转细粒棘球绦虫Eg95-EgA31融合基因苜蓿的培育及鉴定

目的培育并鉴定转细粒棘球绦虫Eg95-EgA31融合基因苜蓿，为进一步研究转基因苜蓿疫苗提供依据。方法将构建好的pBI—Eg95-EgA31质粒电穿孔转化根瘤农杆菌（Agrobacterium tumefaciens，At）LBA440d株，利用重组根瘤农杆菌（rAt）侵染苜蓿（alfalfa）叶片，卡那霉素抗性筛选伤愈组织体胚，培育转基因苜蓿．用SDS-PAGE、Western—blot、PCR和RT—PCR鉴定转基因苜蓿。结果SDS—PAGE和Western-blot证实Eg95-EgA31融合基因在苜蓿中得到表达，表达产物分子质量约为37500Mr，表达效率约占苜蓿叶总蛋白的0．05％，且能被感染细粒棘球蚴的鼠血清特异识别。PCR和RT—PCR均扩增出1016bp Eg95-EgA31融合基因片段。结论成功培育了转细粒棘球绦虫Eg95-EgA31融合基因苜蓿，为进一步研究细粒棘球绦虫转基因苜蓿疫苗奠定了基础。

Cultivation and Identification of the Echinococcus granulosus Eg95-EgA31 Transgenic Alfalfa

Objective To cultivate and identify the Echinococcus granulosus Eg95-EgA31 transgenic alfalfa. Method The recombinant pBI-Eg95-EgA31 plasmid was introduced into Agrobacterium tumefaciens by electroporation to construct recombinant Agrobacterium tumefaciens （rAt）. The alfalfa leaves were infected with the recombinant Agrobacterium tumefaciens （rAt）, and the somatic embryo was picked up from the callus with kanamycin resistance. Transgenic alfalfa was onstined from the resistant somatic embryo.The expression of Eg95-EgA31 fusion protein was confirmed by SDS-PAGE and Western-blot assay. The gene was identified by PCR and RT-PCR assay. Result The Eg95-EgA31 fusion gene could be expressed in alfalfa. The expression efficiency was about 0.05% of the total alfalfa leave protein. The expression product with an approximately of 37500 Mr could be recognized by sera from mice infected with E.granulosus. These were confirmed by the SDS-PAGE and Western-blot assay. The 1016bp gene was amplified successfully by PCR and RT-PCR. Conclusion The transgenic alfalfa was constructed successfully. It could provide the basis for further research of the transgenic alfalfa vaccine of Echinococcus granulosus.