西花蓟马FKBP6蛋白多克隆抗体制备、真核表达及亚细胞定位
Polyclonal Antibody Preparation,Eukaryotic Expression and Subcellular Localization of Frankliniella occidentalis FKBP6 Protein
王鑫 1郑立敏 2赵星月 3苗淑月 4叶倩 2张宇 2史晓斌 2张德咏 5陈建斌2
作者信息
- 1. 湖南大学生物学院隆平分院,长沙 410125
- 2. 湖南省农业科学院植物保护研究所,长沙 410125
- 3. 内蒙古农业大学园艺与植物保护学院,呼和浩特 010018
- 4. 河南省农业科学院烟草研究所,许昌 461000
- 5. 湖南大学生物学院隆平分院,长沙 410125;湖南省农业科学院植物保护研究所,长沙 410125
- 折叠
摘要
西花蓟马(Frankliniella occidentalis)是为害全球农业生产的入侵害虫,通过探寻西花蓟马亲免素蛋白FK506-binding protein 6(FKBP6)的生物功能,为防控西花蓟马挖掘新的靶标.本研究运用生物信息学分析西花蓟马FKBP6 蛋白(GenBank No.XP_026292958.1),克隆FKBP6 基因(GenBank No.XM_026437173.1),原核诱导表达FKBP6蛋白并制备其多克隆抗体,利用Bac-to-Bac杆状病毒系统表达FKBP6蛋白,借助荧光免疫(immunofluorescence,IF)标记和Western blot检测多克隆抗体及亚细胞定位.结果显示,氨基酸多序列比对与系统进化发育树分析发现,20种昆虫的FKBP6蛋白之间存在保守的区域,但只在种间亲缘性较高.利用杆状病毒系统在Sf9细胞内表达FKBP6蛋白,使用多克隆抗体经IF和Western blot检测显示,该抗体能与FKBP6蛋白发生特异性结合;亚细胞定位发现,FKBP6被定位到细胞质.本研究为FKBP6蛋白的生物功能研究提供分子条件.
Abstract
Frankliniella occidentalis is an invasive pest that harms global agricultural production.Exploring the biological functions of immunophilins FK506-binding protein 6(FKBP6)of F.occidentalis can provide a new target for the prevention and control of F.occidentalis.In this study,FKBP6(GenBank No.XP_026292958.1)of F.occidentalis was analyzed through bioinformatics,the sequence of FKBP6 gene(GenBank No.XM_026437173.1)was cloned,expression of FKBP6 protein was induced through prokaryotic cells,and the polyclonal antibody was prepared.Then,the protein was expressed by the Bac-to-Bac baculovirus system,the polyclonal antibody and its subcellular localization were detected through Western blot and immunofluorescence(IF)labeling.The amino acid sequence alignment and phylogenetic tree analysis showed that FKBP6 proteins among 20 insect species had conserved regions,but they were only highly related among species.FKBP6 protein in Sf9 cells was expressed through baculovirus systems,and then IF and Western blot dection showed that polyclonal antibodies specifically bound to FKBP6 protein.Furthermore,subcellular localization results showed that FKBP6 was localized in the cytoplasm.This study provides molecular conditions for the study of the biological function of FKBP6.
关键词
西花蓟马/FKBP6蛋白/抗体制备/Bac-to-Bac杆状病毒表达系统Key words
Frankliniella occidentalis/FKBP6 protein/Antibody preparation/Bac-to-Bac baculovirus expression system引用本文复制引用
基金项目
国家重点研发计划政府间/港澳台重点专项(2018YFE0112600)
长沙市杰出创新青年培养计划(kq2009006)
长沙市自然科学基金(kq2208140)
国家自然科学基金(3187193532302300)
财政部和农业农村部:国家麻类产业技术体系建设项目(CARS-16-E17)
湖南省农科院科技创新项目(2023CX97)
出版年
2024
国家科技期刊平台
NSTL
万方数据