日本血吸虫中国大陆株性别差异表达基因的研究
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摘要
血吸虫病世界性分布，危害严重，为人畜共患的重大疾病之一。雌雄合抱是血吸虫发育、成熟、产卵的前提，而虫卵是造成病理损害和病原传播的关键。本文针对在我国流行的日本血吸虫中国大陆株开展性别差异表达基因等研究，以深入探索血吸虫发育及生殖机理、开拓免疫预防新途径。
本研究利用双向电泳和质谱技术测定了2种血吸虫雄虫和一种血吸虫雌虫性别差异表达蛋白质的肽质量指纹图谱，并利用生物信息学方法对血吸虫蛋白质进行了初步分析，为进一步利用蛋白质组学方法寻找性别差异表达蛋白质及其相应基因提供了基础。
本研究应用RT-PCR方法和5'RACE技术，首次克隆得到日本血吸虫抱雌沟蛋白的完整编码基因SjGcp，已申报发明专利(申请号：02110673.8；GeneBank登录号：AF519183)。同源性分析提示该蛋白质可能具有发育调节功能。Western印迹分析结果说明该基因在大肠杆菌中的融合表达产物rSjGcp具有良好的抗原性。攻击保护实验显示与保守区相比，血吸虫抱雌沟蛋白全长具更强的免疫保护功能、尤其对减少宿主肝脏卵负荷表现更好效果，有重要意义。
对血吸虫卵壳蛋白的研究有助于阐明雌虫发育机制和研制抗卵胚发育的药物和疫苗。本研究利用RT-PCR、5'RACE和3'RACE技术首次克隆得到日本血吸虫卵壳前体蛋白基因(Sj423)的完整cDNA(GeneBank登录号：D32205)。同源性分析表明该基因编码蛋白质可能具有肝片吸虫卵壳前体蛋白相似的性质和功能。通过从血吸虫基因组DNA中克隆该基因并进行测序分析发现该基因同其他血吸虫卵壳蛋白基因一样没有内含子，这可能有利于卵壳前体蛋白的迅速和大量形成。Western印迹分析结果说明该基因在大肠杆菌中的融合表达产物rSj423具有良好的抗原性。
为查明SjGcp和Sj423基因表达的性别和时相差异，通过Southern blot和Northern blot进行了检测。结果表明SjGcp基因为雄虫特异性表达基因，在尾蚴感染后第14天即雌雄虫将合抱前的雄虫体内可见转录表达；Sj423基因为雌虫特异性表达基因，在尾蚴感染后第11天即雌雄合抱前更早阶段的雌虫体内已见转录
博士学位论文 摘 要 中国农业科学院
表达，显示了血吸虫发育、生殖及其调控机制的复杂性和多样性。
另外，近有研究结果显示IgE抗体在血吸虫病的保护性免疫中起着重要作用，
日本血吸虫菲律宾株一诱导该抗体的抗原编码基因（Sip21．7）己被克隆。我们采
用 RTPCR方法克隆得到日本血吸虫中国大陆株的相应基因 SjCZI．7。软件分析结
果提示其可能与血吸虫的膜相关蛋白及肝片吸虫的钙结合蛋白有相似的结构和功
能，并利用软件对该蛋白质的结构和功能域进行了预测，表明其具有作为血吸虫
候选疫苗抗原的发展潜能。Western印迹分析结果说明该基因在大肠杆菌中的融
合表达产物 rsjcZI．7具有良好的抗原性。
总之，本研究利用双向电泳、质谱结合生物信息学技术对日本血吸虫雌雄蛋
白质差异进行了初步分析和探索，首次克隆了日本血吸虫中国大陆株性别差异表
达基因SjGCp和Sj423及在血吸虫病免疫保护中起重要作用的抗原基因SjCZI．7，
并对其抗原性、SjGCp和 Sj423基因的表达差异性及 SjGCp基因在大肠杆菌中融
合表达产物的免疫保护功能进行了研究。本项研究为进一步阐明血吸虫性别发育。
生殖机制，提供了一定的理论基础，对开发血吸虫病疫苗候选抗原基因和研制新
药物，具有重要意义。
Schistosomiasis is an important helminthic disease of human being and animals in the world. The pairing between male and female is necessary for the development, maturing and egg production of schistosome, and the eggs laid by the mature female parasite is the main pathogenic source for the disease. Focusing in the schistosoma japonicum (Chinese strain) spreading in China, the study on the sex-specific expressing genes "is important in theorical and practical significance for elucidating the schistosome mechanism of development and reproduction, developing novel vaccines and drugs against schistosomiasis.
2-DE and mass spectrometry methods were employed to identity sex-differencial expression proteins, the peptide mass fingerprintings of two male-specific proteins and one female-specific protein of schistosome were got, and these proteins were initially analyzed by bioinformatics. The results provided basic knowladge for the following studies.
