应用SSH技术研究氧化胁迫下细粒棘球蚴基因的表达
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摘要
细粒棘球蚴(Echinococcus granulosus, Eg)是囊性包虫病的病原体,棘球蚴以包囊形式在人和有蹄兽类等中间宿主的内脏器官中生长。细粒棘球绦虫的生活史中包含了四个不同的时期:卵或六钩蚴,包囊虫,原头蚴和成虫。在棘球蚴的早期发育阶段,包囊自身和宿主的新陈代谢及对感染产生的免疫应答都会产生大量的不稳定的活性氧类物质(reactive oxygen species, ROS),ROS具有很高的反应性,能够对蛋白、膜脂和DNA造成直接的损伤,产生严重的生物学效应,当在机体内大量堆积时可导致细胞和组织的死亡。为了适应高度的氧化胁迫压力的环境及逃避宿主的免疫应答反应,寄生虫通过合成抗氧化酶并大多在宿主—寄生虫的接触面表达以适应及抵抗氧化胁迫的压力。抗氧化酶在保护寄生虫抵御来自宿主新陈代谢和白细胞激活的ROS的氧化损伤起关键作用。
硫氧还蛋白过氧化物酶(thioredoxin peroxidases, TPx)基因家族是一个很大的抗氧化蛋白家族,从原核到真核,广泛存在于生物体中,具有强大的抗氧化功能。TPx在寄生虫逃避氧化性伤害过程中起着重要的作用。若能将此功能阻断,则可以减弱或使包虫的生理机能丧失,从而导致其死亡,这将为疫苗的研制和特效药的筛选奠定基础。
原头蚴在天然情况下可以向两个方向发育分化:一个是无性繁殖方向分化形成包囊,另一个是有性繁殖方向分化形成能产卵的成虫。本研究首先利用双相培养方法,掌握了原头蚴体外培养及无性发育至包囊阶段的技术,为建立药物筛选的体外模型,研究虫体发育及生活各阶段的基因功能打下基础。
应用抑制性消减杂交技术(suppression subtractive hybridization,SSH)研究H2O2胁迫下细粒棘球蚴基因的表达。以H2O2胁迫后的细粒棘球蚴cDNA为试验方(tester),正常生长的细粒棘球蚴cDNA为驱动方(driver),构建了H2O2胁迫下与正常细粒棘球蚴差异表达的消减cDNA文库。文库扩增后得到124个阳性克隆,菌落PCR分析,均得到200～1000 bp插入片段。将整个文库克隆进行测序,测得序列结果利用BLAST在线软件与GenBank数据库进行同源序列比对分析和BlastX分析。结果获得重要基因的cDNA序列,如氧化还原酶、蛋白激酶、生长因子等。另有部分克隆在GenBank中无法查到对应的同源基因,可能代表了新基因。利用定量PCR的方法初步获得了原头蚴在H2O2胁迫24h条件下部分差异表达基因片段在mRNA水平上的表达情况,为研究细粒棘球蚴在抗氧化过程中的相关靶基因筛选奠定基础。
采用RT-PCR的方法,根据GenBank已发表的细粒棘球蚴硫氧还蛋白过氧化物酶(EgTPx)基因的序列设计引物,获取EgTPx的cDNA片段并克隆到原核表达载体pET41-b中,构建原核表达质粒pET41-b-EgTPx,经过IPTG诱导,在原核表达系统大肠杆菌E.coli (BL21)中获得可溶性表达的融合蛋白,经过谷胱甘肽Sepharose4B层析柱分离纯化融合蛋白,免疫小鼠获得高效价的特异性抗血清。ELISA和Western blot结果表明融合蛋白免疫小鼠产生了高效价和特异性强的抗血清。为深入开展EgTPx基因功能研究和免疫学诊断试剂,及疫苗的开发分析奠定了物质基础。
利用重组EgTPx多克隆抗体,经消化处理后的原头蚴制备石蜡切片,以间接免疫荧光法确定抗氧化蛋白EgTPx在原头蚴内的分布。结果表明,EgTPx多克隆抗体能够特异性地结合天然EgTPx抗原表位, EgTPx广泛分布在原头蚴的体表皮层、皮下层和钙颗粒细胞内。构建了原头蚴EgTPx基因与绿色荧光蛋白(GFP)融合的虫体表达载体,命名为pPD-EgTPx,通过脂质体转染活性状态良好的原头蚴,在倒置荧光显微镜下观察该基因在原头蚴中的瞬时表达。结果表明,虫体表达载体pPD-EgTPx构建正确,而且能够在原头蚴中进行瞬时表达。
总之,本研究为细粒棘球蚴在抗氧化过程中相关靶基因的筛选和功能研究提供了重要的参考,为进一步利用抗氧化基因开展包虫病的预防治疗和疫苗研制提供了物质基础,具有重要的现实意义。
Echinococcu granulosus, the cause of cystic echinococcosis, is a small tapeworm that resides in the intestine of carnivore definitive hosts,generally dogs or wolves.The life cycle of E. granulosus consists of four stages: egg or oncosphere, hydatid cyst, protoscolex and the adult worm. Survival of the infectious agent in the mammalian hosts requires adaptive mechanisms, such as producing antioxidant enzymes against reactive oxygen species (ROS) generated by the host immune responses. ROS are generally considered to be cytotoxic because high concentrations of ROS can have seriously deleterious effects on membrane lipids, nucleic acids and proteins.To avoid