摘要
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 obtain expression of Eg95 -Eg. ferritin fusion gene of Echinococcus granulosus in insect cell/baculovirus expression system and to provide materials for developing Eg novel vaccine and establishing serological diagnostic method of echinococcosis. Methods The total RNA was extracted from hydatid cyst protoscoleces shattered by ultrasound breaking and the gene encoding Eg95 -Eg. ferritin antigen was amplified by RT - PCR from the template of total RNA. The Eg95 - Eg. ferritin fusion gene was obtained with gene SOEing, inserted into pFast Bac DUAL vector, then transferred the recombinant plasmid pFastBac DUAL -Eg95 -Eg.ferritin into DH10Bac to get recombinant Bacmid. Subsequently, the recombinant Bacmid was transfected into Sf- 9 cells. The expressed recombinant protein was identified by western blot after three cell passages. Results The Eg95 and Eg.ferritin gene were successfully cloned and the fusion gene Eg95 -Eg. ferritin was obtained by the flexible amino acid linker. The recombinant plasmid pFastBac DUAL - Eg95 - Eg.ferritin was constructed successfully by enzyme digestion and PCR identification. The obtained recombinant Eg95- Eg. ferritin protein reacted with Echinococcosispatients -positive serum that was substantiated by Western -blot analysis. Conclusion The Eg95 -Eg. ferritin fusion gene of Echinococcus granulosus is successfully expressed in Bac - to - Bac baculovirus expression system and shows good reactivity with positive ser-um of Echinococcosis patients.
出处
《宁夏医科大学学报》
2015年第2期120-124,F0004,共6页
Journal of Ningxia Medical University
基金
宁夏教育厅项目(NGY2013070)
校级大学生科技创新项目
