日本血吸虫雌性特异基因及排泄—分泌抗原的筛选
发布时间：2019-06-03 10:56
【摘要】： 研究背景：血吸虫病是由血吸虫感染引起的一种分布广泛、危害严重的人畜共患寄生虫病。据世界卫生组织专家估计，目前仍有76个国家和地区流行血吸虫病，有2亿人受感染，6亿人受威胁。人体血吸虫病的病原为日本血吸虫(Schistosoma japonicum)，埃及血吸虫(S．haematobium Bilharz)，曼氏血吸虫(S．mansoni Sambon)，间插血吸虫(S．intercalatum Fisher)，湄公血吸虫(S．mekongi Voge et al)和马来血吸虫(S．malayensis Greer et al)。在我国流行的血吸虫病为日本血吸虫(中国大陆株)。日本血吸虫病曾流行于我国南方12个省(市)、自治区，至今仍有8个省127个县，至少有2300万人受血吸虫病的威胁。尽管血吸虫病的防治主要依靠化疗及杀灭钉螺，但是血吸虫病的控制并不理想。因而血吸虫病的研究转向了对血吸虫基础原理的研究，尤其是对其基因组的研究。血吸虫基因组计划(Schistosoma Genome Project，SGP)始创于1992年，，“血吸虫基因发现计划”是SGP的一部分，该计划目的是通过对血吸虫(曼氏血吸虫和同本血吸虫)新基因的研究，搜集血吸虫生理活动、耐药机制和免疫逃避方面的资料，为找到新的治疗药物和疫苗奠定基础。尽管血吸虫病的防治主要依靠药物治疗及杀灭钉螺，但是血吸虫病的控制却并不乐观，其中的原因有：治疗药物的毒性、行政管理的不善、寄生虫抗药性等治疗上的限制，频繁的重复感染，有效疫苗的使用和研制进程受到限制及对血吸虫生物学知识的了解不够深入等等。因而在这个血吸虫病防治研究的瓶颈期，大规模地从基因水平了解血吸虫基本的生物学、抗药性机制、决定免疫逃避机制的抗原变异等等都是非常重要的。 关于血吸虫雌性特异性基因方面的研究过去报道很少，这些基因都是用传统方法逐个鉴定的，不仅耗时费力，而且单个基因的克隆表达，无法得到基因之间变化的相关性，因此利用高效的分离、分析技术研究雌虫特异基因的表达，就有重要的意义(如性成熟相关基因、产卵相关基因、雌雄合抱信息传递相关基因等等)，对筛选相关重要功能、阐明其结构与功能，不仅对解读血吸虫生长发育机理有重要意义，更可为合理的设计抗病疫苗或新药物等提供坚实的理论基础，有助于开拓防治血吸虫病的新途径。本研究拟将抑制性消减杂交(suppression subtractive hybridization SSH)和表达序列标签(ExpressionSequence Tag，EST)这一有效的技术方法应用于血吸虫雌性特异基因的筛选。SSH是一种筛选差异表达基因的技术，它是以抑制性PCR(suppression PCR)为基础，将消减杂交(subtractive hybridization，SH)和cDNA单链检测标准化(normalization)合为一体，具有假阳性低、敏感性高、速度快、效率高等特点。而EST是一些从基因文库中随机挑取的cDNA短序列(约300bp)，利用已有的数据库搜索进行DNA或蛋白质的同源性分析，可以鉴定这些基因的起源，从而达到发现基因的目的。我们在建立日本血吸虫雌成虫扣除雄成虫消减cDNA文库的基础上利用SSH技术筛选出雌性特异表达基因，利用EST从日本血吸虫成虫cDNA文库中筛选雌成虫特异的cDNA序列，并对其功能进行研究。从而找到一些有价值的性别特异性基因，为血吸虫病疫苗的研制及防治方法的选择提供依据，将对血吸虫病的防治具有重要的意义。 血吸虫的排泄-分泌(exretory-secretory，E-S)抗原是血吸虫童虫进入人体后在生长发育过程中的排泄分泌产物，包括童虫、成虫及雌虫产出的虫卵在发育成熟过程中的排泄分泌物。这些抗原直接与宿主免疫系统接触，与宿主的抗体形成、免疫调节及肉芽肿的形成密切相关，因而对血吸虫排泄-分泌抗原进行蛋白质谱的鉴定研究，不仅可以掌握更多的疫苗及诊断候选分子，还能更全面地了解血吸虫-宿主的免疫关系，包括所产生的免疫反应、宿主肉芽肿形成的机制等等。本研究通过对日本血吸虫成虫、成虫体外产出的虫卵进行体外培养，收集其E-S抗原。双向凝胶电泳后，切取目标蛋白，LC-MS／MS进行多肽测序。测得的多肽序列利用NCBI的Blast网站的Blast P及Blast N进行同源性搜索，并对血吸虫不同阶段的E-S抗原谱进行分析。这是首次对血吸虫的E-S抗原进行系统的蛋白质谱鉴定分析的研究。我们还发现通过体外培养收集E-S抗原具有简单易行、纯度高、不易污染、不受宿主成分的影响等优点。 目的： 1．发现日本血吸虫雌成虫特异性基因，通过GenBank数据库的软件与所有已知基因和蛋白做同源性分析，初步了解这些基因的功能；找到一些与雌虫的生殖发育和产卵有关的基因，并对其功能进行验证。 2．本研究利用蛋白质组学的研究技术对日本血吸虫的E-S抗原尽可能多地进行鉴定和分析，获得尽可能完善的E-S抗原蛋白质谱。为发现新的疫苗候选分子提供充分的数据和理论依据。 方法： 1．应用Clontech PCR-Select~(TM) cDNA Subtraction Kit构建日本血吸虫雌虫的消减性cDNA库。此库中的cDNA与T载体连接环化成质粒，转化细菌，筛选有插入片段的克隆。 2．随机挑选一些阳性克隆提取质粒DNA，经聚合酶链反应(polymerase chainreaction PCR)扩出插入片段，琼脂糖凝胶电泳后转移到尼龙膜上，然后分别与~(32)p标记的雌雄成虫第一链cDNA探针杂交，筛选出含雌虫特异性的基因片段的质粒DNA。 3．将得到的雌性特异性基因用Blast X及Blast N(http://www.ncbi.him.nih.gov/blast/)程序进行同源性搜索，对基因的功能进行深入的生物信息学分析。 4．通过对日本血吸虫成虫、虫卵体外培养方法的建立来收集E-S抗原。 结果： 1．成功构建了日本血吸虫雌虫的消减cDNA文库。 2．得到正向消减及反向消减cDNA文库，斑点杂交筛选出50个雌虫特异性表达的克隆。 3．将随机挑选出的50个与正向消减文库探针杂交信号值明显高于与反向消减文库探针杂交信号值的克隆进行测序。测序得到了42个EST，其中17个基因与已知日本血吸虫卵壳蛋白基因高度同源；17个基因与日本血吸虫未知基因高度同源、且有一小段与卵壳蛋白基因高度同源；有8个基因与日本血吸虫其他未知基因高度同源。 4．成功的进行了日本血吸虫成虫和虫卵的体外培养，并获得了E-S抗原。 结论： 1．构建了雌、雄虫正向及反向消减cDNA文库。用抑制性消减杂交技术(Suppression subtractive hybridization，SSH)筛选了日本血吸虫雌性特异性表达基因，并对基因的功能进行了预测分析。 2．E-S抗原经SDS-PAGE分析，出现了比较明显的7条蛋白带，其中主带有4条分别为：20.1DKa、31.0DKa、43.0DKa、90.0DKa证明是特异的蛋白带。为下一步试验奠定了坚实的基础。
[Abstract]:Background of the study: Schistosomiasis is a widespread and serious parasitic disease caused by the infection of Schistosoma japonicum. According to the experts of the World Health Organization, there are still 76 countries and regions with endemic schistosomiasis, and 200 million people are infected and 600 million people are under threat. The pathogenic factors of schistosomiasis in the human body are Schistosoma japonicum, S. haerobic Bilharz, S. mansoni Samon, S. intercalaum Fisher, S. mekongi Voge et al. and S. malayensis Greer et al. The prevalence of schistosomiasis in China is Schistosoma japonicum (Chinese mainland strain). Schistosomiasis in Japan has been popular in 12 provinces (cities) and autonomous regions in the south of China, and there are still 8 provinces and 127 counties, with at least 23 million people receiving the threat of schistosomiasis. Although the prevention and control of schistosomiasis mainly depends on the chemotherapy and the