the oxygen toxicity, parasites have developed a mechanism of antioxidant defenses, including enzymes that decompose peroxides and superoxide anion (O2-) and compounds that sequester metal ions. Parasites have adapted to considerable oxidative stress by synthesizing high levels of antioxidant enzymes and, in many cases, expressing them at the host–parasite interface.The levels of these defense enzymes are correlated with the survival of a number of parasitic helminths in host tissues.
Antioxidants including thioredoxin peroxidas have been identified and characterized in parasitic nematodes e.A new class of antioxidant enzyme has recently been described, which reduces hydroperoxides with thioredoxin as an immediate hydrogen donor. These enzymes are known as thioredoxin peroxidases (TPx). TPx, one of the peroxiredoxin (Prx) gene superfamily, functions as an antioxidant to remove ROS and H2O2 derived from normal cellular metabolism using thioredoxin as the electron donor. TPx have been found in diverse organisms ranging from prokaryotes to mammals. EgTPx may play a role in protecting the parasite from oxidative damage.
The larval stages (protoscoleces) are exceptional in having the potential to differentiate in either of two directions. The protoscolex can develop asexually, into the hydatid cyst, or sexually into the adult worm. In vitro culture procedures, PSC were aspirated and pooled from sheep liver hydatid cysts collected from a slaughterhouse in Urumqi, Xinjiang. PSC were collected under sterile conditions from intact cysts, washed three times in phosphate-buffered saline (PBS) prior to digestion in pepsin to release PSC from capsules and remove immature PSC by digestion. After the digestion procedure, the PSC were washed three times with PBS and incubated in glass culture vessels in a liquid DMEM medium placed above a solid-phase base prepared by heating new born calf serum. The solid-phase significantly enhanced PSC growth. After 40 days cultivation, about 10% of the PSC had developed into cysts. In paraffin section of the protoscolex there were many calcareous corpuscle cells, which could provide energy for the protoscolex.The aims of the project are to fully establish and exploit an in vitro culture model that can be used to assess the functions and interactions of a panel of selected E. granulosus genes during differentiation and development of the larval protoscolex stage in an asexual direction to form a hydatid cyst.
