无形体分泌蛋白Ats-1与宿主细胞互作蛋白筛选及其生物信息分析

Identification and bioinformation analysis of host target protein (s)interacted with Ats-1 of Anaplasma phagocytophilum

目的筛选与无形体Ats-1互作的THP-1宿主细胞蛋白,为揭示病原体侵袭宿主的致病机理提供科学数据。方法利用分子克隆法构建p GBKT7-ats-1诱饵质粒并转化酵母Y2HGold,验证诱饵质粒是否有自激活、毒性现象;制备THP-1细胞c DNA并连接到p GADT7-Rec上,转化酵母Y187并鉴定c DNA文库质量;酵母双杂交筛选与Ats-1互作的宿主细胞靶蛋白,通过GO、String生物信息分析细胞靶蛋白可能的生物学过程。结果成功构建诱饵质粒;cDNA文库容量4×10~6克隆,插入片段100~3 000 bp,且无污染,达到建库质控要求;酵母双杂交结果显示,与Ats-1蛋白互作的宿主细胞靶蛋白7个,分别是真核延伸因子1复合体α1亚基(EEF1A1)、TIMP金属蛋白酶抑制子1(TIMP1)、锌指蛋白36,C3H样2 (ZFP36L2)、半乳糖凝集素1(LGALS1)、前胸腺素α(PTMA)、WBSCR22、吸引素(ATRN)。生物信息分析显示,与Ats-1互作的宿主靶蛋白主要参与细胞分化增殖、细胞凋亡、自噬、炎症等生物学过程。结论病原诱饵蛋白与宿主细胞靶蛋白互作是一种细胞-细胞间发生信号联系的分子水平生物学过程,可能影响病原粘附、侵袭及胞内生存,最终导致疾病发生及临床症状出现。

Objective To identify target proteins from THP-1 interation with Ats-1 protein from Anaplasma phagocytophilum,and contribute to understanding pathogenic molecular mechanism. Methods Constructed bait plasmid p GBKT7-ats-1 by molecular cloning methods was transfomated into yeast Y2 HGold,and tested bait for autoactivation and toxicity. c DNAlibrary with SMARTTMtechnology were constructed into vector p GADT7-Rec by homologous recombination,which was transfomated into yeast Y187.To screen targets from THP-1 interation with Anaplasma phagocytophilum protein Ats-1 by yeast two-hybrid system.Biology processes of THP-1 targets were analysised by bioinformatics.Results Content of c DNA library were 4×10~6 clones,c DNA size of the library were 100 ~ 3 000 bp.Seven target proteins interation with Ats-1 protein included Homo sapiens eukaryotic translation elongation factor 1 alpha 1( EEF1 A1),Homo sapiens TIMP metallopeptidase inhibitor 1( TIMP1),Homo sapiens ZFP36 ring finger protein-like 2( ZFP36L2),Homo sapiens lectin galactoside-binding soluble 1( LGALS1),Homo sapiens prothymosin,alpha( PTMA),Homo sapiens Williams Beuren syndrome chromosome region 22( WBSCR22),Homo sapiens attractin( ATRN). According to bioinformatic analysis,The target proteins interaction with Ats-1 participated in biological processes including cell proliferation and differentiation,apoptosis,autophagy,and inflammation etc.Conclusion Interactions between A. phagocytophilum Ats-1 and interacting partners demonstrate molecular cross-talk on cellular processes in pathogen-host interaction.These ndings further elucidate the role of Ats-1 as virulence factor that promote intracellular survival by interfering cell signal pathway and evading the immune system responds in host cell.