白纹伊蚊唾液蛋白Alb-AG5-3的基因特征分析及真核表达  

Characteristic analysis and eukaryotic expression of Aedes albopictus saliva protein Alb-AG5-3

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作  者:高敏 吴家红 程金芝[2,3] 张霞 牟小会[2,3] 聂映 彭哲慧 李志强 赵久刚 商正玲 刘磊 蓝静 刘红美 GAO Min;WU Jiahong;CHENG Jinzhi;ZHANG Xia;MU Xiaohui;NIE Ying;PENG Zhehui;LI Zhiqing;ZHAO Jiugang;SHANG Zhengling;LIU Lei;LAN Jing;LIU Hongmei(Department of Biotechnology School of Biology and Engineering(School of Modern Industry of Health Medicine),Guizhou Medical University,Guiyang 550025,China;Department of Parasitology,School of Basic Medical Sciences,Guizhou Medical University;Characteristic and Key Laboratory of Modern Pathogenic Biology,Guizhou Medical University;Department of Immunology,School of Basic Medical Sciences,Guizhou Medical University)

机构地区:[1]贵州医科大学生物与工程学院/健康医药现代产业学院生物技术教研室,贵州贵阳550025 [2]贵州医科大学基础医学院人体寄生虫学教研室 [3]贵州医科大学现代病原生物学特色重点实验室 [4]贵州医科大学基础医学院免疫学教研室

出  处:《中国病原生物学杂志》2023年第9期1039-1046,共8页Journal of Pathogen Biology

基  金:国家自然科学基金项目(No.32160185;81971972);贵州省科学技术基金项目[黔科合基础-ZK(2021)一般481;黔科合基础ZK(2022)一般366]。

摘  要:目的 初探白纹伊蚊唾液蛋白Antigen5-3(Alb-AG5-3)的结构与功能特征,并采用果蝇S2真核表达体系表达Alb-AG5-3。方法 采取同源比对的方式,从NCBI数据库中钓取Antigen5-3序列,根据白纹伊蚊分泌蛋白Antigen5-3基因参考序列(GenBank:AY826105.1)设计特异性引物,从白纹伊蚊安顺株中扩增基因全长。经序列比对后利用美国国家生物技术信息中心(NCBI)和瑞士生物信息学研究所的蛋白分析专家系统(Expasy)以及生物信息学在线软件SignalP、TMHMM、SOPMA、SWISS-MODEL、AlphaFold等对该蛋白进行生物信息学分析。Trizol法提取白纹伊蚊雌蚊与雄蚊整蚊以及吸血前后雌蚊唾液腺RNA,qRT-PCR检测Alb-AG5-3在RNA相对表达量;以白纹伊蚊整蚊cDNA为模板,特异性扩增除信号肽外的Alb-AG5-3编码区,PCR产物经切胶回收纯化后与昆虫真核表达载体pMT/BiP/V5连接,构建真核重组质粒pMT/BiP/V5-Alb-AG5-3。随后转入果蝇S2细胞中,500μmol/L CuSO4诱导重组蛋白Alb-AG5-3的表达。收集上清,采用Western blot鉴定V5表达标签,镍柱亲和层析纯化重组蛋白Alb-AG5-3。结果 生物信息学分析该基因CDS(Coding sequence)区扩增全长为768 bp,编码255个氨基酸,二、三级结构预测以无规则卷曲和a-螺旋为主,具CAP超家族经典的PR-1结构域,该基因编码的蛋白是一种强亲水性(GRAVY:-0.864)的富含半胱氨酸的分泌蛋白。Alb-AG5-3 mRNA在雌蚊中表达量高于雄蚊且吸血后的表达量显著下调。成功构建了白纹伊蚊唾液蛋白pMT/BiP/V5-Alb-AG5-3真核表达质粒并通过果蝇S2表达体系表达出真核蛋白,镍柱亲和纯化后的Alb-AG5-3经Western blot检测能被V5标签蛋白抗体识别。结论 成功构建白纹伊蚊Alb-AG5-3真核质粒并在果蝇S2细胞中表达真核蛋白Alb-AG5-3,镍柱亲和纯化后获得单一的Alb-AG5-3蛋白以备后续分析。Objective To investigate the structural and functional characteristics of the saliva protein Antigen5-3(Alb-AG5-3)of Aedes albopictus,and to express Alb-AG5-3 protein in Drosophila S2 eukaryotic expression system.Methods Antigen5-3 sequences were extracted from NCBI database by homologous comparison.Specific primers were designed according to the reference sequence of the secreted protein Antigen5-3 gene(GenBank:AY826105.1),and the full length of the gene was amplified from Aedes albopictus.After sequence comparison,the National Center for Biotechnology Information(NCBI),the protein analysis expert system(Expasy)of the SWISS Bioinformatics Institute and the online bioinformatics software SignalP,TMHMM,SOPMA,Swiss-Model and AlphaFold were used for bioinformatics analysis of the protein.The total RNAwas extracted by Trizol method fromthe whole body of the female and male Aedes albopictus and the sugar-feeding and the blood-feeding female mosquitoes salivary glands.Relative expressions of Alb-AG5-3 were detected by qRT-PCR.Using the cDNA as template,the Alb-AG5-3 coding region except the signal peptide was amplified specifically.The PCR products were purified and then ligated to the insect eukaryotic expression vector pMT/BiP/V5 to construct eukaryotic recombinant plasmid pMT/BiP/V5-Alb-AG5-3.Therecombinant plasmid subsequently transferred into Drosophila S2 cells.500μmol/L CuSO4 were used to induced express the recombinant protein Alb-AG5-3.The cell supernatant was collected and the V5 expression label was identified by Western blot.The recombinant protein Alb-AG5-3 was purified by nickel column affinity chromatography.Results Bioinformatics analysis showedthat the total length of CDS(Coding sequence)region of the gene was 768 bp,encoding 255 amino acids.The secondary and tertiary structure prediction showed thatthe protein was dominated by random curl and alpha-helix,with the classic PR-1 domain of CAP superfamily.The gene encodes a secretedprotein with strong hydrophilia(GRAVY:-0.864)and richin cysteine.The expr

关 键 词:白纹伊蚊 唾液腺 Alb-AG5-3蛋白 真核表达 

分 类 号:R384.1[医药卫生—医学寄生虫学]

 

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