贵州医科大学学报

2020, v.45;No.233(02) 145-150

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家蝇三龄幼虫Mdctl基因原核表达及生物信息学分析
Prokaryotic Expression and Bioinformatics Analysis of Mdctl in the Housefly Third Instar Larva

常振策;李忠旬;贾利娜;修江帆;国果;吴建伟;
CHANG Zhence;LI Zhongxun;JIA Lina;XIU Jiangfan;GUO Guo;WU Jianwei;Key Laboratory of Modern Pathogenic Biology of Guizhou Higher Education Institutions,Guizhou Medical University;Department of Parasitology,Guizhou Medical University;

摘要(Abstract):

目的:构建家蝇C型凝集素基因(Mdctl)原核表达体系,对Mdctl基因及编码蛋白进行生物信息学分析。方法:采用生物信息学方法,分析Mdctl基因及编码蛋白;将构建的pET28a(+)/Mdctl重组表达质粒转化至大肠杆菌BL21-DE3中,以异丙基硫代半乳糖苷(IPTG)诱导表达重组蛋白并通过SDS-PAGE对表达产物进行分析;Ni-IDA法纯化重组蛋白,Western-Blot对其鉴定。结果:生物信息学分析显示,Mdctl基因开放阅读框全长333 bp,共编码110个氨基酸,理论分子量为1313 kDa,等电点为449,信号肽位于1~22位氨基酸之间;Mdctl蛋白中存在Clect结构域,属于C型凝集素超家族;Mdctl重组蛋白在大肠杆菌BL21-DE3中主要以包涵体形式表达,重组蛋白的相对分子质量与Mdctl蛋白两端融合6×His标签后的总分子量相一致。结论:成功构建Mdctl原核表达系统,并获得重组蛋白。
Objective: To construct a prokaryotic expression system of Musca domestica C-type lectin gene( Mdctl) and perform bioinformatics analysis ofMdctlgene and Mdctl-encoded protein.Methods: The Mdctl gene and the encoded protein were analyzed by bioinformatics approach. The constructed recombinant plasmid pET28 a( + )/Mdctltagged with 6 xHis was transformed into E. coli BL21-DE3 and Mdctl expression was induced by isopropyl thiogalactoside(IPTG). The expression of recombinant Mdctl protein was analyzed by SDS-PAGE. The recombinant Mdctl protein was purified by Ni-IDA purification kit and verified by Western Blot.Results: Bioinformatics analysis showed that the open reading frame ofMdctlgene is 333 bp in length and encodes 110 amino acids. The theoretical molecular weight is 13.13 kDa, and its the isoelectric point is 4.49. Moreover, the signal peptide in Mdctl is located between 1 and 22 amino acids. Mdctl protein has a Clect domain, which belongs to the C-type lectin superfamily. Furthermore, the Mdctl recombinant protein is mainly expressed in the form of inclusion bodies inE. coliBL21-DE3. Western blot analysis revealed that the molecular weight of purified recombinant Mdctl protein is the same as that of the recombinant Mdctl protein recognized by anti-His antibody.Conclusion: The prokaryotic expression system of Mdctl was successfully constructed and recombinant Mdctl protein was obtained.

关键词(KeyWords): 家蝇;Mdctl基因;原核表达;蛋白纯化;生物信息学;基因表达
Musca domestica;Mdctlgene;prokaryotic expression;protein purification;bioinformatics;gene expression

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基金项目(Foundation): 国家自然科学基金项目(81360254);; 国家科技支撑计划(2011BAC06B12);; 贵州省农业攻关[黔科合NY(2014)3054];; 贵州省科学技术基金[黔科合J字(2012)2038]

作者(Author): 常振策;李忠旬;贾利娜;修江帆;国果;吴建伟;
CHANG Zhence;LI Zhongxun;JIA Lina;XIU Jiangfan;GUO Guo;WU Jianwei;Key Laboratory of Modern Pathogenic Biology of Guizhou Higher Education Institutions,Guizhou Medical University;Department of Parasitology,Guizhou Medical University;

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DOI: 10.19367/j.cnki.1000-2707.2020.02.004

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