killing of the snail, the control of the schistosomiasis is not ideal. Therefore, the study of schistosomiasis has shifted to the study of the basic principle of the schistosome, in particular the study of its genome. The genome project of the Schistosoma japonicum (SGP) was established in 1992, and the "SCHISTOSOMA GENE discovery program" was part of the SGP. The purpose of the program was to collect the information on the physiological activities, the resistance mechanism and the immune escape of the Schistosoma japonicum by the study of the new gene of the Schistosoma japonicum and the same. Lays the foundation for finding new therapeutic drugs and vaccines. Although the prevention and control of the schistosomiasis mainly depends on the medicine treatment and the killing of the oncomelania, the control of the schistosomiasis is not optimistic, the causes are as follows: the toxicity of the treatment medicine, the poor administration of the administration, the limitation of the drug resistance of the parasite and the like, and the frequent repeated infection, The use and development of effective vaccines are limited and the knowledge of the biological knowledge of the schistosome is not well understood. So it is very important to know the basic biology and drug-resistance mechanism of the schistosome from the gene level in the bottle-neck of the schistosomiasis control and control, and to determine the antigenic variation of the immune escape mechanism. The studies on the female specific genes of the Schistosoma japonicum have been reported in the past few years, all of which are identified by a conventional method, not only time-consuming, but also the cloning and expression of a single gene, which is not able to obtain a correlation between the genes, and therefore, It is of great significance to study the expression of female worm-specific gene by high-efficiency separation and analysis (such as sex-related genes, oviposition-related genes, sex-related genes, and so on). The structure and function of the invention can not only provide a solid theoretical foundation for interpreting the growth and development mechanism of the schistosome, but also provide a solid theoretical foundation for rational design of disease-resistant vaccines or new drugs and the like, The effective technique of suppression subtractive hybridization (SSH) and expression sequence tag (EST) is proposed in this study. SSH is a technique for screening differential expression genes, which is based on suppression PCR, and has the advantages of low false positive, high sensitivity and high speed. High efficiency and that like. EST is a short sequence of cDNA (about 300 bp) randomly selecte from the gene library, and the homology analysis of the DNA or protein is carried out by using the existing database search, so that the origin of the genes can be identified, and the generation of the gene can be achieved. The purpose of the present gene is to screen the female specific expression gene from the cDNA library of the adult Schistosoma japonicum adult by using the SSH technology on the basis of establishing the subtractive cDNA library of the female adult of the Schistosoma japonicum, and screening the specific cDNA sequence of the female adult from the cDNA library of the adult Schistosoma japonicum adult cDNA library, so as to find some valuable sex-specific genes, provide the basis for the preparation of the schistosomiasis vaccine and the selection of the prevention and control method, It is of great significance that the excretion-secretion (E-S) antigen of the Schistosoma japonicum is the excretion and secretion product of the schistosome of Schistosoma japonicum in the process of growth and development, including the eggs of the insect, the adult and the female worm. These antigens are directly contacted with the host's immune system, and are closely related to the formation of the host's antibody, the