摘要
本研究通过构建含有3拷贝羊C3d基因与细粒棘球蚴EG95s基因的重组杆状病毒,旨在通过昆虫细胞表达EG95-(C3d)3重组融合蛋白,为原核表达和真核表达EG95-(C3d)3重组融合蛋白的免疫效果的比较奠定了基础。将细粒棘球蚴EG95s基因与3拷贝羊C3d基因串联,插入pTarget载体,获得重组质粒pTarget-EG95-(C3d)3,利用BamHⅠ和XbaⅠ双酶切位点将目的基因EG95-(C3d)3克隆至Bac-to-Bac系统的转移载体pFastBac-Hta中,获得pFastBacHta-EG95-(C3d)3重组质粒,将该重组质粒转化含穿梭载体Bacmid的感受态细胞DH10Bac中进行转座重组,获得携带3拷贝羊C3d基因与细粒棘球蚴EG95s基因的重组转座子rBacmid-EG95-(C3d)3,转染Sf9昆虫细胞后获得EG95-(C3d)3重组杆状病毒,并表达EG95-(C3d)3重组融合蛋白,采用SDS-PAGE和Western blotting进行重组蛋白的鉴定。同时利用昆虫细胞表达蛋白对羊棘球蚴病特异性抗体进行检测。结果表明,获得了EG95-(C3d)3重组杆状病毒,且EG95-(C3d)3重组融合蛋白在Sf9昆虫细胞得到了正确表达,大小约为132ku,Western blotting鉴定结果显示表达的EG95-(C3d)3重组融合蛋白能与细粒棘球蚴阳性血清产生特异性反应,表明EG95-(C3d)3基因表达产物具有免疫活性。同时表达蛋白对羊棘球蚴病特异性抗体有良好的敏感性。EG95-(C3d)3基因在昆虫细胞获得表达,为原核表达和真核表达EG95-(C3d)3重组融合蛋白的免疫效果的比较,及细粒棘球蚴病的高效检测方法的建立奠定了基础。
This study was conducted to prepare for comparison of the prokaryotic and eukaryotic EG95-（C3d）3proteins in the immune effects and for development of efficient method of detection Echinococcosis by building recombinant baculovirus containing three copies of C3dgene and EG95s gene of Echinococcus granulosus.EG95s gene of and three tandem copies of goat C3dgene were inserted to pTarget vector to obtain recombinant plasmid,named pTarget-EG95-（C3d）3,then the target gene,EG95-（C3d）3,were cloned into the pFastBac-Hta of Bac-to-Bac system by BamH I and XbaI,the recombinant plasmid,pFastBacHta-EG95-（C3d）3,were obtained and then was transformed into the DH10 Bac for transposon reorganization.The recombinant transposon,rBacmid-EG95-（C3d）3,were transfected into Sf9insect cells for obtain the EG95-（C3d）3 recombinant baculovirus and expression of EG95-（C3d）3 recombinant proteins.The proteins were identified by SDS-PAGE and Western blotting.rUsing the recombinant proteins detected sheep antibodies against Echinococcosis granulosa.The results showed that the EG95-（C3d）3 recombinant baculovirus was obtained,and the EG95-（C3d）3recombinant protein with the size of about 132ku were correctly expressed in insect cells in Sf9,the results of Western blotting showed that this EG95-（C3d）3 protein could response to Echinococcus granulosus-specific positive serum,indicating that EG95-（C3d）3protein has immune activity.The recombinant proteins had well sensitivity with Echinococcosis granulosa.The EG95-（C3d）3 gene was correctly expressed in insect cells,which built the bases for comparison of the immune effects of prokaryotic and eukaryotic EG95-（C3d）3 proteins,and for development of efficient method of detection Echinococcosis.
出处
《中国畜牧兽医》
CAS
北大核心
2010年第11期44-50,共7页
China Animal Husbandry & Veterinary Medicine
基金
国家科技支撑计划(2008BAK51B08)
