家蚕热休克蛋白HSP90相互作用蛋白的筛选与鉴定  

Screening and Identification of HSP90 Interacting Proteins in Silkworm(Bombyx mori)

作　　者：龙艳碧 吴云飞[1] 张倩[1] 陈鹏[1,2] 潘敏慧[1,2] LONG YanBi;WU YunFei;ZHANG Qian;CHEN Peng;PAN MinHui(State Key Laboratory of Silkworm Genomic Biology,Southwest University,Chongqing 400716;Key Laboratory of Sericulture Biology and Genetic Breeding,Ministry of Agriculture and Rural Affairs,Southwest University,Chongqing 400716)

机构地区：[1]西南大学家蚕基因组生物学国家重点实验室,重庆400716 [2]西南大学农业农村部蚕桑生物学与遗传育种重点实验室,重庆400716

出　　处：《中国农业科学》2022年第6期1253-1262,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31872427);重庆市自然科学基金(cstc2020jscx-msxmX0045);国家蚕桑产业技术体系(CARS-18)。

摘　　要：【目的】HSP90 是热休克蛋白家族的成员之一,在昆虫的抗逆性和变态发育中发挥重要作用,已有研究表明 HSP90能够促进家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)的增殖,但作用机制尚不清楚。本研究通过鉴定 BmHSP90 的相互作用蛋白,为其促进 BmNPV 增殖的作用机制解析提供参考。【方法】构建连接在 pIZ/V5-His 的BmHSP90;真核过表达载体,在家蚕 BmN-SWU1 细胞中转染 48 h 后,感染 BmNPV 继续培养 48 h 后收集蛋白,保留总蛋白后将这些蛋白均分两管,进行免疫共沉淀,分别用抗 HA 抗体和 IgG 抗体钓取相互作用蛋白,对蛋白胶进行硝酸银染色后,获取差异条带并做质谱分析,将质谱结果与信息分析结合进行候选互作蛋白筛选,并克隆鉴定互作蛋白。通过免疫荧光验证 HSP90 与互作蛋白的共定位情况,并进一步采用免疫共沉淀试验确定其是否存在相互作用关系。【结果】硝酸银染色结果显示,试验组与对照组在 90、70 和 60 kD 附近处存在差异条带,并验证了 90 kD 处的差异条带为诱饵蛋白;将另外两条差异带进行质谱分析,共鉴定到 7 个候选相互作用蛋白,通过分析选择其中两个候选蛋白作后续研究,分别为Tubulin-specific chaperone E(Tbce)和 Golgin subfamily A member 5(Golga5)。BmTbce 的最大开放阅读框长度为 1 728bp,编码 576 个氨基酸,BmGolga5 的最大开放阅读框长度为 1 854 bp,编码 618 个氨基酸;同源比对和系统进化树显示 BmTbce的微管结合结构域(cytoskeleton-associated protein-glycine-rich,CAP-Gly)位于 N 端且在不同物种之间保守性较高,BmGolga5 的跨膜区(transmembrane domain,TMD)位于 C 端,也较为保守;荧光共定位显示 BmHSP90 与 BmTbce 和 BmGolga5在细胞质中发生共定位,并进一步通过免疫共沉淀证明 BmHSP90;和 BmTbce;、BmHSP90;和 BmGolga5;具有相互作用关系。【结论】经过筛选与鉴定,在 BmNPV 感染家蚕细胞的过程中,与家�【Objective】HSP90 is a member of the heat shock protein family and plays an important role in insect resistance and metamorphosis. Studies have shown that HSP90 can promote the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV),but the mechanism of action is unclear. The objective of this study is to identify the interacting proteins of BmHSP90, and to provide a reference for the analysis of its mechanism of promoting BmNPV proliferation.【Method】The BmHSP90;eukaryotic overexpression vector linked to pIZ/V5-His was constructed. After transfection in BmN-SWU1 cells for 48 h, BmNPV was infected and cultured for 48 h to collect the proteins. After the total protein was retained, the proteins were divided into two tubes and co-immunoprecipitation was performed. The interacting protein was caught with anti-HA antibody and IgG antibody, respectively,after staining the protein gel with silver nitrate, the different bands were obtained and mass spectrometry analysis was performed.The mass spectrometry results were combined with the information analysis to screen candidate interacting proteins, and then the interacting proteins were cloned and identified. The co-localization of HSP90 and the interacting protein was verified by immunofluorescence, and the co-immunoprecipitation experiment was further used to determine whether they had an interaction relationship.【Result】The results of silver nitrate staining showed that the experimental group and the control group had different bands near 90, 70 and 60 kD, and verified that the different bands at 90 kD were bait proteins. The other two different bands were analyzed by mass spectrometry, and a total of 7 candidate interacting proteins were identified. Two of the candidate proteins were selected for follow-up study through analysis, namely Tubulin-specific chaperone E (Tbce) and Golgin subfamily A member 5(Golga5). The maximum open reading frame length of the BmTbce is 1 728 bp, which encodes 576 amino acids, and the maximum open reading frame length of the

关 键 词：家蚕 家蚕核型多角体病毒 HSP90 Tbce Golga5 

分 类 号：S881.2[农业科学—特种经济动物饲养]