嗜吞噬无形体Msp2蛋白与宿主互作靶蛋白筛选及生物信息分析

目的:筛选与嗜吞噬无形体Msp2蛋白互作的THP-1细胞靶蛋白,有助于理解病原体侵袭宿主的分子机理。方法:PCR获得无形体msp2基因克隆到pGBKT7载体上构建诱饵质粒并转化酵母菌株Y2HGold,营养缺陷培养基及蓝白斑实验验证诱饵质粒是否有自激活、毒性;抽提THP-1总RNA,经反转录、Long-distance PCR、同源重组等构建到pGADT7-Rec载体上并转化酵母菌株Y187,鉴定cDNA文库质量;酵母双杂交筛选与无形体Msp2互作的宿主细胞靶蛋白,生物信息学分析蛋白互作可能导致的信号通路变化。结果:成功构建诱饵质粒;cDNA文库容量达4×106克隆,插入片段大小100-3000 bp,且无污染;酵母双杂交获得宿主靶蛋白7个,分别是NADH脱氢酶(泛醌)1α亚体13(NDUFA13)、锌指蛋白36, C3H样2(ZFP36L2)、核糖体蛋白11(RPL11)、前胸腺素α(PTMA)、C19orf10、组织蛋白酶G(CTSG)、核糖体蛋白S25(RPS25);生物信息学分析,互作的宿主靶蛋白主要参与细胞增殖、细胞凋亡、溶酶体成熟及其它一些信号通路等生物学过程。结论:应用酵母双杂交系统初步筛选出与嗜吞噬细胞无形体表面蛋白Msp2互作的宿主细胞靶蛋白,并利用生物信息学初步分析了其参与的生物学过程,为进一步研究病原菌胞内生存分子机制奠定了基础。

Screening and Bioinformatic Analysis of THP-1 Target Protein(s) Interacted with Msp2 of Anaplasma phagocytophilum

ABSTRACT Objective: To screen target proteins from THP-1 interation with Msp2 protein from Anaplasma phagocytophilum, and contribute to understanding pathogenic molecular mechanism. Methods: Bait msp2 cloned into vector pGBKT7 was transfomated into yeast Y2HGold, and tested bait for autoactivation and toxicity. Generating cDNA library with SMARTTM technology were constructed into vector pGADT7-Rec by homologous recombination, which was transfomated into yeast Y187, and assessed the library. To screen targets from THP-1 interation with Anaplasma phagocytophilum protein Msp2 by yeast two-hybrid system(YTH). Biology processes of THP-1 targets were analysised by bioinformatics. Results: Bait were successfully constructed, content of cDNA library were 4×106 clones, inserts size of the library were 100-3000bp. Seven proteins interaction with Msp2 protein included Homo sapiens NADH dehydrogenase (ubiquinone)1 alpha subcomplex 13 (NDUFA13), Homo sapiens ZFP36 ring finger protein-like 2 (ZFP36L2), Homo sapiens ribosomal protein L11 (RPL11), Homo sapiens prothymosin, alpha(PTMA), Homo sapiens chromosome 19 open reading frame 10 (C19orf10), Homo sapiens cathepsin G (CTSG), Homo sapiens ribosomal protein S25 (RPS25). According to bioinformatic analysis, the targets interaction with Msp2 participated in biological processes including cell proliferation, apoptosis, lysosomes mature, and some signal pathways etc. Conclusion: Some host proteins which may interact with A. phagocytophilum Msp2 have been screened out by YTH, the screened proteins may participate in biological processes by bioinformatics. These results provids important basis for further investigating intracellular pathogen survival mechanism.