结核分枝杆菌融合基因Rv0071/74编码蛋白的生物信息学分析与制备  

Bioinformatics analysis and preparation of protein encoded by Mycobacterium tuberculosis fusion gene Rv0071/74

作　　者：杨玲玲 秦莲花 黄劲[2] YANG Lingling;QIN Lianhua;HUANG Jing(School of Public Health,the Key Laboratoryof Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,Guiyang 561113,China;Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Guizhou Medical University;Shanghai Key Laboratory of Tuberculosis,Shanghai Pulmonary Hospital,Tongji UniversitySchool of Medicine)

机构地区：[1]贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵州贵阳561113 [2]贵州医科大学基础医学院生物化学与分子生物学教研室 [3]同济大学医学院附属上海市肺科医院上海市结核病(肺)重点实验室

出　　处：《中国病原生物学杂志》2024年第12期1391-1396,1403,共7页Journal of Pathogen Biology

基　　金：贵州省科技厅科技计划项目(黔科合支撑项目[2023]一般233);上海市结核病(肺)重点实验室开放基金(2022-KF-06)。

摘　　要：目的 探究融合基因编码的蛋白Rv0071/74在北京型结核分枝杆菌的流行和耐药中的作用。方法 运用NCBI获取并下载Rv0071、Rv0074的蛋白氨基酸序列,采用在线软件Protparam、ProtScale、PSORT分析蛋白的理化性质、亲疏水性和亚细胞定位;使用在线软件SignaIP6.0、TMHMM、NetNGlyc1.0、NetPhos-3.1分析蛋白的信号肽数量、跨膜区域、糖基化和磷酸化位点;运用软件SPOMA、SWISS-MODEL进行蛋白的二级结构和三级结构的预测;使用ABCpred、SYFPEITHI软件进行蛋白的T、B细胞抗原表位预测;采用软件STRING进行预测分析Rv0074蛋白的相互作用蛋白。结果 Rv0071/74基因全长1 032 bp,编码的蛋白氨基酸序数为343,分子式为C1548H2458N450O481S9,为稳定的疏水蛋白,亚细胞可能定位于细胞质。该蛋白有21个磷酸化位点,无糖基化位点、信号肽及跨膜区域;二级结构预测结果显示α-螺旋(38.78%)、无规卷曲(37.90%)为该蛋白的主要元件,三级结构建模与羧肽酶有较高相似性。融合蛋白Rv0071/74含有32个B细胞抗原表位,17个T细胞抗原表位。Rv0074蛋白的相互作用蛋白为Rv0073、Rv0075、Rv0072、Rv0071、Rv3346c、pks15、PPE38、Rv0552、Rv2052c、Rv2628。重组质粒Pet28a-Rv0071/74在表达宿主E.coli BL21中为不可溶性表达;SDS-PAG证实重组质粒Pet28a-Rv0071/74在E.coli BL21表达成功。结论 生物信息学预测Rv0071/74可能为羧肽酶家族,T、B细胞抗原表位有两个区域重叠,可能是结核病新型疫苗开发新靶点,可为探索其在北京型结核分枝杆菌致病性中的功能作用提供理论基础。Objective Exploring the role of the fusion gene-encoded protein Rvo071/74 in the prevalence and drug resistance ofBeijing Mycobacterium tuberculosis strainM.ethods NCBI was used to obtain and download the protein amino acid sequences of Rv0071 and Rv0074,Online software Protparam,ProtScale and PSORT were used to analyze the physical and chemical properties,hydrophilicity and subcellular localization of proteins;the number of signal peptides,transmembrane regions,glycosylation and phosphorylation sites of the proteins were analyzed by using the online software SignaIP6.0,TMHMM,NetNGlycl,O,NetPhos-3.1 to analyze the number of signal peptides,transmembrane regions,glycosylation and phosphorylation sites of proteins;the software SPOMA and SWISS-MODEL were used for the prediction of the secondary and tertiary structures of proteins;ABCpred and SYFPEITHI software were used for the prediction of the T and B cell antigenic epitopes of proteins;the software STRING was used for the prediction and analysis of Rvo074 protein interacting proteins.R1esultsThe Rv0071/74 gene is 1032 bp in length and encodes a protein with an amino acid sequence of 343 and a molecular formula of Ci548 H2458 N450 O481 Sg,which is a stabilized hydrophobic protein with a subcellular possible localization in the cytoplasm.The protein has 21 phosphorylation sites and no glycosylation sites,signal peptide or transmembrane regions;secondary structure prediction showedα-helix(38.78%)and random coiling(37.90%)as the main components of the protein,and tertiary structure modeling showed high similarity with carboxypeptidase.The fusion protein Rv0071/74 contains 32 B-cell antigenic epitopes and 17 T-cell antigenic epitopes.The interacting proteins of the Rv0074 protein are Rv0073,Rv0075,Rv0072,Rv0071,Rv3346c,pks15,PPE38,Rv0552,Rv2052c,Rv2628.The recombinant plasmid Pet28a-Rv0071/74 was insoluble expressed in the expression host E.coli BL21;SDS-PAG confirmed that the recombinant plasmid Pet28a-Rv0071/74 was successfully expressed in E.coli BL21.Conclusion B

关 键 词：结核分枝杆菌 Rv0071/74 生物信息学 

分 类 号：R378.911[医药卫生—病原生物学]