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

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

【目的】探讨非洲猪瘟病毒(African swine fever virus,ASFV)衣壳蛋白E120R在病毒感染中的作用,确定与E120R蛋白互作的宿主蛋白。【方法】将ASFV分离株CADC_HN09株E 120 R基因合成并克隆于pGBKT7表达载体,获得pGBKT7-E120R诱饵质粒,同时构建E 120 R基因截短表达质粒pGBKT7-E120R-1(1-61位氨基酸)和pGBKT7-E120R-2(62-122位氨基酸)。经毒性和自激活性检测后,通过酵母双杂交技术对骨髓巨噬细胞(BMDMs)cDNA均一化酵母文库进行初步筛选,以获得与ASFV E120R蛋白互作的蛋白。通过NCBI数据库进行序列比对并经免疫共沉淀试验验证,确定互作的宿主蛋白。筛选的宿主蛋白经webgenstal在线分析网站初步进行GO功能和KEGG通路富集分析,以确定所筛选的宿主蛋白参与的生物过程与信号通路。【结果】诱饵质粒pGBKT7-E120R-2无毒性和自激活性,可用于文库筛选。通过酵母双杂交系统从BMDMs cDNA均一化酵母文库中初步筛选到46个阳性克隆并进行回转验证,经NCBI数据库序列比对分析共获得29个宿主蛋白。免疫共沉淀试验结果显示,多聚胞嘧啶结合蛋白2(PCBP2)和干扰素刺激基因15(ISG15)均与E120R蛋白存在互作。GO功能富集分析表明,所筛选的宿主蛋白可参与代谢过程、生物调节、应激反应等生物过程;KEGG通路富集分析表明,这些宿主蛋白可参与抗原呈递、铁死亡和坏死性凋亡等多条信号通路。【结论】ASFV E120R蛋白可与宿主免疫应答、细胞死亡等信号通路相关的多种宿主蛋白互作,为进一步研究ASFV E120R蛋白在病毒感染过程中的作用提供了重要理论依据。

【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 E 120 R 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 E 120 R 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.