To isolate specific genes expressed in PSC of E. granulosus under H2O2 stresses, we used suppression subtractive hybridization (SSH) technique with cDNA from PSC treated with H2O2 as tester and cDNA from PSC in normal growth as driver. mRNA was isolated from the tester and driver respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the products were subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search. The subtractive library of genes regulated by H2O2 stresses was constructed successfully. The amplified library contained 124 clones, of which all clones had 200-1000 bp inserts. Sequence analysis indicated that 124 clones containing the coding sequences, of which some had homology in the GenBank and other were unknown. The obtained sequences may be target genes regulated by H2O2, which provided foundation for the further identify differentially expressed genes in PSC of E. granulosus exposed to oxidative stress.
The cDNA sequence of thioredoxin peroxidase from E. granulosus (EgTPx) was cloned from PSC by RT-PCR and subcloned into the expressive vector pET41-b after sequence analysis。A recombinant plasmid carrying EgTPx gene from was constructed. The resulting recombinant plasmid pET41-b- EgTPx was transformed into the competent cells BL21 and the recombinant plasmid was expressed by IPTG induction.The TPx fusion protein was purified through a Glutathione Sepharose 4B affinity column, and high titer of polyclonal antiserum was obtained by the immunizing the mice using the TPx fusion protein. SDS- PAGE analysis showed that the EgTPx was expressed in E. coli and the relative molecular weight of expressed fusion protein was 54 000Da. ELISA and Western blot indicated the high titer and specifically antiserum produced by protein immune strategy. Construction of pET41-b- EgTPx recombinant plasmid and preparation of the high titer and specifica antiserum were provided as the experimental materials for the EgTPx function analysis.
To study the localization of the antioxidant protein–thioredoxin peroxidase (TPx) of E. granulosus (EgTPx) in the protoscolex of the parasite, protoscoleces of E. granulosus were aspirated and pooled from sheep liver hydatid cysts collected from a slaughterhouse in Urumqi, Xinjiang. After digested by pepsin, the sedimented protoscoleces were used for paraffin sections and indirect immunofluorescence staining analysis with polyclonal antibody against rEgTPx. The results indicated that the EgTPx distributed mainly in the tegument, subtegument and calcareous corpuscle cells of the protoscolex. The wide distribution and large sites of EgTPx in the parasite were clearly demonstrated. In the present study, we reported on the cloning, overexpression, immunogenicity, localisation and functional characterisation of E. granulosus TPx, which established a significant clue for further studies of biologic functions and application of TPx protein.
In summary, this study could provide primary reference for antioxidant enzymes and genes of E. granulosus. Further studies to elucidate the precise role of antioxidant enzymes may pave the way for considering the enzyme as an alternative target for hydatid disease chemotherapy and present a novel strategy to develop vaccine in human.
