家蚕热休克蛋白HSP90相互作用蛋白的筛选与鉴定

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(Bombyxmorinucleopolyhedrovirus,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...

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

【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 ^HA 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 BmGolga5 is 1 854 bp, which encodes 618 amino acids. The homology alignment and phylogenetic tree showed that the microtubule binding domain of BmTbce (cytoskeleton-associated protein-glycine-rich, CAP-Gly) was located at the N-terminus and was highly conserved among different species. The transmembrane domain (TMD) of BmGolga5 was located at the C-terminus and was also conservative. The fluorescence co-localization showed that BmHSP90 co-localized with BmTbce and BmGolga5 in the cytoplasm, and it was further proved by co-immunoprecipitation that BmHSP90 ^HA and BmTbce^Flag, BmHSP90^HA and BmGolga5^Flag had an interaction relationship.【Conclusion】After screening and identification, in the process of BmNPV infection of B. mori cells, the proteins that interact with the B. mori heat shock protein HSP90 are BmTbce and BmGolga5.