摘要
本研究旨在利用草地贪夜蛾Spodoptera frugiperda昆虫细胞Sf9-杆状病毒表达系统筛选禾谷镰孢菌Fusarium graminearum β-1,3-葡聚糖合成酶催化亚基GLS2在昆虫细胞中的异源表达载体和分离纯化所用的去污剂,为后续研究该蛋白与药剂的结合模型提供基础。通过对杆状病毒表达质粒pFastBac进行设计和改造、利用同源重组的方法构建质粒、使用昆虫细胞表达系统对重组蛋白进行异源表达、筛选适合提取目的蛋白的去污剂等方法,得到适合禾谷镰孢菌β-1,3-葡聚糖合成酶催化亚基GLS2异源表达的载体和分离目的蛋白的方法。结果表明,载体pFastBac-GP67-8×His-GFP-TEV-FgGLS2、pFastBac-HA-8×His-GFP-TEV-FgGLS2、pFastBacGP67-6×His-2×Strep-TEV-FgGLS2和pFastBac-FgRHO-TEV-8×His均可在草地贪夜蛾昆虫细胞Sf9表达系统中进行表达,其中:GP67-8×His-GFP-TEV-FgGLS2融合蛋白可以用去污剂十二烷基二甲胺氧化胺(dodecyldimethylamine oxide,DDAO)从细胞膜上分离,HA-8×His-GFPTEV-FgGLS2融合蛋白可以用去污剂十二烷基-β-D-麦芽糖苷(n-dodecyl-β-maltoside, DDM)、十烷基-β-D-麦芽糖苷(n-decyl-β-maltoside, DM)或DDAO从细胞膜上分离,GP67-6×His-2×Strep-TEV-FgGLS2融合蛋白可以用去污剂DM或DDAO从细胞膜上分离。本研究首次成功地在草地贪夜蛾昆虫细胞Sf9表达系统中表达了β-1,3-葡聚糖合成酶催化亚基GLS2,FgRHO蛋白可促进FgGLS2的稳定表达,并筛选到适合FgGLS2提取的去污剂DDAO。
This study aims to screen the heterologous expression vector of Fusarium graminearumβ-1,3-glucan synthetase catalytic subunit GLS2(FgGLS2)and the purification detergents by Spodoptera frugiperda cell-baculovirus expression system,and will provide the basis for the study of the binding model between the protein and its inhibitor.The plasmid was constructed based on the design and modification of the baculovirus expression plasmid pFastBac by homologous recombination.The recombinant protein was heterologously expressed in the insect cell expression system and suitable detergents were screened for the extraction of the target protein.We expected to obtain the suitable vector and purification method for heterologous expression of the subunit GLS2 ofβ-1,3-glucan synthetase from F.graminearum.The results showed that the proteins from plasmids pFastBac-GP67-8×His-GFP-TEV-FgGLS2,pFastBac-HA-8×His-GFP-TEV-FgGLS2,pFastBac-GP67-6×His-2×Strep-TEV-FgGLS2,and pFastBac-FgRHO-TEV-8×His could be expressed in insect cell expression system of S.frugiperda.GP67-8×His-GFP-TEV-FgGLS2 fusion protein could be separated from cell membrane with detergent dodecyldimethylamine oxide(DDAO).HA-8×His-GFP-TEV-FgGLS2 fusion protein can be separated from cell membrane with detergent n-dodecyl-β-maltoside(DDM),n-decyl-β-maltoside(DM)and DDAO.GP67-6×His-2×Strep-TEV-FgGLS2 fusion protein can be separated from cell membrane with detergent DM and DDAO.Ultimately,for the first time,we successfully express the catalytic subunit GLS2 ofβ-1,3-glucan synthase from F.graminearum in S.frugiperda cell expression system.In addition,the protein FgRHO could stabilize the expression of FgGLS2 and the detergent DDAO was suitable for the extraction of GLS2.
作者
延扬帆
张峰
YAN Yangfan;ZHANG Feng(College of Plant Protection,Nanjing Agricultural University,Nanjing 210095,China)
出处
《农药学学报》
CAS
CSCD
北大核心
2023年第3期611-620,共10页
Chinese Journal of Pesticide Science
基金
国家重点研发计划(2022YFD1700200)
江苏省六大人才高峰高层次人才项目(NY-035)
教育部霍英东教育基金会高等学校青年教师基金(161022)。
