家蚕热休克蛋白HSP90相互作用蛋白的筛选与鉴定

doi:10.3864/j.issn.0578-1752.2022.06.016

Abstract

Abstract:

【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.

Key words: Bombyx mori, BmNPV, HSP90, Tbce, Golga5

LONG YanBi,WU YunFei,ZHANG Qian,CHEN Peng,PAN MinHui. Screening and Identification of HSP90 Interacting Proteins in Silkworm (Bombyx mori)[J].Scientia Agricultura Sinica, 2022, 55(6): 1253-1262.

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References 38

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引物名称Primer name	引物序列Primer sequence
BmHSP90^HA-Sac I-F	5′-CGAGCTCATGTACCCATACGATGTTCCAGATTACGCTATGCCGGAAGAAATGGAGACA-3′
BmHSP90^HA-Sac II-R	5′-TCCCCGCGGGGATTAAGCGTAATCTGGAACATCGTATGGGTAATCAACCTCCTCCATGCGA-3′
BmGolga5^Flag-Xho I-F	5′-CCGCTCGAGCATGGATTACAAGGATGACGACGATAAGATGGCTTGGTTTGCGGACTT-3′
BmGolga5^Flag-Sac II-R	5′-TCCCCGCGGCTTATCGTCGTCATCCTTGTAATCTGACTTCATCGCTCTCGTGATAT-3′
BmTbce^Flag-BamH I-F	5′-CGGGATCCATGGATTACAAGGATGACGACGATAAGATGAGTAAAGTTTTTGTGCAAG-3′
BmTbce^Flag-Not I-R	5′-GAATGCGGCCGCTCTTATCGTCGTCATCCTTGTAATCTCTGAATTTAACTAGAATAACATCG-3′

蛋白ID Protein ID	描述 Description	分子量 Molecular weight (kD)	功能 Function
XP_012553114	Serine/threonine-protein phosphatase 5 (PP5)	53.90	信号转导机制、能量代谢 Signal transduction mechanisms, energy metabolism
NP_001268820	Juvenile hormone esterase-like precursor	58.41	控制细胞周期、细胞分裂 Cell cycle control, cell division
XP_004923154	Tubulin-specific chaperone E	58.52	控制细胞周期、细胞分裂、染色体分配 Cell cycle control, cell division, chromosome partitioning
XP_004923957	60 kD heat shock protein (HSP60)	62.91	翻译后修饰、蛋白质折叠、伴侣 Posttranslational modification, protein turnover, chaperones
XP_012550337	Golgin subfamily A member 5	67.87	碳水化合物的运输和代谢 Carbohydrate transport and metabolism
XP_012545391	SAM and SH3 domain-containing protein 1-like	70.73	信号转导机制Signal transduction mechanisms
NP_001036892	Heat shock cogn protein	71.39	翻译后修饰、蛋白质折叠、伴侣 Posttranslational modification, protein turnover, chaperones

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

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