【作者】 张亮；

【导师】 林矫矫；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2002， 硕士

【摘要】 血吸虫病是一种分布广泛，危害严重的人畜共患寄生虫病。利用基因工程技术发展血吸虫病疫苗已成为当今寄生虫学者研究的热点。目前已鉴定出数个有免疫保护潜能的曼氏血吸虫及日本血吸虫疫苗分子。尽管血吸虫病疫苗候选抗原分子的研究有了很大的进展，但至今保护效果都不够理想，因此开辟新途径，不断寻找新的有效的候选抗原分子，以提高免疫保护作用，仍是当前血吸虫病研究的重点。 研究表明，东方田鼠作为日本血吸虫的非相容性宿主对日本血吸虫病具有先天遗传的天然抵抗力，野生东方田鼠和实验室繁殖传代的东方田鼠同样具有这一特性。已报道有39种哺乳动物可自然感染血吸虫，东方田鼠是迄今发现的唯一一种具有抗日本血吸虫病现象的啮齿类动物。机制研究表明体液免疫在其中发挥重要作用，其血清IgG3抗体与东方田鼠抗日本血吸虫病相关。据此我们用东方田鼠阴性血清免疫筛选日本血吸虫成虫cDNA文库，以期分离东方田鼠血清IgG3抗体针对的日本血吸虫特异性抗原基因并进一步研究其生物学功能，为研制和开发新的血吸虫病疫苗或治疗药物开辟新思路。 本研究利用实验室繁殖传代的东方田鼠阴性血清和辣根过氧化物酶标记的羊抗小鼠IgG3免疫筛选日本血吸虫成虫cDNA文库，经过初筛和复筛获得两个阳性克隆。应用RACE技术对两个阳性克隆的cDNA片段进行扩增，获得两个东方田鼠血清IgG3抗体相关的日本血吸虫抗原编码基因。同源序列分析表明，获得的两个基因为新的日本血吸虫抗原编码基因，国内外未见相关报道。其中N-1基因的开放阅读框长780bp，编码259个氨基酸，N-2基因的开放阅读框长336bp，编码111个氨基酸。将N-1基因克隆到pET-28a(+)原核表达载体，转化大肠杆菌BL21感受态细胞，经IPTG诱导在E.coli中表达，并以werstern-blotting试验对重组蛋白的抗原性进行分析，结果在预测位置出现了明显的识别条带，显示N-1基因重组抗原具有良好的抗原性。为了探讨该基因真核表达产物在血吸虫病防治中的作用，我们成功将此基因克隆到昆虫病毒转移载体pBacPAK- HIS 中，为进一步探讨真核表达产物作为血吸虫候选疫苗的可行性奠 定了基础。 为了评估N－1 基因做为日本血吸虫核酸疫茵候选分子的可行性， 我们将此基因克隆到真核表达载体pCDNA3 中，构建成DNA 疫苗，并 进行动物免疫保护实验，结果实验组小鼠的减虫率达28．64％，肝脏 组织减卵率为21．73％，与对照组相比差异显著，表明此核酸疫苗应 用于日本血吸虫病的防治很有发展潜力。 本研究通过免疫筛选及RACE 技术首次克隆到两个新的东方田鼠 血清IgG3抗体相关的日本血吸虫特异性抗原编码基因，其中N－1基 因在大肠杆菌中获得表达，同时构建成核酸疫苗进行动物兔疫实验， 获得显著的免疫保护效果，为发展新的抗日本血吸虫病疫苗奠定了基 石出。更多还原

【Abstract】 Schistosomiasis, caused by Schistosomes, is a serious parasitic zoonosis afflicting both human and animals in the world. In recent years, a great attention was paid in the study of vaccines against Schistosomiasis by genetic engineering techniques. Now several candidate antigens with potential immune protection of Schistosomiasis have been obtained.Although a significant progress has been made in the screening of candidate antigen genes of Schistosomes, protective effects of all available antigens obtained by conventional gene engineering techniques are not so satisfied. Therefore, establishment of new techniques and strategies to screen effectively protective antigen genes would be an alternative to resolution of the above problem.Recent studies have shown that Microtus fortis is naturally genetous resistance to Schistosomiasis and humoral immunity, especially the specific IgG3 antibody in sera of Microtus fortis could play a relatively important role for Microtus fortis against Schistosomiasis japonicum. To screen the antigen genes of Schistosotna japonicum that related to the specific IgG3 antibody and provide valuable information for searching Schistosomiasis vaccine candidates, the Schistosoma Japonicum adult worm cDNA library was immunoscreened with the sera of uninfected Microtus fortis and Goat anti-mouse IgG3-HRP.Two positive clones were obtained and then were amplified using RACE technique (rapid amplification of cDNA ends), which resulted in acquiring two of novel genes of Schistosoma japonicum. One gene is 780 base pairs in open reading frame, encoding 259 amino acids and the other is 1169 bp.By cloning the N-l gene into pET~28a(+) expression vector, a recombinant of pET-28a(+)/N-l was constructed and expressed in E.coli. The expression product was analyzed by SDS-PAGE and Western-blotting. The result revealed that the molecular weight of recombinant protein was approximately 35KD and can be specifically recognized by mouse serumagainst Schistosoma japonicum adult worm antigen preparation (SWAP), suggesting that the recombinant protein possesses good antigenicity. Furthermore, the N-l gene was successfully cloned into plasmid pBacPAK-His, an insect virus transfer vector, which may pave the way for further study on feasibility of application on Schistosomiasis using eukaryotic expression product of N-l gene.In order to evaluate the feasibility of N-l gene as DNA vaccine candidate in the controlling Schistosomiasis, the gene was cloned into eukaryotic expression vector pcDNAS and then animal protection experiment was carried out. 28.64% worm burden reduction and 21.73% eggs number reduction were obtained in Kunming mice immunized with recombinant plasmid, indicating the prospect for N-l gene DNA vaccine as a potential vaccine against Schistosomiasis.In conclusion, two novel genes of Schistosoma japonicum related to the IgG3 antibody of Microtus fortis were firstly cloned by immunoscreening the Schistosoma Japonicum adult worm cDNA library and RACE technique. Furthermore, the N-l gene was expressed in E. coli successfully. The DNA vaccine contain N-l sequence was constructed and used to vaccinate the mice, significant protection was obtained when comparing with control group. Therefore, this study has provided the theoretical and methodological basis for the development of an effective vaccine against Schistosomiasis.更多还原