RT-PCR and 5'RACE techniques were used to clone the SjGCP gene (GenBank accerssion number: AF519183) encoding gynecophoral canal protein of Schistosoma japonicum (Chinese strain). Homology analysis indicated that the protein is identical to some factors with developmental-regulated functioa It was found that the fusion protein has good antigenicity by Western-blot. Comparison with peptide of the conservative region rSjGCPl, rSjGCP induced relatively high protective effect, especially on greatly reducing the egg number in liver.
The research on eggshell protein is helpful to elucidate the mechanism of female development and the development of new drugs or vaccines against egg development. In this study, RT-PCR, 5'RACE and 3'RACE techniques were used to clone the full-length cDNA of the precursor eggshell protein gene (sj423, GenBank accerssion number: AF519182). Homology analysis indicated that the protein is identical to the eggshell precursor protein of F. hepatica. Sequencing analysis showed that the gene, like that of other eggshell protein of schistosoma, has no introns. Western blotting showed that the recombinant protein had good antigenicity.
Southern blotting and Northern blotting detected the sex-specific and stage-differential expression of sjGCP and sj423. The results showed that the SjGcp is a male-specific expressing gene and the gene was found to be transcripted on day 14 post infection; the Sj423 is a female-specific expressing gene and the gene was found to be transcripted on day 11 post infection. The results also showed the complexity and diversity of schistosome development, reproduction and regulated machinism.
In addition, it is reported that specific IgE could play an important role in the immunological response against schistosome. The gene Sjc21.7 (GenBank accerssion number: AF519184) corresponding to Sjp21.7 (Philippines strain) that immunoscreened by IgE antibodies from high-IgE/SWAP responders was cloned. Homology analysis suggested that the protein could have the structure and function similar to tegumental protein of schistosome and calcuim-binding protein of F. hepatica. The prediction results of construction and function domains indicated that the protein has good potentiality as novel viccine against Schistosomiasis. Western blotting showed that the recombinant protein had good antigenicity.
In conclusion, the expression difference of protein between male and female of Schistosoma japonicum (Chinese strain) was analyzed by 2-DE, MS and bioinformatics techniques. The male-specific gene SfGcp, female-specific gene Sj423 and important immunologic antigen gene Sfc2L7 of Schistosoma japonicum (Chinese strain) were cloned. The studies on their antigenicity, the sex-specific and stage-differential ofsjGCP and sj423 and the protective effect of rSjGcp were studied. This study provides an important foundation in theory and practice for further studying on the mechanism of sex maturing, as well as the development of novel viccines or new drugs against Schistosomiasis.
本研究利用双向电泳和质谱技术测定了2种血吸虫雄虫和一种血吸虫雌虫性别差异表达蛋白质的肽质量指纹图谱，并利用生物信息学方法对血吸虫蛋白质进行了初步分析，为进一步利用蛋白质组学方法寻找性别差异表达蛋白质及其相应基因提供了基础。
本研究应用RT-PCR方法和5'RACE技术，首次克隆得到日本血吸虫抱雌沟蛋白的完整编码基因SjGcp，已申报发明专利(申请号：02110673.8；GeneBank登录号：AF519183)。同源性分析提示该蛋白质可能具有发育调节功能。Western印迹分析结果说明该基因在大肠杆菌中的融合表达产物rSjGcp具有良好的抗原性。攻击保护实验显示与保守区相比，血吸虫抱雌沟蛋白全长具更强的免疫保护功能、尤其对减少宿主肝脏卵负荷表现更好效果，有重要意义。
对血吸虫卵壳蛋白的研究有助于阐明雌虫发育机制和研制抗卵胚发育的药物和疫苗。本研究利用RT-PCR、5'RACE和3'RACE技术首次克隆得到日本血吸虫卵壳前体蛋白基因(Sj423)的完整cDNA(GeneBank登录号：D32205)。同源性分析表明该基因编码蛋白质可能具有肝片吸虫卵壳前体蛋白相似的性质和功能。通过从血吸虫基因组DNA中克隆该基因并进行测序分析发现该基因同其他血吸虫卵壳蛋白基因一样没有内含子，这可能有利于卵壳前体蛋白的迅速和大量形成。Western印迹分析结果说明该基因在大肠杆菌中的融合表达产物rSj423具有良好的抗原性。
为查明SjGcp和Sj423基因表达的性别和时相差异，通过Southern blot和Northern blot进行了检测。结果表明SjGcp基因为雄虫特异性表达基因，在尾蚴感染后第14天即雌雄虫将合抱前的雄虫体内可见转录表达；Sj423基因为雌虫特异性表达基因，在尾蚴感染后第11天即雌雄合抱前更早阶段的雌虫体内已见转录
博士学位论文 摘 要 中国农业科学院
表达，显示了血吸虫发育、生殖及其调控机制的复杂性和多样性。
另外，近有研究结果显示IgE抗体在血吸虫病的保护性免疫中起着重要作用，
日本血吸虫菲律宾株一诱导该抗体的抗原编码基因（Sip21．7）己被克隆。我们采
用 RTPCR方法克隆得到日本血吸虫中国大陆株的相应基因 SjCZI．7。软件分析结
果提示其可能与血吸虫的膜相关蛋白及肝片吸虫的钙结合蛋白有相似的结构和功
能，并利用软件对该蛋白质的结构和功能域进行了预测，表明其具有作为血吸虫
候选疫苗抗原的发展潜能。Western印迹分析结果说明该基因在大肠杆菌中的融
合表达产物 rsjcZI．7具有良好的抗原性。
总之，本研究利用双向电泳、质谱结合生物信息学技术对日本血吸虫雌雄蛋
白质差异进行了初步分析和探索，首次克隆了日本血吸虫中国大陆株性别差异表
