论文部分内容阅读
目的利用昆虫细胞/杆状病毒系统表达细粒棘球蚴Eg95-Eg.ferritin融合蛋白,用于开发包虫病新型疫苗以及建立相关血清学诊断方法等研究。方法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扩增细粒棘球蚴Eg95和Eg.ferritin基因,采用基因拼接法将Eg95和Eg.ferritin融合,将该融合基因Eg95-Eg.ferritin插入到p Fast Bac DUAL载体中,构建重组转座载体后转化DH10Bac感受态细胞,获得重组Bacmid质粒后转染Sf-9昆虫细胞,传毒3代,对表达蛋白进行Western blot鉴定。结果成功克隆了Eg95和Eg.ferritin基因,通过柔性氨基酸linker成功获得了融合基因Eg95-Eg.ferritin,经PCR和酶切鉴定成功构建了重组质粒p Fast Bac DUAL-Eg95-Eg.ferritin,Western blot结果证实表达蛋白能够被包虫病人标准阳性血清识别。结论在Bac-to-Bac杆状病毒表达系统中成功表达了细粒棘球蚴Eg95-Eg.ferritin融合蛋白,与包虫病人标准阳性血清具有良好的反应性。
OBJECTIVE: To express Echinococcus granulosus Eg95-Eg.ferritin fusion protein using insect cell / baculovirus system for the development of new hydatid disease vaccine and to establish related serological diagnostic methods. Methods The protoscoleces were isolated from the cysts of Echinococcus granulosus and the whole RNA was extracted by sonication. The Eg95 and Eg.ferritin genes of Echinococcus granulosus were amplified by RT-PCR. Eg95 and Eg .ferritin fusion, the fusion gene Eg95-Eg.ferritin inserted into the p Fast Bac DUAL vector, construct a transposon vector after transformation DH10Bac competent cells obtained recombinant Bacmid plasmid transfected Sf-9 insect cells, poison 3 Generation, Western blot identification of the expressed protein. Results The Eg95 and Eg.ferritin genes were successfully cloned. The fusion gene Eg95-Eg.ferritin was successfully obtained by flexible amino acid linker. The recombinant plasmid pFacE-Bac95 was successfully constructed by PCR and restriction enzyme digestion. Western Blot The result confirmed that the expressed protein can be recognized by the standard positive serum of hydatid disease patients. Conclusion The Eg95-Eg.ferritin fusion protein of Echinococcus granulosus was successfully expressed in Bac-to-Bac baculovirus expression system, which showed good reactivity with standard positive serum of hydatid disease patients.