【作者】 黎万奎；

【导师】 徐恒；

【作者基本信息】 四川大学 ， 微生物学， 2003， 硕士

【摘要】 肝片吸虫（Fasciola hepatica）是牛羊及其它数十种哺乳类动物胆道内的常见寄生虫，人体亦可感染。肝片吸虫病是我国危害最严重、覆盖面最广的牲畜寄生虫病之一。随着转基因植物可食疫苗（edible vaccine，oral vaccine）技术的逐渐成熟，利用牧草生产抗肝片吸虫可食疫苗成为防治肝片吸虫病的可行手段。我们将本实验室通过构建肝片吸虫cDNA文库进行免疫印迹筛选克隆得到的肝片吸虫主要保护性抗原基因FH3转入草食性牲畜喜食的“牧草之王”——紫花苜蓿（Medicago Sative L.）中，得到稳定表达的转基因苜蓿，以此研究肝片吸虫可食疫苗。 将肝片吸虫保护性抗原基因FH3克隆到设计有一段根瘤农杆菌T-DNA边界序列（T-DNA borders）的质粒pBI121上，构建植物表达中间载体pBI121-FH3。用pBI121-FH3经冻融法直接转化预先用Cacl₂诱导成感受态的根瘤农杆菌株EHA105，并经抗性平板筛选得到阳性EHA105；苜蓿种子经乙醇、升汞消毒处理并用无菌水清洗后接种到MS培养基上，于25℃左右，16h／d光照条件下进行无菌实生苗的培养。选取7日苗龄苜蓿无菌实生苗的子叶和下胚轴经预处理后浸入离心后去上清并用液体MS₀重悬的阳性EHA105菌液中摇床培养30分钟后转入UM培养基培养，经愈伤组织诱导培养、不定芽诱导培养、生根诱导培养、炼苗后移栽至土壤，完成转基因首稽植株的再生。转基因首稽经Southern blot表明:约80%转基因首蓓株基因组中可检测到FH3基因;提取转基因首稽植株总蛋白质，SDS一PAGE结果显示转基因首稽株表达了重组抗原蛋白（分子量²2KD）;Western blot免疫检测验证了转基因首稽植株表达的重组抗原有免疫原性。 本项研究为研制肝片吸虫转基因植物可食疫苗奠定了基础。更多还原

【Abstract】 Fasciolasis is a serious parasitic disease over the world. Fosciola hepatica widely parasite in many kinds of economic herbivores such as sheep, yak, cattle, horse, deer, camel, ...etc., and hinder the development of the productive of the livestock husbandry. Moreover, the economic losses caused by fascioliasis can be very huge. So it is very important to develop an effective vaccine against fasciolisis. With the rapid development in the field of edible vaccine (oral vaccine), it becomes an available approach to use recombinant DNA methods to generate a cheap and plentiful supply of antigens. We report here the preliminary study on edible vaccine of Medicago sativa L. containing protective antigenic gene FH3 of F. hepatica.Antigenic gene FH3 of Fasciola hepatica was cloned into vector pBI121, recombinant plant expression vector was gotten, named pBI121-FH3. Agrobacteriwn tumefaciens EHA105 was transformed using pBI121-FH3. Positive EHA105 strains containing gene FH3 were picked out by plating. FH3 was introduced into alfalfa cells by co-cultivating alfalfa leaf discs with positive EHA105. Transgenic alfalfa plants were selected or gotten through methods such as tissue culture and selected culture. About 80% transgenic alfalfa plants were found containing FH3 gene by PCR and Southern blotting. Among these transgenic alfalfa plants, 5-10% plants were confirmed expressing FH3 recombinant protein, which molecular weight was about 22KD through SDS-PAGE. Based on ELISA and Western blotting, results showed that recombinant protein expressed by transgenic alfalfa plants could make responses to F. hepatica rabbit antisera.This project could explore out a new approach for studying F. hepatica plant edible vaccine.更多还原