硫氧还蛋白过氧化物酶(thioredoxin peroxidases, TPx)基因家族是一个很大的抗氧化蛋白家族,从原核到真核,广泛存在于生物体中,具有强大的抗氧化功能。TPx在寄生虫逃避氧化性伤害过程中起着重要的作用。若能将此功能阻断,则可以减弱或使包虫的生理机能丧失,从而导致其死亡,这将为疫苗的研制和特效药的筛选奠定基础。
原头蚴在天然情况下可以向两个方向发育分化:一个是无性繁殖方向分化形成包囊,另一个是有性繁殖方向分化形成能产卵的成虫。本研究首先利用双相培养方法,掌握了原头蚴体外培养及无性发育至包囊阶段的技术,为建立药物筛选的体外模型,研究虫体发育及生活各阶段的基因功能打下基础。
应用抑制性消减杂交技术(suppression subtractive hybridization,SSH)研究H2O2胁迫下细粒棘球蚴基因的表达。以H2O2胁迫后的细粒棘球蚴cDNA为试验方(tester),正常生长的细粒棘球蚴cDNA为驱动方(driver),构建了H2O2胁迫下与正常细粒棘球蚴差异表达的消减cDNA文库。文库扩增后得到124个阳性克隆,菌落PCR分析,均得到200～1000 bp插入片段。将整个文库克隆进行测序,测得序列结果利用BLAST在线软件与GenBank数据库进行同源序列比对分析和BlastX分析。结果获得重要基因的cDNA序列,如氧化还原酶、蛋白激酶、生长因子等。另有部分克隆在GenBank中无法查到对应的同源基因,可能代表了新基因。利用定量PCR的方法初步获得了原头蚴在H2O2胁迫24h条件下部分差异表达基因片段在mRNA水平上的表达情况,为研究细粒棘球蚴在抗氧化过程中的相关靶基因筛选奠定基础。
采用RT-PCR的方法,根据GenBank已发表的细粒棘球蚴硫氧还蛋白过氧化物酶(EgTPx)基因的序列设计引物,获取EgTPx的cDNA片段并克隆到原核表达载体pET41-b中,构建原核表达质粒pET41-b-EgTPx,经过IPTG诱导,在原核表达系统大肠杆菌E.coli (BL21)中获得可溶性表达的融合蛋白,经过谷胱甘肽Sepharose4B层析柱分离纯化融合蛋白,免疫小鼠获得高效价的特异性抗血清。ELISA和Western blot结果表明融合蛋白免疫小鼠产生了高效价和特异性强的抗血清。为深入开展EgTPx基因功能研究和免疫学诊断试剂,及疫苗的开发分析奠定了物质基础。
利用重组EgTPx多克隆抗体,经消化处理后的原头蚴制备石蜡切片,以间接免疫荧光法确定抗氧化蛋白EgTPx在原头蚴内的分布。结果表明,EgTPx多克隆抗体能够特异性地结合天然EgTPx抗原表位, EgTPx广泛分布在原头蚴的体表皮层、皮下层和钙颗粒细胞内。构建了原头蚴EgTPx基因与绿色荧光蛋白(GFP)融合的虫体表达载体,命名为pPD-EgTPx,通过脂质体转染活性状态良好的原头蚴,在倒置荧光显微镜下观察该基因在原头蚴中的瞬时表达。结果表明,虫体表达载体pPD-EgTPx构建正确,而且能够在原头蚴中进行瞬时表达。
总之,本研究为细粒棘球蚴在抗氧化过程中相关靶基因的筛选和功能研究提供了重要的参考,为进一步利用抗氧化基因开展包虫病的预防治疗和疫苗研制提供了物质基础,具有重要的现实意义。
Echinococcu granulosus, the cause of cystic echinococcosis, is a small tapeworm that resides in the intestine of carnivore definitive hosts,generally dogs or wolves.The life cycle of E. granulosus consists of four stages: egg or oncosphere, hydatid cyst, protoscolex and the adult worm. Survival of the infectious agent in the mammalian hosts requires adaptive mechanisms, such as producing antioxidant enzymes against reactive oxygen species (ROS) generated by the host immune responses. ROS are generally considered to be cytotoxic because high concentrations of ROS can have seriously deleterious effects on membrane lipids, nucleic acids and proteins.To avoid the oxygen toxicity, parasites have developed a mechanism of antioxidant defenses, including enzymes that decompose peroxides and superoxide anion (O2-) and compounds that sequester metal ions. Parasites have adapted to considerable oxidative stress by synthesizing high levels of antioxidant enzymes and, in many cases, expressing them at the host–parasite interface.The levels of these defense enzymes are correlated with the survival of a number of parasitic helminths in host tissues.
Antioxidants including thioredoxin peroxidas have been identified and characterized in parasitic nematodes e.A new class of antioxidant enzyme has recently been described, which reduces hydroperoxides with thioredoxin as an immediate hydrogen donor. These enzymes are known as thioredoxin peroxidases (TPx). TPx, one of the peroxiredoxin (Prx) gene superfamily, functions as an antioxidant to remove ROS and H2O2 derived from normal cellular metabolism using thioredoxin as the electron donor. TPx have been found in diverse organisms ranging from prokaryotes to mammals. EgTPx may play a role in protecting the parasite from oxidative damage.
