细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗构建及鉴定

Construction and identification of the recombinant fusion gene vaccine Bb-Eg95-EgA31 of Echinococcus granulosus

目的构建和鉴定细粒棘球绦虫(Eg)重组双歧杆菌(Bb)-Eg95-EgA31融合基因疫苗。方法自行设计引物,从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA为模板,通过RT-PCR分别扩增Eg95和EgA31抗原编码基因,然后采用基因拼接法(gene SOEing)剪接Eg95和EgA31,得到Eg95-EgA31融合基因,经BamHⅠ和EcoRⅠ双酶切,定向克隆到大肠杆菌-双歧杆菌穿梭表达载体pGEX-1λT中,转化大肠杆菌BL21(DE3)感受态细胞,构建重组质粒pGEX-Eg95-EgA31,抽提质粒进行双酶切鉴定,电穿孔法转化两歧双歧杆菌(Bifidobacteria bifidum,Bb),构建细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗,抽提质粒进行PCR扩增鉴定。结果RT-PCR扩增出约1 016bp的Eg95-EgA31融合基因;重组质粒用双酶切鉴定可切出预期大小片段,以具有氨苄青霉素抗性的rBb中抽提的质粒为模板进行PCR扩增可得到约1016bp的Eg95-EgA31融合基因片段。结论成功构建了细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗,为该疫苗的开发利用奠定了实验基础。

In this study, the total RNA was extracted from hydatid cyst protoscoleces shattered by ultrasound breaking and the gene encoding Eg95-EgA31 antigen was amplified by RT-PCR from template of the total RNA using the primers designed according to DNA sequence of Eg95 and EgA31. The Eg95-EgA31 fusion gene was obtained with gene SOEing , cloned to E. coli-Bifidobacteria shuttle plasmid pGEX-1λT and transformed into E. coli BL21（DE3） competent cells to construct plasmid pGEX-Eg95-EgA31 by using BamHI and EcoRI. This recombinant plasmid was identified by restriction endonuelease digestion and electroporated into Bifidobacterium bifiduzn to construct fusion gene vacciner rBb-Eg95-EgA31. The extracted plasmid was identified by PCR. Experimental result showed that the fusion gene Eg95-EgA31 of 1 016 bps in length was amplified by RT-PCR and the size of the products obtained by restriction endonuclease digestion was just the same as expected. It is evident that the recombinant gene vacciner Bb-Eg95-EgA31 of Echinococcus granulosus has been successively constructed and identified, which may lay experimental foundation for this vaccine.