细粒棘球绦虫转基因植物载体重组pBI-Eg95-EgA31质粒构建及鉴定

目的构建并鉴定细粒棘球绦虫转基因植物载体重组pBI-Eg95-EgA31质粒。方法从细粒棘球蚴包囊中分离原头节,超声粉碎后抽提总RNA为模板,采用RT-PCR方法分别扩增Eg95和EgA31编码基因,然后采用基因拼接法(gene SOEing)扩增Eg95-EgA31融合基因;将该融合基因定向克隆到植物表达载体pBI121中构建pBI-Eg95-EgA31重组质粒;电穿孔转化根癌农杆菌(Agrobacterium tumefaciens,At)LBA4404株,抽提质粒进行酶切及PCR鉴定。结果电泳及测序证实1 016bpEg95-EgA31融合基因克隆成功。经酶切及PCR证实重组质粒pBI-Eg95-EgA31成功转入根癌农杆菌。结论成功构建了细粒棘球绦虫转基因植物载体重组pBI-Eg95-EgA31质粒,为进一步构建细粒棘球绦虫转基因植物疫苗奠定了基础。

Construction and identification for the transgenic plant vector of recombinant plasmid pBI-Eg95-EgA31 of Echinococcus granulosus

To construct and identify the transgenic plant vector for recombinant plasmid pBI-Eg95-EgA31 of Echinococcus granulosus,the total RNA was extracted from hydatid cyst pro-toscoleces shattered by supersound,and the Eg95 and EgA31-coding genes were amplified by RT-PCR.The Eg95-EgA31-fusion gene was obtained with GOEing.and then the fusion gene was cloned into the plant expression vector pBI-121 to construct the recombinant plasmid pBI-Eg95-EgA31.As demonstrated by electrophoresis and sequencing,the Eg95-EgA31 fusion gene with 1016 bps was successfully amplified and the recombinant plasmid pEgBI-Eg95-EgA31 was also successivelly constructed,as confirmed by restriction endonuclease digestion and PCR.The results in the present investigation would provide a basis for the further study on the trangenic plant vaccine for E granulosus infection.