【摘要】： 研究背景：血吸蟲病是由血吸蟲感染引起的一種分布廣泛、危害嚴重的人畜共患寄生蟲病。據(jù)世界衛(wèi)生組織專家估計，目前仍有76個國家和地區(qū)流行血吸蟲病，有2億人受感染，6億人受威脅。人體血吸蟲病的病原為日本血吸蟲(Schistosoma japonicum)，埃及血吸蟲(S．haematobium Bilharz)，曼氏血吸蟲(S．mansoni Sambon)，間插血吸蟲(S．intercalatum Fisher)，湄公血吸蟲(S．mekongi Voge et al)和馬來血吸蟲(S．malayensis Greer et al)。在我國流行的血吸蟲病為日本血吸蟲(中國大陸株)。日本血吸蟲病曾流行于我國南方12個省(市)、自治區(qū)，至今仍有8個省127個縣，至少有2300萬人受血吸蟲病的威脅。盡管血吸蟲病的防治主要依靠化療及殺滅釘螺，但是血吸蟲病的控制并不理想。因而血吸蟲病的研究轉向了對血吸蟲基礎原理的研究，尤其是對其基因組的研究。血吸蟲基因組計劃(Schistosoma Genome Project，SGP)始創(chuàng)于1992年，，“血吸蟲基因發(fā)現(xiàn)計劃”是SGP的一部分，該計劃目的是通過對血吸蟲(曼氏血吸蟲和同本血吸蟲)新基因的研究，搜集血吸蟲生理活動、耐藥機制和免疫逃避方面的資料，為找到新的治療藥物和疫苗奠定基礎。盡管血吸蟲病的防治主要依靠藥物治療及殺滅釘螺，但是血吸蟲病的控制卻并不樂觀，其中的原因有：治療藥物的毒性、行政管理的不善、寄生蟲抗藥性等治療上的限制，頻繁的重復感染，有效疫苗的使用和研制進程受到限制及對血吸蟲生物學知識的了解不夠深入等等。因而在這個血吸蟲病防治研究的瓶頸期，大規(guī)模地從基因水平了解血吸蟲基本的生物學、抗藥性機制、決定免疫逃避機制的抗原變異等等都是非常重要的。 關于血吸蟲雌性特異性基因方面的研究過去報道很少，這些基因都是用傳統(tǒng)方法逐個鑒定的，不僅耗時費力，而且單個基因的克隆表達，無法得到基因之間變化的相關性，因此利用高效的分離、分析技術研究雌蟲特異基因的表達，就有重要的意義(如性成熟相關基因、產(chǎn)卵相關基因、雌雄合抱信息傳遞相關基因等等)，對篩選相關重要功能、闡明其結構與功能，不僅對解讀血吸蟲生長發(fā)育機理有重要意義，更可為合理的設計抗病疫苗或新藥物等提供堅實的理論基礎，有助于開拓防治血吸蟲病的新途徑。本研究擬將抑制性消減雜交(suppression subtractive hybridization SSH)和表達序列標簽(ExpressionSequence Tag，EST)這一有效的技術方法應用于血吸蟲雌性特異基因的篩選。SSH是一種篩選差異表達基因的技術，它是以抑制性PCR(suppression PCR)為基礎，將消減雜交(subtractive hybridization，SH)和cDNA單鏈檢測標準化(normalization)合為一體，具有假陽性低、敏感性高、速度快、效率高等特點。而EST是一些從基因文庫中隨機挑取的cDNA短序列(約300bp)，利用已有的數(shù)據(jù)庫搜索進行DNA或蛋白質(zhì)的同源性分析，可以鑒定這些基因的起源，從而達到發(fā)現(xiàn)基因的目的。我們在建立日本血吸蟲雌成蟲扣除雄成蟲消減cDNA文庫的基礎上利用SSH技術篩選出雌性特異表達基因，利用EST從日本血吸蟲成蟲cDNA文庫中篩選雌成蟲特異的cDNA序列，并對其功能進行研究。從而找到一些有價值的性別特異性基因，為血吸蟲病疫苗的研制及防治方法的選擇提供依據(jù)，將對血吸蟲病的防治具有重要的意義。 血吸蟲的排泄-分泌(exretory-secretory，E-S)抗原是血吸蟲童蟲進入人體后在生長發(fā)育過程中的排泄分泌產(chǎn)物，包括童蟲、成蟲及雌蟲產(chǎn)出的蟲卵在發(fā)育成熟過程中的排泄分泌物。