Screening and Identifing the Interacting Proteins of Grouper（Epinephelus coioides）EcBAG3 Using Yeast Two-hybrid System

doi:10.13560/j.cnki.biotech.bull.1985.2021-1368

Abstract

Abstract:

The EcBAG3 protein involved in the antiviral immune response of grouper（Epinephelus coioides）was used as the research object,and the interacting proteins of EcBAG3 were screened and identified by yeast two-hybrid technology. Firstly,a yeast two-hybrid cDNA library of grouper（Epinephelus coioides）spleen cells was constructed. The capacity of the library was 5.6×10⁶ CFU,the average length of the inserted cDNA fragments was over 750 bp,and the recombination rate was 100%. Furthermore,the interaction factors of Ec-BAG3 were screened from this library and 83 positive clones were initially obtained. Finally 7 candidate interacting proteins were confirmed through sequencing and one-to-one interaction verification. The functional prediction showed that the interacting proteins were involved in immune regulation,transcriptional regulation,stress response,signal transduction,etc.,which laid a foundation to further understand the mechanism of EcBAG3 during viral infection.

Key words: grouper（Epinephelus coioides）, yeast two hybrid, BAG3, interacting proteins

CAI Jia, LIANG Zhen-yu, HUANG Yu, LU Yi-shan, SHI gang, JIAN Ji-chang. Screening and Identifing the Interacting Proteins of Grouper（Epinephelus coioides）EcBAG3 Using Yeast Two-hybrid System[J]. Biotechnology Bulletin, 2022, 38(8): 77-83.

Figures/Tables 6

Fig. 1 Agarose gel electrophoresis for the detection of total RNA M：DL2000 DNA marker. 1：Total RNA extracted from GS cells

Fig. 2 Agarose gel electrophoresis for the detection of partial inserted cDNA fragments in library M：DL2000 DNA marker. 1-24：PCR products of inserted cDNA fragments

Fig. 3 Self-activation verification of pGBKT7-EcBAG3

Fig. 4 Partial positive clones screened from SD/-Leu-Trp-Ade-His/X-α-Gal/Aba plates

Table 1 Alignments of positive clones screened through yeast two hybrid

候选蛋白 Candidate protein	NCBI登录号 Accession number of NCBI
Heat Shock Cognate 71 kD Protein（Hsc71）	XP_033491860.1
Heat Shock Protein 70（Hsp70）	AWD76391.1
Filamin-A	XP_033484911.1
Collagen,Type V,Alpha 2a	XP_033473099.1
Zinc Finger Protein 217（ZNF217）	XP_033484552.1
CCN Family Member 1（CCN1）	XP_033476535.1
Suppressor Of Cytokine Signaling 9（SOCS9）	XP_033486547.1

Fig. 5 Verification of EcBAG3 and candidate interacting protein Positive control：pGBKT7-53+pGADT7-T. Negative control：pGBKT7-lam+pGADT7. Negative control：pGBKT7-EcBAG3+pGADT7

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