and identification of in vivo induced antigens of T. gondii in pigs
T. gondii RH strain tachyzoites were collected and purified, and then total RNA was extracted. A cDNA expression library was constructed based on a phage vector using SMARTTM technology. The titers of initial and amplified library were2.1x106pfu/mL and1.6x109pfu/mL, respectively. The recombinant rate was more than98%. Equal amounts of the positive sera from the infected pigs were pooled and successively adsorbed against the in vitro-grown tachyzoites, ultrasonic lysates and heat-denatured lysates of the in vitro-grown tachyzoites.3264clones from the library were screened using the adsorbed sera and yielded14positive clones containing8functional proteins which involved in ion/protein binding, signal transduction, protein folding, cell invasion, biosynthesis and metabolism, and6hypothetical proteins.
(2) Differential expression between in vivo and in vitro and prokaryotic expression of the five in vivo induced antigen
Five in vivo induced antigens genes (CaM, dense granule protein5(GRA5), MIC11,18kDa cyclophilin (C-18) and serine proteinase inhibitor (PI)) were selected and detected the differential expression under routine in vitro culture conditions or in the host by real-time PCR. Differences in the degree of up-regulation of the five genes were detected between the in vivo tachyzoites collected from BALB/c mice and the tachyzoites grown in vitro in BHK cells (p<0.05). CaM transcripts were detected at approximately15.04-fold higher levels in the in vivo samples than in the in vitro samples, which showed the most significant differences. Five pairs of primers of the genes were designed for prokaryotic expression. The ORFs were amplified and inserted into pGEX-6p-1, resulting in the recombinant plasmids expressing GST-fusion proteins, respectively. All the4recombinant proteins could be expressed efficiently in E. coli BL21, except pGEX-6p-1/GRA5. In the Western-blot analysis, both the positive sera against T. gondii and anti-GST antibodies recognized the recombinant proteins encoding the four in vivo induced antigens, suggesting the good antigenicity. The antigenic responses of CaM and C-18were stronger.
(3) Phenotypic consequences of the down-regulated expression of T. gondii CaM
Three pairs of siRNA (siRNA900, siRNA865and siRNA707) targeting different sites of CaM gene were used to suppress CaM expression. A certain degree of suppression by each siRNA on CaM expression was detected using real-time PCR. No effect was shown on the other proteins homologous with T. gondii CaM, suggesting the strong specific suppression by the designed siRNA. Most efficient suppression was found in tachyzoites treated with200nmol/L of siRNA900for24h. The phenotypic consequences of the parasite after treatment with siRNA900were investigated in BHK cells, and the treated tachyzoites showed decreased abilities of cell adherence, invasion and egress, but un-correlation with replication.
(4) Immunogenicity of in vivo induced antigen MIC11
The DNA fragment of T. gondii MIC11a-chain was inserted into pcDNA3.1vector, resulting in the recombinant plasmid pcDNA3.1/MIC11. Expression of MIC11from this vector was confirmed by indirect immune-fluorescence assay following transfection into BHK cells. The BALB/c mice were received the intramuscular injection of100μg pcDNA3.1/MIC11. The mice injected with100μg pcDNA3.1and100μL sterile PBS only were served as the control groups. Antibodies against TLA were first detected in the mice vaccinated with pcDNA3.1/MIC11two weeks after the first immunization. More anti-TLA IgG was accumulated and antibody titers increased dramatically in mice group vaccinated with pcDNA3.1/MIC11after the second and third immunization (p<0.05). In the ELISA analysis for cytokines, mice immunized with pcDNA3.1/MIC11produced highly significant levels of IFN-y, IL-12, IL-2compared with those immunized with PBS or pcDNA3.1(p<0.05). Mice immunized with pcDNA3.1/MIC11also induced higher level of lymphocyte proliferation compared with control groups (p<0.05). Mice were challenged with T. gondii RH strain tachyzoites at day14after the final immunization. All mice in the control groups died within10days post infection, while17%of the mice immunized with pcDNA3.1/MIC11were survived to day15after the parasite challenging. All the results demonstrated that the constructed DNA vaccine efficiently induced high levels of antibodies against T. gondii, Thl immune responses, and mediated good protection in BALB/c mice.
