家蚕热休克蛋白HSP60相互作用蛋白筛选与鉴定

doi:10.3864/j.issn.0578-1752.2019.02.016

摘要/Abstract

摘要：

【目的】 HSP60是热休克蛋白中重要的一员,在昆虫先天性免疫过程中起重要作用,同时也是昆虫生长发育的必要因子。前期研究证明家蚕BmHSP60参与家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus,BmNPV）入侵细胞过程,本研究通过鉴定BmHSP60相互作用蛋白,为其在家蚕先天性免疫中作用机制研究打下基础。【方法】利用免疫共沉淀和银联-质谱分析技术（LC-MS/MS）,首先构建BmHSP60过表达载体并融合Flag标签,转染到家蚕细胞中48 h后,收集蛋白,进行免疫共沉淀,用Flag抗体孵育磁珠调取相互作用蛋白,银染处理后,能够检测到明显的差异条带,把差异条带切胶保存在-80℃,然后质谱分析差异条带的多肽;根据质谱结果和差异条带的大小与NCBI数据库进行比对,筛选到候选相互作用蛋白。最后,构建候选相互作用蛋白的克隆载体并融合HA标签,分别和BmHSP60表达载体共转到家蚕细胞,免疫共沉淀和荧光共定位验证BmHSP60和候选蛋白之间的相互作用。并通过Mito-Tracker-Green分析BmHSP60候选相互作用蛋白与线粒体的共定位。【结果】免疫共沉淀银染结果显示BmHSP60相互作用蛋白在110、90、75、60和35 kD附近有5条明显的差异条带,将这5条条带混成蛋白池进行质谱分析,通过银联-质谱分析结果和家蚕基因组数据库以及NCBI数据库进行比对分析共鉴定到32个差异蛋白,根据多肽数量和蛋白分子量的大小共筛选到5个相互作用候选蛋白与银染结果差异条带一致,分别为ADP/ATP translocase(ANT)、Actin、Alpha-tubulin、Elongation factor 1-alpha(EF1α)和HSP90。构建BmHSP60候选相互作用蛋白克隆表达载体并融合HA标签,与BmHSP60共同转染到家蚕细胞,免疫共沉淀immunoprecipitated（IP）用α-Flag抗体孵育,immunoblotting（IB）再用α-Flag抗体孵育,均能够检测到BmHSP60表达。而IB用α-HA抗体孵育显示只有BmANT和BmHSP90能够检测到相应的条带,而Alpha-tubulin和EF1α与BmHSP60没有检测到相应的条带,说明只有BmANT和BmHSP90与BmHSP60具有直接的相互作用,而Alpha-tubulin和EF1α与BmHSP60没有相互作用。通过荧光共定位进一步分析表明BmANT和BmHSP90能够与BmHSP60共定位在细胞质中。线粒体标记物Mito-Tracker-Green共定位结果显示BmHSP60、BmANT和BmHSP90均能够与线粒体共定位到一起。【结论】鉴定到家蚕热休克蛋白BmHSP60能够与BmANT和BmHSP90相互作用,并共定位于线粒体,可用于研究BmHSP60在家蚕抗病毒免疫中的作用机制。

关键词: 家蚕, BmHSP60, 相互作用蛋白, 免疫共沉淀, BmANT1, BmHSP90

Abstract:

【Objective】 HSP60 is an important member of heat shock proteins, which plays an important role in the innate immunity of insects, and is also an essential factor for insect growth and development. Previous studies have proved that BmHSP60 is involved in the invasion process of Bombyx mori nucleopolyhedrovirus (BmNPV). The objective of this study is to identify the interaction proteins of BmHSP60, and to provide a basis for further exploring the mechanism in the innate immunity of B. mori.【Method】 BmHSP60 overexpression vector was constructed and fused with the Flag tag for co-immunoprecipitation and silver-linked-mass spectrometry (LC-MS/MS). After transfected into the B. mori cells for 48 h, the cell lysates was collected for co-immunoprecipitation. The antibody was incubated with the magnetic beads to capture the interaction proteins. After silver staining, the distinct bands could be detected. The differential strips were stored at -80℃, and then the differentially expressed peptides were analyzed by mass spectrometry. The size of the bands was aligned with the NCBI database to screen for candidate interaction proteins according to the mass spectrometry results. Finally, the cloning vector of candidate interaction proteins was constructed and fused with HA tag, and transferred into B. mori cells with BmHSP60, respectively. Co-immunoprecipitation and fluorescence co-localization confirmed the interaction between BmHSP60 and candidate proteins. The co-localization of BmHSP60 candidate interaction proteins with mitochondria was analyzed by Mito-Tracker-Green.【Result】 Mass spectrometry showed that there were 5 distinct bands in the vicinity of 110, 90, 75, 60 and 35 kD. A total of 32 differentially expressed proteins were identified by comparing the results of silver-linked mass spectrometry with those of silkworm genome database and NCBI database. The 5 bands were mixed into a protein pool for mass spectrometry, and 5 interaction proteins were screened consistent with the differential bands in silver staining according to the number of peptides and molecular weight of proteins. Candidate proteins were ADP/ATP translocase (ANT), Actin, Alpha-tubulin, Elongation factor 1-alpha (EF-1α) and HSP90, respectively. The BmHSP60 candidate interaction protein clone expression vector was constructed and fused with HA tag, and co-transfected with BmHSP60 into B. mori cells. Immunocoprecipitation (IP) was incubated with α-Flag antibody, immunoblotting (IB) was incubated with α-Flag antibody, and BmHSP60 expression was detected by IP. IB incubated with α-HA antibody showed that only BmANT and BmHSP90 could detect the corresponding bands, while Alpha-tubulin and EF1α did not detect the corresponding bands with BmHSP60, indicating that only BmANT and BmHSP90 had direct interaction with BmHSP60, and Alpha-tubulin and EF1α had no direct interaction with BmHSP60. Further analysis of fluorescence co-localization showed that BmANT and BmHSP90 could co-locate with BmHSP60 in cytoplasm. Mitochondrial marker Mito-Tracker-Green co-localization results showed that BmHSP60, BmANT and BmHSP90 were co-localized with mitochondria.【Conclusion】 Heat shock protein BmHSP60 of B. mori was identified to interact with BmANT and BmHSP90, and co-localized in mitochondria. It can be used to study the mechanism of BmHSP60 in antiviral immunity of B. mori.

