摘要
利用生物信息学方法预测cry1C蛋白的抗原表位区,并命名为Δcry1C。优化Δcry1C的核苷酸序列并进行人工合成,构建重组表达载体pET28b(+)-Δcry1C。在E.coli中诱导表达His6-Δcry1C蛋白,纯化后免疫新西兰大白兔,分离、纯化获得cry1C多抗血清。ELISA测定结果表明,抗体效价最高可达1∶512 000。Western Blot分析结果表明,cry1C多克隆抗体能够与cry1C转基因抗虫水稻蛋白特异性结合。利用生物信息学筛选抗原表位区并优化序列、原核表达重组蛋白、免疫制备cry1C多克隆抗体,为深入研究抗鳞翅目害虫植物提供了前提条件。
In this study,cry1 C antigenic epitope region of cry1 C was predicted by bioinformatics analysis and designated as Δcry1 C.The nucleotide sequence of the Δcry1 C was synthesized after codon optimization,and then recombinant expression vector pET28 b（＋）-Δcry1 Cwas constructed. To prepare cry1 C polyclonal antibody,His6-Δcry1 C recombinant protein was purified after induction in Escherichia coli competent cell BL21（DE3）,and then was used to immunize New Zealand white rabbits as antigen. ELISA assay showed that the titer of cry1 Cpolyclonal antibody reached 1 ：512 000.Western Blot analysis showed that cry1 C polyclonal antibody was able to specifically detect the targetprotein of cry1 C in transgenic pest-resistant rice. In summary,cry1 C antigenic epitope region was screened by bioinformatics analysis and its polyclonal antibody was prepared successfully. This study provides a basis for further studying Lepidopteran pest-resistant transgenic plants.
作者
金永梅
陈莫军
刘笑笑
林秀峰
JIN Yong-mei;CHEN Mo-jun;LIU Xiao-xiao;LIN Xiu-feng(Jilin Academy of Agricultural Sciences,Changchun 130124)
出处
《生物技术通报》
CAS
CSCD
北大核心
2018年第9期224-229,共6页
Biotechnology Bulletin
基金
国家转基因重大项目(2016ZX08001001-001-007)
吉林省科技发展计划重点科技攻关项目(20160204033NY)
吉林省农业科技创新工程项目(CXGC2017TD009)
作者简介
金永梅，女，研究员，研究方向：水稻生物技术育种；E-mail：ymjin0303@163．com;通讯作者：林秀峰，女，研究员，研究方向：水稻生物技术育种；E-mail：linxiufeng8581@163@.com