The results of the present studies of identification of T. gondii in vivo induced antigen by IVIAT and the functional analysis improve our understanding of T. gondii pathogenesis and provide vaccine candidates against T. gondii infection.
为此,本研究构建了RH株弓形虫速殖子cDNA表达文库,利用体内诱导抗原技术(IVIAT),鉴定出14个猪弓形虫体内诱导抗原；利用实时荧光定量PCR(Real-time PCR)对其中5个抗原基因进行了体内、外表达差异的验证和体外原核表达；对弓形虫钙调蛋白(CaM)基因进行了RNA干扰(RNAi),评价其表达下调对弓形虫速殖子滑行运动、细胞粘附、入侵和溢出及胞内繁殖等表型的影响；以弓形虫体内诱导抗原微线体蛋白11(MIC11)α链构建了DNA疫苗,并在BALB/c鼠模型中检测到保护性免疫应答的产生。(1)猪弓形虫体内诱导抗原基因的筛选与鉴定
大量收集并纯化RH株弓形虫速殖子,提取总RNA,利用商业化SMARTTM技术构建以噬菌体为载体的cDNA表达文库。构建的文库重组率达到98%以上,初始文库和扩增文库滴度分别为2.1x106pfu/mL和1.6x109pfu/mL,文库容量为3.2×1011个重组子。混合10份猪弓形虫阳性血清并通过逐级吸附弓形虫速殖子全细胞、超声破碎裂解液和热裂解物,去除血清中体外弓形虫抗体。运用吸附后血清对表达文库的3264个克隆进行免疫筛选,得到14个阳性克隆。对筛选得到的体内诱导抗原进行EST序列分析和功能预测,14个抗原中包括涉及离子/蛋白结合、信号转导、蛋白折叠、细胞入侵和生物合成与代谢等8个功能蛋白,同时包括6个未知功能的假定蛋白。
(2)体内诱导抗原体内外表达差异的验证及原核表达
选择其中5个体内诱导抗原基因,即CaM、致密颗粒蛋白5(GRA5)、MIC11、18KDa亲环蛋白(C-18)和丝氨酸蛋白酶抑制剂(PI),针对各自的ORF设计引物,real-time PCR检测感染BALB/c鼠72h和在BHK细胞中培养72h的速殖子中,5个基因mRNA相对表达水平的差别。结果显示,宿主体内速殖子中5个基因mRNA表达量均高于体外培养速殖子中表达量,统计学分析差异显著(p<0.05)。其中CaM基因体内、外mRNA相对表达量差异最大,可达15.04倍。利用5个基因全长CDS序列设计原核表达引物,分别克隆各自ORF片段,连接原核表达载体pGEX-6p-1,形成重组GST融合表达质粒。除pGEX-6p-1/GRA5表达失败以外,其余4个重组质粒均可在E. coli BL21系统中高效表达。分别利用弓形虫阳性猪血清和商业化GST一抗血清对表达产物进行Western-blot分析,4个体内诱导抗原均具有良好抗原性,且CaM和C-18抗原性较强。
(3)弓形虫CaM的下调表达对速殖子表型的影响
针对弓形虫CaM基因序列不同位点设计3对siRNA (siRNA707, siRNA865, siRNA900),对CaM的表达进行体外干扰。利用real-time PCR检测转录水平干扰效果,结果显示,3对siRNA对弓形虫CaM的表达均有一定程度的干扰,对其他与弓形虫CaM有同源性的基因无干扰作用,表明干扰特异性强,无脱靶效应。确定siRNA最优作用时间为24h,最佳工作浓度为200nmol/L。用鼠抗弓形虫CaM多克隆抗体对干扰前后速殖子进行Western-blot,检测蛋白质水平干扰效果。三对siRNA中,siRNA900在转录和蛋白表达水平上干扰效果最显著。在BHK细胞模型中,观察CaM被干扰后弓形虫速殖子表型的变化。结果显示,弓形虫CaM的表达下调显著降低速殖子的细胞粘附、入侵和溢出能力(p<0.05),但对其胞内繁殖无影响(p>0.05)。
(4)体内诱导抗原MIC11免疫原性的研究
将弓形虫MIC11-a链基因片段与真核表达载体pcDNA3.1连接,构建DNA疫苗pcDNA3.1/MIC11。间接免疫荧光(IFA)证明重组质粒在BHK细胞中可高效表达,并能被弓形虫多克隆抗血清特异性识别。重组质粒100μg免疫BALB/c小鼠,同时,设相同剂量pcDNA3.1空载体和100μL PBS阳性对照组。首次免疫后两周,利用TLA-ELISA可在pcDNA3.1/MIC11免疫鼠血清中检测到特异性TLA抗体,随免疫时间延长,抗体水平持续升高,对照组无抗体产生。细胞因子ELISA结果证明,构建的DNA疫苗可有效刺激机体产生高水平的IL-2、IL-12及IFN-y,但对IL-4无刺激作用。淋巴细胞增殖实验显示,pcDNA3.1/MIC11免疫鼠淋巴细胞增殖水平显著高于空载体组(p<0.05)和PBS对照组(p<0.05)。免疫后第8周,小鼠腹腔接种RH株弓形虫速殖子,对照组小鼠在10天内全部死亡,而疫苗组小鼠在攻虫后15天仍有17%的存活率。以上结果表明,DNA疫苗pcDNA3.1/MIC11可有效诱导机体产生特异性弓形虫抗体和Thl型细胞免疫反应,同时产生一定保护力。
本研究通过利用IVIAT对弓形虫体内诱导抗原的鉴定及功能分析,深化了人们对弓形虫病致病机制的理解,并为弓形虫疫苗的研究提供了理论基础。
Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite infecting all kinds of eukaryocyte. The parasite has a complex life cycle and distributes worldwide due to the extensive host rang and the various routs of transmission. T. gondii usually causes abortion in pregnant women and congenital diseases in neonates, it also causes life-threatening diseases in immune-compromised individuals. Toxoplasmosis in livestock always results in abortion and mal-development which brings serious economic losses. People become infection with T. gondii by ingesting the undercooked meat contaminated with cysts. Pork is one of main meat sources for human, thus, toxoplasmosis in pigs is thought to be a threat for human health. Understanding the pathogenic mechanism and development of vaccine are important for preventing and controlling T. gondii.
In the present study, a cDNA expression library of T. gondii RH strain tachyzoites was constructed. Then in vivo induced antigen technology (TVIAT) was used for screening the constructed library by anti-T. gondii convalescence sera from pigs and yielded14in vivo induced antigens of T. gondii in pigs. The differential expressions of five of the identified antigens between in vivo and in vitro were confirmed using real-time PCR. Prokaryotic expression of the five antigens was also performed. RNA interference (RNAi) was carried out on T. gondii calmodulin (CaM) to evaluate the function in cell events of tachyzoites such as gliding movement, cell attachment, invasion, egress and intracellular replication. Finally, a DNA vaccine against T. gondii encoding micromene protein11(MIC11) was constructed and the protective immunity was detected in BALB/c mice model.(1) Screening and identification of in vivo induced antigens of T. gondii in pigs
T. gondii RH strain tachyzoites were collected and purified, and then total RNA was extracted. A cDNA expression library was constructed based on a phage vector using SMARTTM technology. The titers of initial and amplified library were2.1x106pfu/mL and1.6x109pfu/mL, respectively. The recombinant rate was more than98%. Equal amounts of the positive sera from the infected pigs were pooled and successively adsorbed against the in vitro-grown tachyzoites, ultrasonic lysates and heat-denatured lysates of the in vitro-grown tachyzoites.3264clones from the library were screened using the adsorbed sera and yielded14positive clones containing8functional proteins which involved in ion/protein binding, signal transduction, protein folding, cell invasion, biosynthesis and metabolism, and6hypothetical proteins.