這些抗原直接與宿主免疫系統(tǒng)接觸，與宿主的抗體形成、免疫調(diào)節(jié)及肉芽腫的形成密切相關，因而對血吸蟲排泄-分泌抗原進行蛋白質(zhì)譜的鑒定研究，不僅可以掌握更多的疫苗及診斷候選分子，還能更全面地了解血吸蟲-宿主的免疫關系，包括所產(chǎn)生的免疫反應、宿主肉芽腫形成的機制等等。本研究通過對日本血吸蟲成蟲、成蟲體外產(chǎn)出的蟲卵進行體外培養(yǎng)，收集其E-S抗原。雙向凝膠電泳后，切取目標蛋白，LC-MS／MS進行多肽測序。測得的多肽序列利用NCBI的Blast網(wǎng)站的Blast P及Blast N進行同源性搜索，并對血吸蟲不同階段的E-S抗原譜進行分析。這是首次對血吸蟲的E-S抗原進行系統(tǒng)的蛋白質(zhì)譜鑒定分析的研究。我們還發(fā)現(xiàn)通過體外培養(yǎng)收集E-S抗原具有簡單易行、純度高、不易污染、不受宿主成分的影響等優(yōu)點。 目的： 1．發(fā)現(xiàn)日本血吸蟲雌成蟲特異性基因，通過GenBank數(shù)據(jù)庫的軟件與所有已知基因和蛋白做同源性分析，初步了解這些基因的功能；找到一些與雌蟲的生殖發(fā)育和產(chǎn)卵有關的基因，并對其功能進行驗證。 2．本研究利用蛋白質(zhì)組學的研究技術對日本血吸蟲的E-S抗原盡可能多地進行鑒定和分析，獲得盡可能完善的E-S抗原蛋白質(zhì)譜。為發(fā)現(xiàn)新的疫苗候選分子提供充分的數(shù)據(jù)和理論依據(jù)。 方法： 1．應用Clontech PCR-Select~(TM) cDNA Subtraction Kit構建日本血吸蟲雌蟲的消減性cDNA庫。此庫中的cDNA與T載體連接環(huán)化成質(zhì)粒，轉化細菌，篩選有插入片段的克隆。 2．隨機挑選一些陽性克隆提取質(zhì)粒DNA，經(jīng)聚合酶鏈反應(polymerase chainreaction PCR)擴出插入片段，瓊脂糖凝膠電泳后轉移到尼龍膜上，然后分別與~(32)p標記的雌雄成蟲第一鏈cDNA探針雜交，篩選出含雌蟲特異性的基因片段的質(zhì)粒DNA。 3．將得到的雌性特異性基因用Blast X及Blast N(http://www.ncbi.him.nih.gov/blast/)程序進行同源性搜索，對基因的功能進行深入的生物信息學分析。 4．通過對日本血吸蟲成蟲、蟲卵體外培養(yǎng)方法的建立來收集E-S抗原。 結果： 1．成功構建了日本血吸蟲雌蟲的消減cDNA文庫。 2．得到正向消減及反向消減cDNA文庫，斑點雜交篩選出50個雌蟲特異性表達的克隆。 3．將隨機挑選出的50個與正向消減文庫探針雜交信號值明顯高于與反向消減文庫探針雜交信號值的克隆進行測序。測序得到了42個EST，其中17個基因與已知日本血吸蟲卵殼蛋白基因高度同源；17個基因與日本血吸蟲未知基因高度同源、且有一小段與卵殼蛋白基因高度同源；有8個基因與日本血吸蟲其他未知基因高度同源。 4．成功的進行了日本血吸蟲成蟲和蟲卵的體外培養(yǎng)，并獲得了E-S抗原。 結論： 1．構建了雌、雄蟲正向及反向消減cDNA文庫。用抑制性消減雜交技術(Suppression subtractive hybridization，SSH)篩選了日本血吸蟲雌性特異性表達基因，并對基因的功能進行了預測分析。 2．E-S抗原經(jīng)SDS-PAGE分析，出現(xiàn)了比較明顯的7條蛋白帶，其中主帶有4條分別為：20.1DKa、31.0DKa、43.0DKa、90.0DKa證明是特異的蛋白帶。為下一步試驗奠定了堅實的基礎。
[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鏋勫緩鏃ユ湰琛

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