鸟分枝杆菌MAV-2921基因编码蛋白的生物信息学分析及其对巨噬细胞凋亡的影响  

Bioinformatic analysis of theMycobacterium avium MAV-2921 protein and its effect on apoptosis of macrophages

作　　者：何会姣 田爽 宁雪萍 龙倩 凌敏[1] HE Hui-jiao;TIAN Shuang;NING Xue-ping;LONG Qian;LING Min(Key Laboratory of Biological Molecular Medicine Research of Guangxi University,Department of Biotechnology,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学长寿与老年相关疾病教育部重点实验室广西高校生物分子医学研究重点实验室广西医科大学生物技术教研室,广西南宁530021

出　　处：《中国病原生物学杂志》2022年第1期75-80,共6页Journal of Pathogen Biology

基　　金：广西自然科学基金项目(No.2018GXNSFAA281023)。

摘　　要：目的应用生物信息学软件分析鸟分枝杆菌MAV-2921蛋白的结构和功能,观察其对人源THP-1巨噬细胞凋亡的影响。方法在NCBI数据库中获取鸟分枝杆菌MAV-2921蛋白编码基因及其氨基酸序列信息,运用ProtParam、ProtScale、TMPred、SignalIP、SOPMA、SWISS-MODEL等在线工具对鸟分枝杆菌MAV-2921蛋白进行生物信息学分析。提取全基因组DNA,进行PCR扩增、载体构建得到鸟分枝杆菌MAV-2921基因的重组表达质粒,经诱导表达和分离纯化后获得重组MAV-2921蛋白。将重组蛋白MAV-2921作用于人源THP-1巨噬细胞,ELISA法检测细胞上清液TNF-α表达量的变化,流式细胞术检测巨噬细胞凋亡率。结果鸟分枝杆菌MAV-2921基因全长285 bp,为编码94个氨基酸的蛋白质。该MAV-2921蛋白是稳定的亲水性蛋白,有1个跨膜区段,无信号肽;二级结构以α-螺旋为主,三级结构模型构建显示该蛋白为单体蛋白,不形成二聚体。利用大肠埃希菌表达系统制备了重组鸟分枝杆菌MAV-2921蛋白,将重组MAV-2921蛋白作用于人源THP-1巨噬细胞,与对照组相比细胞上清液TNF-α含量升高(P<0.05),细胞凋亡率升高(P<0.05)。结论鸟分枝杆菌MAV-2921蛋白为稳定的、亲水性跨膜蛋白,对人源THP-1巨噬细胞具有促凋亡作用。Objective To bioinformatically analyze the structure and function of the MAV-2921 protein of Mycobacterium avium and to investigate the effect of the MAV-2921 protein of M.avium on the apoptosis of human THP-1 macrophages.Methods Genetic information and the amino acid sequence of the MAV-2921 protein of M.avium were retrieved from the NCBI database.ProtParam,ProtScale,TMpred,SignalP,SOPMA,and SWISS-MODEL were used to bioinformatically analyze the characteristics of the MAV-2921 protein of M.avium.After genomic DNA was extracted,a recombinant expression plasmid to express the M.avium MAV-2921 gene was constructed via amplification with PCR and vector construction.Expression of the protein was induced,and the protein was separated and purified to prepare recombinant MAV-2921 protein.After human THP-1 macrophages were treated with the recombinant MAV-2921 protein,ELISA was utilized to determine the level of TNF-αin culture supernatant,and flow cytometry was used to determine the rate of macrophage apoptosis.Results The MAV-2921 gene is 285 bp in length,and the MAV-2921 protein of M.avium contains 94 amino acids.The MAV-2921 protein is a stable hydrophilic protein with a transmembrane domain(from intracellular to extracellular)located at amino acids 35-57,and it is a transmembrane protein with no signal peptides.The protein’s secondary structure mainly consists of alpha-helices.A model of its tertiary structure indicated that MAV-2921 was a monomer protein that does not form a dimer.The recombinant MAV-2921 protein of M.avium was successfully prepared using an E.coli expression system.After human THP-1 macrophages were treated with the recombinant MAV-2921 protein,the level of TNF-αin culture supernatant increased(P<0.05),and the rate of macrophage apoptosis increased(P<0.05).Conclusion The MAV-2921 protein of M.avium is a stable and hydrophilic transmembrane protein that can enhance the apoptosis of human THP-1 macrophages.

关 键 词：鸟分枝杆菌 MAV-2921蛋白 生物信息学分析 THP-1巨噬细胞 细胞凋亡 

分 类 号：R378.91[医药卫生—病原生物学]