(2) Differential expression between in vivo and in vitro and prokaryotic expression of the five in vivo induced antigen
Five in vivo induced antigens genes (CaM, dense granule protein5(GRA5), MIC11,18kDa cyclophilin (C-18) and serine proteinase inhibitor (PI)) were selected and detected the differential expression under routine in vitro culture conditions or in the host by real-time PCR. Differences in the degree of up-regulation of the five genes were detected between the in vivo tachyzoites collected from BALB/c mice and the tachyzoites grown in vitro in BHK cells (p<0.05). CaM transcripts were detected at approximately15.04-fold higher levels in the in vivo samples than in the in vitro samples, which showed the most significant differences. Five pairs of primers of the genes were designed for prokaryotic expression. The ORFs were amplified and inserted into pGEX-6p-1, resulting in the recombinant plasmids expressing GST-fusion proteins, respectively. All the4recombinant proteins could be expressed efficiently in E. coli BL21, except pGEX-6p-1/GRA5. In the Western-blot analysis, both the positive sera against T. gondii and anti-GST antibodies recognized the recombinant proteins encoding the four in vivo induced antigens, suggesting the good antigenicity. The antigenic responses of CaM and C-18were stronger.
(3) Phenotypic consequences of the down-regulated expression of T. gondii CaM
Three pairs of siRNA (siRNA900, siRNA865and siRNA707) targeting different sites of CaM gene were used to suppress CaM expression. A certain degree of suppression by each siRNA on CaM expression was detected using real-time PCR. No effect was shown on the other proteins homologous with T. gondii CaM, suggesting the strong specific suppression by the designed siRNA. Most efficient suppression was found in tachyzoites treated with200nmol/L of siRNA900for24h. The phenotypic consequences of the parasite after treatment with siRNA900were investigated in BHK cells, and the treated tachyzoites showed decreased abilities of cell adherence, invasion and egress, but un-correlation with replication.
(4) Immunogenicity of in vivo induced antigen MIC11
The DNA fragment of T. gondii MIC11a-chain was inserted into pcDNA3.1vector, resulting in the recombinant plasmid pcDNA3.1/MIC11. Expression of MIC11from this vector was confirmed by indirect immune-fluorescence assay following transfection into BHK cells. The BALB/c mice were received the intramuscular injection of100μg pcDNA3.1/MIC11. The mice injected with100μg pcDNA3.1and100μL sterile PBS only were served as the control groups. Antibodies against TLA were first detected in the mice vaccinated with pcDNA3.1/MIC11two weeks after the first immunization. More anti-TLA IgG was accumulated and antibody titers increased dramatically in mice group vaccinated with pcDNA3.1/MIC11after the second and third immunization (p<0.05). In the ELISA analysis for cytokines, mice immunized with pcDNA3.1/MIC11produced highly significant levels of IFN-y, IL-12, IL-2compared with those immunized with PBS or pcDNA3.1(p<0.05). Mice immunized with pcDNA3.1/MIC11also induced higher level of lymphocyte proliferation compared with control groups (p<0.05). Mice were challenged with T. gondii RH strain tachyzoites at day14after the final immunization. All mice in the control groups died within10days post infection, while17%of the mice immunized with pcDNA3.1/MIC11were survived to day15after the parasite challenging. All the results demonstrated that the constructed DNA vaccine efficiently induced high levels of antibodies against T. gondii, Thl immune responses, and mediated good protection in BALB/c mice.
The results of the present studies of identification of T. gondii in vivo induced antigen by IVIAT and the functional analysis improve our understanding of T. gondii pathogenesis and provide vaccine candidates against T. gondii infection.
引文
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