The larval stages (protoscoleces) are exceptional in having the potential to differentiate in either of two directions. The protoscolex can develop asexually, into the hydatid cyst, or sexually into the adult worm. In vitro culture procedures, PSC were aspirated and pooled from sheep liver hydatid cysts collected from a slaughterhouse in Urumqi, Xinjiang. PSC were collected under sterile conditions from intact cysts, washed three times in phosphate-buffered saline (PBS) prior to digestion in pepsin to release PSC from capsules and remove immature PSC by digestion. After the digestion procedure, the PSC were washed three times with PBS and incubated in glass culture vessels in a liquid DMEM medium placed above a solid-phase base prepared by heating new born calf serum. The solid-phase significantly enhanced PSC growth. After 40 days cultivation, about 10% of the PSC had developed into cysts. In paraffin section of the protoscolex there were many calcareous corpuscle cells, which could provide energy for the protoscolex.The aims of the project are to fully establish and exploit an in vitro culture model that can be used to assess the functions and interactions of a panel of selected E. granulosus genes during differentiation and development of the larval protoscolex stage in an asexual direction to form a hydatid cyst.
To isolate specific genes expressed in PSC of E. granulosus under H2O2 stresses, we used suppression subtractive hybridization (SSH) technique with cDNA from PSC treated with H2O2 as tester and cDNA from PSC in normal growth as driver. mRNA was isolated from the tester and driver respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the products were subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search. The subtractive library of genes regulated by H2O2 stresses was constructed successfully. The amplified library contained 124 clones, of which all clones had 200-1000 bp inserts. Sequence analysis indicated that 124 clones containing the coding sequences, of which some had homology in the GenBank and other were unknown. The obtained sequences may be target genes regulated by H2O2, which provided foundation for the further identify differentially expressed genes in PSC of E. granulosus exposed to oxidative stress.
The cDNA sequence of thioredoxin peroxidase from E. granulosus (EgTPx) was cloned from PSC by RT-PCR and subcloned into the expressive vector pET41-b after sequence analysis。A recombinant plasmid carrying EgTPx gene from was constructed. The resulting recombinant plasmid pET41-b- EgTPx was transformed into the competent cells BL21 and the recombinant plasmid was expressed by IPTG induction.The TPx fusion protein was purified through a Glutathione Sepharose 4B affinity column, and high titer of polyclonal antiserum was obtained by the immunizing the mice using the TPx fusion protein. SDS- PAGE analysis showed that the EgTPx was expressed in E. coli and the relative molecular weight of expressed fusion protein was 54 000Da. ELISA and Western blot indicated the high titer and specifically antiserum produced by protein immune strategy. Construction of pET41-b- EgTPx recombinant plasmid and preparation of the high titer and specifica antiserum were provided as the experimental materials for the EgTPx function analysis.
To study the localization of the antioxidant protein–thioredoxin peroxidase (TPx) of E. granulosus (EgTPx) in the protoscolex of the parasite, protoscoleces of E. granulosus were aspirated and pooled from sheep liver hydatid cysts collected from a slaughterhouse in Urumqi, Xinjiang. After digested by pepsin, the sedimented protoscoleces were used for paraffin sections and indirect immunofluorescence staining analysis with polyclonal antibody against rEgTPx. The results indicated that the EgTPx distributed mainly in the tegument, subtegument and calcareous corpuscle cells of the protoscolex. The wide distribution and large sites of EgTPx in the parasite were clearly demonstrated. In the present study, we reported on the cloning, overexpression, immunogenicity, localisation and functional characterisation of E. granulosus TPx, which established a significant clue for further studies of biologic functions and application of TPx protein.
In summary, this study could provide primary reference for antioxidant enzymes and genes of E. granulosus. Further studies to elucidate the precise role of antioxidant enzymes may pave the way for considering the enzyme as an alternative target for hydatid disease chemotherapy and present a novel strategy to develop vaccine in human.
引文
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