中文摘要：

目的培育并鉴定细粒棘球绦虫转Eg95基因苜蓿疫苗。方法利用转pBI—Eg95质粒的根癌农杆菌LBA440d株介导的苜蓿子叶浸染法，将Eg95基因导入紫花苜蓿基因组，转Eg95基因苜蓿外植体在含有卡那霉素的选择培养基上经愈伤、出芽和生根阶段生长出小苗，最后移栽到装有营养土的花盆中，生长2～3个月，获得完整的转Eg95基因苜蓿疫苗。提取转Eg95基因苜蓿的DNA、RNA及叶蛋白，采用PCR、RT—PCR、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）及Westernblot法进行鉴定。结果PCR、RT—PCR法在471bp处均扩增出目的条带；SDS—PAGE及Westernblot法可见转E驴5基因苜蓿蛋白在相对分子质量约16．5×10^3处出现特异条带，与预期结果相符：Bio—Rad Quantityone系统分析表达效率约占提取总苜蓿叶蛋白的0．06％。结论成功培育出细粒棘球绦虫转Eg95基因苜蓿疫苗。

英文摘要：

Objective To cultivate and identify the transgenic alfalfa containing Echinococcus granulosus Eg95 gene. Methods The alfalfa plants were transformed by co-cultivating alfalfa cotyledons via recombinant Agrobacterium tumefaciens LBA4404 harboring pBI-Eg95. The transgenic alfalfa explants were selected by kanamycin after calli formation, shoots and roots regeneration in the selective medium, the seedlings of transgenic plants were obtained which were finally transplanted into pots containing nutrient soil. After 2 - 3 months growth, the complete transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene were obtained. To identify the transgenic alfalfa plants, the total DNA, RNA and leaf protein were extracted from fresh leaf tissue of the transgenic alfalfa plants and confirmed by PCR, RT-PCR, SDS-PAGE and Western blot assay. Results A specific band around 471 bp was amplified by PCR with total DNA, and the same band was obtained by RT-PCR with total RNA, which confirmed that the Eg95 gene was stably integrated into the transformed alfalfa genome. SDS-PAGE analysis showed that the relative molecular mass（Mr） of the expressed protein was about 16.5 × 10^3, consistent with the Eg95 protein, and the level of Eg95 expression was up to 0.06% of total soluble leaf protein by Bio-Rad Quantity one assay. Western blot verified the expressed protein was reactive with the sera of mice infected with Echinococcus granulosus. Conclusion The transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene are successfully cultivated.