羊C3d基因与细粒棘球蚴EG95s基因在杆状病毒中的串联表达

›› 2010, Vol. 37 ›› Issue (11): 44-49.

羊C3d基因与细粒棘球蚴EG95s基因在杆状病毒中的串联表达

于琳琳1,2，贾红1，侯绍华1，刘丹1，丁敏1，郭晓宇1，朱鸿飞1

（1.中国农业科学院北京畜牧兽医研究所，北京 100193； 2.中国农业科学院生物技术研究所，北京 100081)

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-11-20 发布日期:2010-11-20
通讯作者: 朱鸿飞

Tandem Expression and Identification of EG95s Gene of Echinococcusgranulosus and C3d Gene Using Baculovirus Expression System

YU Lin-lin1,2, JIA Hong1, HOU Shao-hua1, LIU Dan1, DING Min1, GUO Xiao-yu1, ZHU Hong-fei1

（1.Institute of Animal Sciences, Chineses Academy of Agricultural Sciences, Beijing 100193, China;2.Biotechnology Research Institute,Chinese Academy of Agricultral Sciences,Beijing 100081,China)

Received:1900-01-01 Revised:1900-01-01 Online:2010-11-20 Published:2010-11-20
Contact: ZHU Hong-fei

摘要/Abstract

摘要： 本研究通过构建含有3拷贝羊C3d基因与细粒棘球蚴EG95s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EG95s基因与3拷贝羊C3d基因串联，插入pTarget载体，获得重组质粒pTarget-EG95-(C3d)3，利用BamH Ⅰ和Xba Ⅰ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C3d基因与细粒棘球蚴EG95s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132 ku，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。

关键词: 包虫病; EG95s基因; C3d基因; 重组杆状病毒

Abstract:

This study was conducted to prepare for comparison of the prokaryotic and eukaryotic EG95-(C3d)3 proteins in the immune effects and for development of efficient method of detection Echinococcosis by building recombinant baculovirus containing three copies of C3d gene and EG95s gene of Echinococcus granulosus. EG95s gene of and three tandem copies of goat C3d 〖JP2〗gene 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 BamH I and Xba I, the recombinant plasmid,pFastBacHta-EG95-(C3d)3, were obtained and then〖JP〗 was transformed into the DH10Bac for transposon reorganization. The recombinant transposon, rBacmid-EG95-(C3d) 3, were transfected into Sf9 insect cells for obtain the EG95-(C3d)3 recombinant baculovirus and expression〖JP2〗 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)3 recombinant protein with the size of about 132 ku 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)3 protein 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.

Key words: Echinococcosis; EG95s gene; C3d gene; recombinant baculovirus

中图分类号:

于琳琳;;贾红;侯绍华;刘丹;丁敏;郭晓宇;朱鸿飞. 羊C3d基因与细粒棘球蚴EG95s基因在杆状病毒中的串联表达[J]. , 2010, 37(11): 44-49.

YU Lin-lin;;JIA Hong;HOU Shao-hua;LIU Dan;DING Min;GUO Xiao-yu;ZHU Hong-fei. Tandem Expression and Identification of EG95s Gene of Echinococcusgranulosus and C3d Gene Using Baculovirus Expression System [J]. , 2010, 37(11): 44-49.

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