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2025年8月11日 星期一
  2018, Vol. 26 Issue (2): 311-318    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
布鲁氏菌ManB蛋白的生物信息学分析及免疫原性研究
徐锦凤1,王震2,易德武3,童志霞3,王月丽3,陈创夫4,许志平5
1. 石河子大学生命科学学院
2. 石河子大学动物科技学院
3.
4. 石河子大学
5. 新疆石河子大学 动物科技学院
Bioinformatic Analysis and Immunogenic Study of Brucella ManB Protein
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摘要 布鲁氏菌(Brucella)是一种革兰氏阴性兼胞内寄生菌,是多种动物和人布氏杆菌病的病原。 布鲁氏菌磷酸甘露糖变位酶(phosphomannomutase, manB)基因编码磷酸甘露糖变位酶,是布鲁氏菌毒力因子脂多糖的重要组成成分。为了研究布鲁氏菌毒力因子脂多糖的ManB蛋白的生物学功能和免疫原性,本文构建磷酸甘露糖变位酶manb基因原核表达载体,对ManB蛋白进行生物信息学预测分析并鉴定其免疫原性。采用PCR技术对布鲁氏菌manB基因进行特异性扩增,与pET-28a表达载体连接后转化至大肠杆菌(Escherichia coli)BL21(DE3)中,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)和Western blot检测目的蛋白的表达及其反应原性,并通过小鼠(Mus musculus)免疫实验评价其免疫原性。生物信息学分析显示,该蛋白无跨膜区结构,不存在信号肽,二级结构以α-螺旋为主。利用Phyre2服务器成功构建了该蛋白的三维模型。SDS-PAGE和Western blot结果表明获得了约56.3 kD的融合蛋白,且该融合蛋白与羊抗布鲁氏菌阳性血清可发生特异性反应。间接酶联免疫吸附实验(enzyme linked immunosorbent assay, ELISA)结果显示,免疫小鼠7、14、21 d,实验组小鼠均可产生抗体,且随时间增加抗体水平也随之升高,并在免疫21 d后达到最高峰。ManB重组蛋白免疫小鼠后可诱导产生高水平的特异性抗体,具有良好的免疫原性。本研究结果为今后开发布鲁氏菌新型疫苗提供了新材料。
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徐锦凤
王震
易德武
童志霞
王月丽
陈创夫
许志平
关键词 布鲁氏菌磷酸甘露糖变位酶(manB)生物信息学免疫原性    
Abstract:Brucella is a Gram-negative facultative intracellular bacterium that is the causative agent of various animal and human brucellosis. The manB gene of Brucella, which encodes mannose phosphate mutase, is an important component of Brucella virulence factor. In order to analyze the biological function and immunogenicity of ManB protein of lipopolysaccharide, this study constructed the prokaryotic expression vector of Mannose phosphate mutase manb gene and predicted the bioinformatics of ManB protein and identified its immunogenicity. The structure and function of ManB protein was analyzed by bioinformatic tools. The manB gene of Brucella was amplified by PCR and ligated with pET-28a expression vector and then transformed into Escherichia coli BL21 (DE3). The expression of target protein was detected by SDS-PAGE and its reaction was detected by Western blot. Then the immunogenicity was analyzed by mouse immunoassay. Bioinformatics analysis showed the protein had no transmembrane domain and had no signal peptide. Its secondary structure was mainly α-helix. The three-dimensional structure of the protein was constructed by Phyre2 Server. SDS-PAGE analysis showed the fusion protein was about 56.3 kD. Western blot showed the protein could react with sheep serum against Brucella, and indicated it possessed reactogenicity. Indirect ELISA results showed that the mice (Mus musculus) in the experimental group produced antibodies in 7th, 14th and 21st d after immunization. The antibody level increased with time and reached the peak in 21st d. The ManB fusion protein had good reactogenicity, immunized mice were able to induce high level of antibody, and had good immunogenicity, which could provide new materials for the development of new vaccine of Brucella in the future.
Key wordsBrucella    Phosphomannomutase (manB)    Bioinformatics    Immunogenicity
收稿日期: 2017-05-15      出版日期: 2018-02-04
基金资助:国家自然科学基金;国家自然科学基金
通讯作者: 陈创夫     E-mail: ccf-xb@163.com
引用本文:   
徐锦凤 王震 易德武 童志霞 王月丽 陈创夫 许志平. 布鲁氏菌ManB蛋白的生物信息学分析及免疫原性研究[J]. , 2018, 26(2): 311-318.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2018/V26/I2/311
 
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