贵州医科大学学报

2018, v.43;No.216(09) 997-1002

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家蝇u93基因的原核表达与体外抗菌活性检测
Prokaryotic Expression of Musca Domestica u93 Gene and Antibacterial Activity in Vitro

马素贞;王涛;龙慧玲;国果;杨阳;常振策;王兵;吴建伟;
MA Suzhen;WANG Tao;LONG Huiling;GUO Guo;YANG Yang;CHANG Zhence;WANG Bing;WU Jianwei;Department of Parasitology,Guizhou Medical University;Department of Microbiology,Guizhou Medical University;Key Laboratory of Pathogenic Biology of Guizhou Higher Education Institutions,Guizhou Medical University;Department of Biotechnology,Guizhou Medical University;

摘要(Abstract):

目的:构建家蝇u93基因原核表达体系,检测重组表达产物的体外抗菌活性。方法:采用生物信息学方法,分析u93基因编码蛋白的理化特性;将前期构建的p ET29a-u93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;纯化重组蛋白,采用微量稀释法检测u93重组蛋白的体外抗菌活性。结果:生物信息学分析显示,u93编码基因开放阅读框全长363 bp,共编码120个氨基酸,理论分子量为13. 35 k D,等电点为7. 52,信号肽位于1~20位氨基酸之间;u93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理论值相一致; u93重组蛋白对白色念珠菌的最小抑菌浓度(MIC)为21 mg/L、对近平滑念珠菌MIC为12. 5 mg/L、对克柔念珠菌MIC为12. 5 mg/L、对热带念珠菌MIC为12. 5 mg/L,但不能抑制金黄色葡萄球菌及大肠埃希菌的生长繁殖。结论:成功构建u93原核表达系统,重组表达的u93蛋白对念珠菌具有较强的抑杀活性。
Objective: To construct the prokaryotic expression system of Musca domestica u93 gene and detect antibacterial activity of recombinant expression product in vitro. Methods: The physicochemical properties of u93 gene encoding protein were analyzed by bioinformatics method. The recombinant pET29 a-u93 expression plasmid was transformed into Escherichia coli BL21/DE3. The recombinant protein was induced by isopropylthiogalactoside( IPTG) and analyzed by SDS-PAGE. Recombinant protein was purified,and microdilution method was used to detect the antibacterial activity of u93 recombinant protein in vitro. Results: Bioinformatics analysis showed that the total length of u93 gene was 363 bp and encoding 120 amino acids,the theoretical molecular weight was 13. 35 kD,isoelectric point was 7. 52,and the signal peptide was between 1 ~ 20 amino acids. The recombinant protein u93 was mainly expressed as inclusion body in escherichia coli BL21/DE3. The molecular weight of the recombinant protein was consistent with the theoretical value. The minimal inhibitory concentration( MIC) of u93 recombinant protein against Candida albicans was 21 mg/L; MIC of Candida parapsilosis was 12. 5 mg/L; MIC of Candida krusei was 12. 5 mg/L; MIC of candida tropicalis was 12. 5 mg/L. But it could not inhibit the growth and reproduction of staphylococcus aureus and Escherichia coli.Conclusion: u93 prokaryotic expression system is successfully constructed,and the recombinant u93 protein has strong inhibitory activity against Candida albicans.

关键词(KeyWords): 家蝇;u93基因;原核表达;抗菌活性
Musca domestica;u93 gene;prokaryotic expression;antimicrobial activity

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基金项目(Foundation): 国家自然科学基金项目(No81360254);; 贵阳市科技局-贵州医科大学联合基金(GY2015-23);; 国家科技支撑计划(2011BAC06B12)

作者(Author): 马素贞;王涛;龙慧玲;国果;杨阳;常振策;王兵;吴建伟;
MA Suzhen;WANG Tao;LONG Huiling;GUO Guo;YANG Yang;CHANG Zhence;WANG Bing;WU Jianwei;Department of Parasitology,Guizhou Medical University;Department of Microbiology,Guizhou Medical University;Key Laboratory of Pathogenic Biology of Guizhou Higher Education Institutions,Guizhou Medical University;Department of Biotechnology,Guizhou Medical University;

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