immunoregulation and the formation of the granuloma. holding more vaccines and diagnostic candidate molecules, and more fully understanding the immune relation of the schistosome-host, including the immune response And the mechanism of the host granuloma formation, and the like. In vitro culture, the E-S antigen is collected. After two-way gel electrophoresis, the target protein, L, The sequencing of the polypeptide by C-MS/ MS was carried out by using the Blast P and Blast N of the Blast site of NCBI. The E-S antigen spectrum of the stage is analyzed. This is the first time for the E-S antigen of the schistosome. It is also found that the E-S antigen can be collected by in vitro culture and has the advantages of simple and feasible process, high purity and no pollution. and is not Objective:1. To find the specific gene of the female adult of Schistosoma japonicum, and to analyze the homology of all known genes and proteins by using the software of the GenBank database. The function of some genes; finding one. Some of the genes related to the reproductive development and the oviposition of the female, and the function of the gene was verified. -S antigen is identified and analyzed as much as possible and is as perfect as possible E- S-antigen protein spectrum. To provide sufficient data and theoretical basis for finding new vaccine candidate molecules. Method:1. Clontech PCR was applied. -Select~(TM) cDNA Subtraction Kit鏋勫缓鏃ユ湰琛
本文编号：2491889
[Abstract]:Background of the study: Schistosomiasis is a widespread and serious parasitic disease caused by the infection of Schistosoma japonicum. According to the experts of the World Health Organization, there are still 76 countries and regions with endemic schistosomiasis, and 200 million people are infected and 600 million people are under threat. The pathogenic factors of schistosomiasis in the human body are Schistosoma japonicum, S. haerobic Bilharz, S. mansoni Samon, S. intercalaum Fisher, S. mekongi Voge et al. and S. malayensis Greer et al. The prevalence of schistosomiasis in China is Schistosoma japonicum (Chinese mainland strain). Schistosomiasis in Japan has been popular in 12 provinces (cities) and autonomous regions in the south of China, and there are still 8 provinces and 127 counties, with at least 23 million people receiving the threat of schistosomiasis. Although the prevention and control of schistosomiasis mainly depends on the chemotherapy and the killing of the snail, the control of the schistosomiasis is not ideal. Therefore, the study of schistosomiasis has shifted to the study of the basic principle of the schistosome, in particular the study of its genome. The genome project of the Schistosoma japonicum (SGP) was established in 1992, and the "SCHISTOSOMA GENE discovery program" was part of the SGP. The purpose of the program was to collect the information on the physiological activities, the resistance mechanism and the immune escape of the Schistosoma japonicum by the study of the new gene of the Schistosoma japonicum and the same. Lays the foundation for finding new therapeutic drugs and vaccines. Although the prevention and control of the schistosomiasis mainly depends on the medicine treatment and the killing of the oncomelania, the control of the schistosomiasis is not optimistic, the causes are as follows: the toxicity of the treatment medicine, the poor administration of the administration, the limitation of the drug resistance of the parasite and the like, and the frequent repeated infection, The use and development of effective vaccines are limited and the knowledge of the biological knowledge of the schistosome is not well understood. So it is very important to know the basic biology and drug-resistance mechanism of the schistosome from the gene level in the bottle-neck of the schistosomiasis control and control, and to determine the antigenic variation of the immune escape mechanism. The studies on the female specific