论文标题:日本血吸虫雌性特异基因及排泄—分泌抗原的筛选
Screening of the Specific Genes of Female Schistosoma Japonicum and the Exretory-secretory Antigen
论文作者
论文导师 彭鸿娟；郑学礼,论文学位 硕士,论文专业 病原生物学
论文单位 南方医科大学,点击次数 83,论文页数 97页File Size4938K
2007-05-25论文网 http://www.lw23.com/lunwen_251781137/
Schistosoma japonicum;; female specific gene;; Suppression subtractive hybridization;; dot blot hybridization;; excreted -secreted antigens
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。
Backgroand: Schistosomiasis, an important verminosis caused by schistosome, is widely and severly hamful to human beings and livestocks. WHO statistics indicated that about 200 million people are infected in 76 countries and prevailing area, about 600 million people are threatened by this parasitic zoonosis. There are many kinds of Schistosomes that can parasitize on human body, such as Schistosoma japonicum, S. haematobium, S. mansoni S. intercalatum, S. mekongi S. malayensis. Schistosoma japonicum is endemic in Southeast Asia including China and Philipine. Schistosomiasis had been epidemic in 12 provinces in the south of China, there"re still 22 million people at the risk of suffering from this disease in 8 provinces and 127 counties. Although chemo and snail control had been applied to prevent this verminosis, it is still insufficient to controll this disease. Consequently, the study of schistosomiasis diverstion to research on foundation principle of schistosome, especially on genome of schistosomiasis. Schistosoma Genome Project(SGP) began in 1992, "Schistosoma Genome Discovery Project" is a part of SGP, this project is to find new drug and vaccine targets through finding and identification of novel genes of S. mansoni and S. japonicum, collecting the datas for physiological functions, drug resistance mechanism and immune evasion. Schistosomiasis prevention and therapy is major through chemocontrol and snail controll, but it is still far away from getting good result, it may ascribe to drug toxicity, administration poverty, drug-resistance, frequent re-infection, the restriction of using and developing effective vaccine, the shortage of deep and wide knowledge about schistosome biology. Therefore, in this hard time for schistosome control, a large scale research on basic schistosome biology, drug-resistance and immune evasion in gene level is very important. There"re not many reports about specific genes of female S. japonicum, these genes were identified one by one in traditional way, it was not only waste of time, but also impossible to get the imformation about the interaction of every single gene. For this reason, it"s important to research on the S. japonicum female-specific gnes by high throughput technique to screen and identify sex-maturity related genes, egg-laying related genes, signal transmission of paroecious embracing genes. It"s ergent to screen and identify the related functions and structures of these genes. It is helpful to understand the growth and development rationale of S. japonicum, and also provide a substantial way to devise reasonable anti-disease vaccines and new drugs, then in the end it will be helpful to expand a new way to prevent and cure schistosomiasis. This study applied Suppression Subtractive Hybridization (SSH) and Expression Sequence Tag (EST) method to screen female specific genes of S. japonicum. SSH is a new and highly effective method, which has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. We constructed the female S. japonicum substrative cDNA library by SSH to screen the female specific genes. The ultimate objective is to find some valuable female specific genes. It"s significant for vaccine development and schistosomiasis control. Secreted antigens of S. japonicum is excreted by development process of schistosomulum after invading human body, including the secreta of schistosomulum, adult worms and development stage of eggs. Host"s immunologic system response to the E-S antigens, it"s related with the provocation of antibody, immunoregulation and the forming of granulomatous. For these reasons, it"s good to palmar grasp much more molecules for vaccine and diagnosis, and understand overall the immune relationship between S.j and host, such as immunoreaction, the mechanism of granuloma and so on. We collected E-S antigens of schistosomulum, adult worms and eggs spawned in vitro, after two-D SDS-PAGE and then got the target bands of protien to sequencing polypeptide by LC-MS/MS. The polypeptide sequence blasted by NCBI for searching the homology. We analyzed the spectrotype of differents stages of E-S antigens. This study of identifying and analyzing protein-spectrum of S.j E-S antigens is never reported at present. It"s simple and easy to manipulate by culture in vitro, and it can get high purity E-S antigens without the effective of host ingredient and avoid contaminating. Objective: 1. To discover the female specific genes of S.japonicum, after homology search through Blast program in GenBank, probably get the message of the gene functions; and to find and authenticate reproductive development gene and egg-laying gene. 2. To gain spectrotype of E-S antigens as perfect as possible, we utilized technology of proteome to identify and analyze E-S antigens of S.j. This basic work provided datas for discovering new molecules for vaccine. Methods: 1. Constructing the female S. japonicum subtractive cDNA library with CLONTECH PCR-SelectTM cDNA Subtraction Kit. This cDNAs were linked with T vector to form a plasmid, and transformed to E. coli, then these plasmids were used to screen the target genes. 2. Some positive clone plasmids were randomly selected, the insers were amplified by PCR, then transferred to nylon membrane after agarose gel electrophoresis, and used to hybridize with the first cDNA chain of male and female worms marked with ~(32)p. Then the female specific genes were screened and identified. 3. The female specific genes were sequenced and searched through Blast X and Blast N (http://www.ncbi.nlm.nih.gov/blast/) program to predict the gene function. 4. Gathering E-S antigens by founding S. japonicum adult and ovum culture in vitro process. Results: 1. The female S. japonicum subtractive cDNA library was constructed. 2. Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. After dot-blot hybridization, 50 clones were tested to be the potential femal differentially expressed genes and were sequenced. 3. 50 randomly selected clones were hybridized with female subtractive and male subtractive libraries, the former hybridization signal were stronger than the latter ones, then 42 expressing sequence tags were got. After bioinformatics analysis, 17 fragments showed high identity with the S. japonicum egg-shell protein genes, 17 sequences were highly homologous to unknown S. japonicum genes and partly homologous to female specific 800 protien. 8 fragments showed high identity with other S. japonicum unknown genes. 4. S. japonicum adult and ovum culture in vitro process is founded, and has gained the E-S antigen. Conclusion: 1. Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. A number of specific genes have been found by SSH. 2. E-S antigens were analyzed by SDS-PAGE, showing 7 relative evident protien bands. There were 4 main bands among them and the corresponding molecular weight of the protein bands respective are 20.1DKa、31.0DKa、43.0DKa、90.0Dka, meanwhile they were tested to be the specific protein bands. This work settled substantial foundation to forward study.
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