达基因SjGCp和Sj423及在血吸虫病免疫保护中起重要作用的抗原基因SjCZI．7，
并对其抗原性、SjGCp和 Sj423基因的表达差异性及 SjGCp基因在大肠杆菌中融
合表达产物的免疫保护功能进行了研究。本项研究为进一步阐明血吸虫性别发育。
生殖机制，提供了一定的理论基础，对开发血吸虫病疫苗候选抗原基因和研制新
药物，具有重要意义。
Schistosomiasis is an important helminthic disease of human being and animals in the world. The pairing between male and female is necessary for the development, maturing and egg production of schistosome, and the eggs laid by the mature female parasite is the main pathogenic source for the disease. Focusing in the schistosoma japonicum (Chinese strain) spreading in China, the study on the sex-specific expressing genes "is important in theorical and practical significance for elucidating the schistosome mechanism of development and reproduction, developing novel vaccines and drugs against schistosomiasis.
2-DE and mass spectrometry methods were employed to identity sex-differencial expression proteins, the peptide mass fingerprintings of two male-specific proteins and one female-specific protein of schistosome were got, and these proteins were initially analyzed by bioinformatics. The results provided basic knowladge for the following studies.
RT-PCR and 5'RACE techniques were used to clone the SjGCP gene (GenBank accerssion number: AF519183) encoding gynecophoral canal protein of Schistosoma japonicum (Chinese strain). Homology analysis indicated that the protein is identical to some factors with developmental-regulated functioa It was found that the fusion protein has good antigenicity by Western-blot. Comparison with peptide of the conservative region rSjGCPl, rSjGCP induced relatively high protective effect, especially on greatly reducing the egg number in liver.
The research on eggshell protein is helpful to elucidate the mechanism of female development and the development of new drugs or vaccines against egg development. In this study, RT-PCR, 5'RACE and 3'RACE techniques were used to clone the full-length cDNA of the precursor eggshell protein gene (sj423, GenBank accerssion number: AF519182). Homology analysis indicated that the protein is identical to the eggshell precursor protein of F. hepatica. Sequencing analysis showed that the gene, like that of other eggshell protein of schistosoma, has no introns. Western blotting showed that the recombinant protein had good antigenicity.
Southern blotting and Northern blotting detected the sex-specific and stage-differential expression of sjGCP and sj423. The results showed that the SjGcp is a male-specific expressing gene and the gene was found to be transcripted on day 14 post infection; the Sj423 is a female-specific expressing gene and the gene was found to be transcripted on day 11 post infection. The results also showed the complexity and diversity of schistosome development, reproduction and regulated machinism.
In addition, it is reported that specific IgE could play an important role in the immunological response against schistosome. The gene Sjc21.7 (GenBank accerssion number: AF519184) corresponding to Sjp21.7 (Philippines strain) that immunoscreened by IgE antibodies from high-IgE/SWAP responders was cloned. Homology analysis suggested that the protein could have the structure and function similar to tegumental protein of schistosome and calcuim-binding protein of F. hepatica. The prediction results of construction and function domains indicated that the protein has good potentiality as novel viccine against Schistosomiasis. Western blotting showed that the recombinant protein had good antigenicity.
In conclusion, the expression difference of protein between male and female of Schistosoma japonicum (Chinese strain) was analyzed by 2-DE, MS and bioinformatics techniques. The male-specific gene SfGcp, female-specific gene Sj423 and important immunologic antigen gene Sfc2L7 of Schistosoma japonicum (Chinese strain) were cloned. The studies on their antigenicity, the sex-specific and stage-differential ofsjGCP and sj423 and the protective effect of rSjGcp were studied. This study provides an important foundation in theory and practice for further studying on the mechanism of sex maturing, as well as the development of novel viccines or new drugs against Schistosomiasis.
引文
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