genes of the Schistosoma japonicum have been reported in the past few years, all of which are identified by a conventional method, not only time-consuming, but also the cloning and expression of a single gene, which is not able to obtain a correlation between the genes, and therefore, It is of great significance to study the expression of female worm-specific gene by high-efficiency separation and analysis (such as sex-related genes, oviposition-related genes, sex-related genes, and so on). The structure and function of the invention can not only provide a solid theoretical foundation for interpreting the growth and development mechanism of the schistosome, but also provide a solid theoretical foundation for rational design of disease-resistant vaccines or new drugs and the like, The effective technique of suppression subtractive hybridization (SSH) and expression sequence tag (EST) is proposed in this study. SSH is a technique for screening differential expression genes, which is based on suppression PCR, and has the advantages of low false positive, high sensitivity and high speed. High efficiency and that like. EST is a short sequence of cDNA (about 300 bp) randomly selecte from the gene library, and the homology analysis of the DNA or protein is carried out by using the existing database search, so that the origin of the genes can be identified, and the generation of the gene can be achieved. The purpose of the present gene is to screen the female specific expression gene from the cDNA library of the adult Schistosoma japonicum adult by using the SSH technology on the basis of establishing the subtractive cDNA library of the female adult of the Schistosoma japonicum, and screening the specific cDNA sequence of the female adult from the cDNA library of the adult Schistosoma japonicum adult cDNA library, so as to find some valuable sex-specific genes, provide the basis for the preparation of the schistosomiasis vaccine and the selection of the prevention and control method, It is of great significance that the excretion-secretion (E-S) antigen of the Schistosoma japonicum is the excretion and secretion product of the schistosome of Schistosoma japonicum in the process of growth and development, including the eggs of the insect, the adult and the female worm. These antigens are directly contacted with the host's immune system, and are closely related to the formation of the host's antibody, the immunoregulation and the formation of the granuloma. holding more vaccines and diagnostic candidate molecules, and more fully understanding the immune relation of the schistosome-host, including the immune response And the mechanism of the host granuloma formation, and the like. In vitro culture, the E-S antigen is collected. After two-way gel electrophoresis, the target protein, L, The sequencing of the polypeptide by C-MS/ MS was carried out by using the Blast P and Blast N of the Blast site of NCBI. The E-S antigen spectrum of the stage is analyzed. This is the first time for the E-S antigen of the schistosome. It is also found that the E-S antigen can be collected by in vitro culture and has the advantages of simple and feasible process, high purity and no pollution. and is not Objective:1. To find the specific gene of the female adult of Schistosoma japonicum, and to analyze the homology of all known genes and proteins by using the software of the GenBank database. The function of some genes; finding one. Some of the genes related to the reproductive development and the oviposition of the female, and the function of the gene was verified. -S antigen is identified and analyzed as much as possible and is as perfect as possible E- S-antigen protein spectrum. To provide sufficient data and theoretical basis for finding new vaccine candidate molecules. Method:1. Clontech PCR was applied. -Select~(TM) cDNA Subtraction Kit鏋勫缓鏃ユ湰琛
本文编号：2491889
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