Key words: Bombyx mori, BmHSP60, interaction protein, co-immunoprecipitation, BmANT1, BmHSP90

董战旗,蒋亚明,潘敏慧. 家蚕热休克蛋白HSP60相互作用蛋白筛选与鉴定[J]. 中国农业科学, 2019, 52(2): 376-384.

DONG ZhanQi,JIANG YaMing,PAN MinHui. Screening and Identification of Candidate Proteins Interacting with BmHSP60 in the Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2019, 52(2): 376-384.

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引物名称Primer name	引物系列Primer sequence (5′-3′)
BmHSP60^Flag-BamH I	CGCGGATCCATGGACTACAAAGACGATGACGACAAGTTGCGTCTACCTCGTGTT
BmHSP60^Flag-Not I	ATAAGAATGCGGCCGCTCACTTGTCGTCATCGTCTTTGTAGTC CATCATGCCTCCCATACCA
BmANT1^HA-KpnI-F	GGGGTACCATGTACCCATACGACGTCCCAGACTACGCTTCGAACCTCGCCGA
BmANT1^HA-SpeI-R	GGACTAGTGGCGTAGTCGGGCACGTCGTAGGGGTACAGGACCTTCTTGATCTCAT
BmTubulin^HA-KpnI-F	GGGGTACCTACCCATACGACGTCCCAGACTACGCTATGAGGGAATGCATCTCC
BmTubulin^HA-SpeI-R	GGACTAGTGGCGTAGTCGGGCACGTCGTAGGGGTAATATTCATTTTCTTCGTCCAACT
BmEF-1α^HA-KpnI-F	GGGGTACCTACCCATACGACGTCCCAGACTACGCTATGGGTAAAGAGAAGACTCATATCA
BmEF-1α^HA-SpeI-R	GGACTAGTGGCGTAGTCGGGCACGTCGTAGGGGTATTTTTTCTTCTGGGCCTTCT
BmHSP90^HA-SpeI-F	GGACTAGTTACCCATACGACGTCCCAGACTACGCTATGAAGGTTGAAACCTTCAC
BmHSP90^HA-XhoI-R	CCGCTCGAGGGCGTAGTCGGGCACGTCGTAGGGGTACTGAATCGGCTCATCTT

蛋白ID Protein ID	描述 Description	分子量Molecular weight (kD)	多肽数量 Peptide number	功能 Function
NP_001037072.1	ADP/ATP translocase (ANT1)	32.9	13	参与ADP/ATP的转运、线粒体稳态以及细胞凋亡Involved in ADP/ATP transport, mitochondrial homeostasis and apoptosis
NP_001119727.1	Actin	41.8	12	肌动蛋白是高度保守的蛋白质,参与各种类型的细胞运动,并在所有核细胞中普遍表达Actin is a highly conserved protein that is involved in various types of cell motility and ubiquitously expressed in all eukaryotic cells
NP_001036884.1	Alpha-tubulin	50.0	8	微管蛋白是微管的主要成分,它结合两个摩尔的GTP,一个在β链上的可交换位点,另一个在α链上的不可交换位点Tubulin is the major constituent of microtubules. It binds two moles of GTP, one at an exchangeable site on the beta chain and one at a non-exchangeable site on the alpha chain
NP_001037510.1	Elongation factor 1-alpha (EF1α)	50.3	7	促进蛋白质生物合成过程中氨基酰-tRNA与核糖体A位点的GTP依赖性结合Promoting the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis
AEB39782.1	HSP90, partial	90.0	4	促进特定靶蛋白的成熟、结构维持和适当调节的分子伴侣,例如参与细胞周期控制和信号转导Molecular chaperone that promotes the maturation, structural maintenance and proper regulation of specific target proteins, for instance involved in cell cycle control and signal transduction