Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (2): 376-384.doi: 10.3864/j.issn.0578-1752.2019.02.016


Screening and Identification of Candidate Proteins Interacting with BmHSP60 in the Silkworm (Bombyx mori)

DONG ZhanQi1,JIANG YaMing1,PAN MinHui1,2()   

  1. 1 State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716
    2 Key Laboratory for Sericulture Functional Genomics and Biotechnology of Ministry of Agriculture, Southwest University, Chongqing 400716
  • Received:2018-08-08 Accepted:2018-09-06 Online:2019-01-16 Published:2019-01-21
  • Contact: MinHui PAN


【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

Table 1

Primers used in this study"

引物名称Primer name 引物系列Primer sequence (5′-3′)

Fig. 1

Co-immunoprecipitation for BmHSP60 interaction candidate proteins"

Table 2

LC-MS/MS analysis of the BmHSP60 interaction proteins"

Protein ID
分子量Molecular weight (kD) 多肽数量
Peptide number
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

Fig. 2

Western blot analysis determining the interaction between BmHsp60 with BmHsp90 and BmANT1 Co-immunoprecipitation of BmHSP60 examined by Western blot. BmN-SWU1 cells are co-transfected with BmHSP60 and candidate BmTubulin, BmEF-1α, BmHSP90, BmANT1 protein. At 96 hours after transfection, cells are lysed and immunoprecipitation performed with α-FLAG, and use α-HA to detect the bound of target protein. The label on the top of each panel shows the antibodies used for immunoprecipitation. The label on the right of each panel shows the antibodies used for analysis of Western blot"

Fig. 3

Immunofluorescence verification of interaction between BmHSP60 with BmHSP90 and BmANT1 Co-localization of BmHSP60 with BmHSP90 and BmANT1 in BmN-SWU1 cells. Alexa 555 labels anti-Flag, FITC labels anti-HA and Hoechst33258 at 48 h post-transfection in the BmN-SWU1 cells. Red fluorescence represents Flag, green fluorescence represents HA, and blue fluorescence represents the nucleus"

Fig. 4

Mitochondria co-localization analysis of BmHSP60, BmANT1 and BmHSP90 BmHSP60, BmANT1 and BmHSP90 are stained with Alexa 555-labeled, mitochondria-tracker and Hoechst33258 at 48 h post-transfection in the BmN-SWU1 cells. Red fluorescence represents BmHSP60, BmANT1 and BmHSP90, green fluorescence represents Mito-Tracker, and blue fluorescence represents the nucleus"

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