【作者】 高阳；

【导师】 王立群；

【作者基本信息】 东北农业大学 ， 微生物， 2013， 硕士

【摘要】 为研究日本血吸虫Wnt基因信号转导蛋白对血吸虫发育的调节作用，对不同期别和性别间Wnt的mRNA表达水平进行了分析。采用定量PCR方法比较了不同发育阶段以及单、复性感染的25天雌雄虫之间Wnt基因的mRNA的表达水平。以23天虫体cDNA为模板，分别扩增编码SjWnt11，SjWnt5和SjWnt1蛋白核苷酸序列，构建了重组表达质粒，并进行了蛋白表达及纯化。以纯化的Wnt基因重组蛋白分别免疫BALB/c小鼠制备了特异性的多抗，用于后续研究。并利用Western blot对SjWnt5蛋白在虫体不同发育阶段的表达水平进行了分析，采用免疫组化（IHC）方法检测SjWnt5蛋白在不同期别虫体的组织定位。利用GenomeWalking技术分析日本血吸虫SjWnt11的基因结构。SjWnt11mRNA在13天表达量的增加，推测SjWnt11可能调节虫体的器官发生过程。SjWnt5mRNA23天生殖器官初步建成开始，SjWnt5mRNA在雌虫中表达逐渐下降，而在雄虫中不断上调。推测SjWnt5引发Wnt信号对虫体发育早期的器官分化有调节作用；对雌虫生殖器官的建成及性成熟雄虫的进一步发育都可能具有调节作用。SjWnt1基因的mRNA在7天时表达量最高，虫卵时期表达量次之，13天以后时期表达量维持在最低水平，推测该蛋白在卵胚发育和虫体早期器官分化过程中发挥调控作用。SjWnt4mRNA水平在7日龄童虫的表达量最高，在其它发育阶段表达量相对较低，推测SjWnt4与卵胚发育、虫体早期器官分化有关。SjWnt2同样在日本血吸虫不同发育阶段表达有差异。在虫卵时期表达量最高，7天童虫细胞分化后表达量出现下调。推测可能与卵胚形成有关。SjWnt11和frizzled7，SjWnt1、frizzled5和frizzled8，SjWnt4和frizzled9表达趋势基本一致，初步推测SjWnt11和frizzled7可能发生相互作用，SjWnt4和frizzled9可能发生相互作用，SjWnt1、frizzled5和frizzled8可能发生相互作用。在25天单性感染雌雄虫和复性感染雌雄虫中Wnt基因mRNA表达水平存在较大差异，推测Wnt信号对雌虫生殖器官的建成及性成熟雄虫的进一步发育可能具有调节作用。成功构建SjWnt11，SjWnt5和SjWnt1基因原核表达质粒在大肠杆菌中成功表达，制备的多抗血清能与虫体天然蛋白的单一条带发生反应，证明多抗是有良好的特异性。通过western blot方法比较日本血吸虫SjWnt5在不同发育阶段的蛋白水平差异，SjWnt5的蛋白表达量的变化趋势与SjWnt5mRNA变化基本一致，结合日本血吸虫发育特征，进一步证实了SjWnt5基因在日本血吸虫器官分化过程起着重要作用。免疫组化（IHC）结果显示SjWnt5蛋白在虫体体壁肌细胞层有阳性着色。并获得了SjWnt11的基因结构，具有5个内含子。对日本血吸虫SjWnt11，SjWnt1和SjWnt5进行了不同期别和性别的mRNA表达水平分析，综合分析推测Wnt通路的调节可能涉及血吸虫的多个发育条件，如细胞分化、器官形成、雌雄虫生殖器官的建成及成熟以及卵胚的发育等。为进一步利用RNA干扰等技术探讨Wnt基因对血吸虫发育的调节作用提供了基础数据。更多还原

【Abstract】 Praziquantel is the only chemotherapy drug to treat the disease. Satisfactory therapeuticeffect, but the long-term single, repeated drug use, there is a risk of drug resistance[1,2]. Fullyunderstand the regulation of growth and development of the schistosome mechanism is the basisof the research and development of new drugs. For the study of Schistosoma japonicum Wntsignal transduction protein gene developmental regulation of the parasite,on the various stagesand sex of Wnt mRNA expression were analyzed.23days parasite cDNA as a template, whichamplified protein and nucleotide sequences of SjWnt11, SjWnt5SjWnt1, and constructedrecombinant expression plasmid of PET28a-SjWnt11, PET28a-SjWnt5and PET28a-SjWnt1,and conduct a protein expression and purification.The purified wnt gene recombinant proteinimmunized BALB/c mice, and specific polyclonal antibody was prepared for the follow-upstudy. Using Western blot analized expression levels of SjWnt5proteins in differentdevelopmental stages of the parasite. Use GenomeWalking technical analysis of the geneticstructure of Schistosoma japonicum SjWnt11.23days genital initially built started and SjWnt5mRNA expression decreased in females, while in males constantly increased.SjWnt11mRNA expression at13days the increase, which presumably SjWnt11may adjustprocess occurs of parasites organ. It inferred SjWnt5triggered Wnt signaling had a moderatingeffect for the early development of organ differentiation parasites. The completion of the femalereproductive organs, and the further development of sexually mature males are likely to haveregulatory role. The mRNA of SjWnt1were the highest expression in7days, and eggs periodwere the second expression.13days later,Period expression remained at the lowest level. Itspeculated that the protein in the egg parasites early embryo development and organdifferentiation play a regulatory role in the process. SjWnt4mRNA level was the highest at7days schistosomula of expression, and in other developmental stages of expression wasrelatively low. The result suggested that SjWnt4and embryo development, body early organdifferentiation. SjWnt2was also expressed in different at developmental stages of Schistosomajaponicum differences. The highest expression level was in the egg period, and7daysschistosomula cell differentiation expression was down-regulated. This was probably related toembryo formation. By comparing the binding receptor frizzled mRNA in different period ofgender expression, SjWnt11and frizzled7, SjWnt1, frizzled5and frizzled8, SjWnt4andfrizzled9expression tendency was consistent.It speculated that SjWnt11and frizzled7may interact, SjWnt4and frizzled9may interact, SjWnt1, frizzled5and frizzled8may interact. Onthe25day sex infection of male and female worms and multiple infection of male and femaleworms, There was a big difference between the level of Wnt gene expression of mRNA. It canbe speculated that The Wnt signal may play a role in the regulation of the female reproductiveorgan built, further development and maturation of male, may play a role in the regulation ofWnt signal. The successful construction of SjWnt11, prokaryotic expression of SjWnt5andSjWnt1gene plasmid was expressed successfully in e.coli, and polyclonal antibody preparationcan be single and the larvae of natural protein with reaction. This proves that polyclonalantibody had good specificity. By Western blot method, comparison of Schistosoma japonicumSjWnt5in protein level in different developmental stages of the differences, the trend of theexpression of SjWnt5protein and SjWnt5mRNA was consistent. Combined with thedevelopment characteristics of Schistosoma japonicum, which furtherly confirmed that SjWnt5gene plays an important role in the process of Schistosoma japonicum organ differentiation.Andgene structure of SjWnt11were obtained, with5introns.Schistosoma japonicum SjWnt11, SjWnt1and SjWnt5had been analysed in Various stagesand gender mRNA. Comprehensive analyed that Wnt pathway may be involved in thedevelopment of Schistosoma japonicum multiple conditions, such as cell differentiation, organformation, male and female reproductive organs built and maturation and embryo development.Studying for the further use of RNA interference technique, Wnt gene and provide basic data toregulate the development of Schistosoma japonicum.更多还原