利用酵母双杂交技术筛选和鉴定非洲猪瘟病毒E120R蛋白的互作宿主蛋白

doi:10.16431/j.cnki.1671-7236.2022.11.004

Abstract

Abstract: 【Objective】 The purpose of this study was to investigate the role of African swine fever virus (ASFV) capsid E120R protein in virus infection, and identify the host proteins that could interact with E120R protein.【Method】 The E120R gene was synthesized according to ASFV CADC_HN09 isolate.The bait plasmid pGBKT7-E120R, truncated plasmids pGBKT7-E120R-1 (amino acids 1 to 61) and pGBKT7-E120R-2 (amino acids 62 to 122) of E120R gene were constructed.After toxicity and auto-activation detected, yeast two-hybrid BMDMs cDNA library was applied to screen the host proteins with E120R protein.Then the host proteins sequences were aligned via NCBI database and verified by co-immunoprecipitation assay.The candidate host proteins were preliminarily analyzed by GO function and KEGG pathway analysis by webgenstal online analysis website, so as to determine the biological process and signal pathway of the screened host proteins.【Result】 The bait plasmid pGBKT7-E120R-2 was non-toxic and not auto-activation, so it was available for cDNA library screening.A total of 46 initial positive clones were screened from the yeast two-hybrid BMDMs cDNA library and verified.After sequence alignment in NCBI database, a total of 29 potential interacting proteins were obtained.Both poly(C) binding protein 2 (PCBP2) and interferon-stimulated gene product 15 (ISG15) were identified to interact with E120R protein by co-immunoprecipitation assay.GO function enrichment analysis showed that these host proteins could participate in metabolic process, biological regulation, response to stimulus and other biological processes.KEGG pathway enrichment analysis indicated that these host proteins could be involved in antigen processing and presentation, ferroptosis, necroptosis and other pathways.【Conclusion】 ASFV capsid protein E120R could interact with a variety of host proteins involved in the host immune response and cell death, which would provide an important theoretical basis for further research on the role of ASFV E120R protein in the process of virus infection.

Key words: African swine fever virus (ASFV); yeast two-hybrid; E120R protein; interacting protein

CLC Number:

S852.65^＋1

CUI Shuai, WANG Yang, GUO Xiaoyu, CHEN Shiyu, JIANG Yajun, PANG Zhongbao, HUANG Jianxin, MENG Fanfeng, JIANG Yitong, ZHU Hongfei, JIA Hong. Screening and Identification of the Host Proteins Interacting with African Swine Fever Virus E120R Protein Using Yeast Two-hybrid[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4139-4149.

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Screening and Identification of the Host Proteins Interacting with African Swine Fever Virus E120R Protein Using Yeast Two-hybrid

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