布鲁氏菌GMD重组蛋白表达及对昆明系小鼠的免疫效果研究

doi:10.6048/j.issn.1001-4330.2017.12.018

新疆农业科学 ›› 2017, Vol. 54 ›› Issue (12): 2304-2313.DOI: 10.6048/j.issn.1001-4330.2017.12.018

布鲁氏菌GMD重组蛋白表达及对昆明系小鼠的免疫效果研究

徐锦凤¹, 王震², 易德武², 易继海², 马旭升¹, 姬祥¹, 张辉², 陈创夫¹

1.新疆石河子大学生命科学学院,新疆石河子 832000;
2.新疆石河子大学动物科技学院,新疆石河子 832000

收稿日期:2017-11-02 出版日期:2017-12-25 发布日期:2018-01-29
通信作者: 陈创夫(1964-),男,广东潮州人,教授,博士生导师,研究方向为病原微生物分子机制,(E-mail)ccf-xb@163.com
作者简介:徐锦凤(1992-),女,甘肃武威人,硕士研究生,研究方向为生物化学与分子生物学,(E-mail)xujinfeng024@qq.com
基金资助:
国家自然科学基金面上项目“TceSR和TcfSR 在布鲁氏菌致病过程中的分子调控机制研究”(31572491)

Expression of GMD Recombinant Protein of Brucella and Its Immune Effect on Kunming Mice

XU Jin-feng¹, WANG Zhen², YI De-wu², YI Ji-hai², MA Xu-sheng¹, JI Xiang¹, ZHANG Hui², CHEN Chuang-fu¹

1.College of Life Technology,Shihezi University,Shihezi Xinjiang 832000,China;
2.College of Animal Science and Technology,Shihezi University,Shihezi Xinjiang 832000,China

Received:2017-11-02 Online:2017-12-25 Published:2018-01-29
Correspondence author: CHEN Chuan-fu(1964-),male, professor, doctoral supervisor, mainly engaged in the study of pathogenic microorganisms pathogenic mechanism, (E-mail)ccf-xb@163.com
Supported by:
National Natural Science Foundation of China "Molecular Mechanism of TceSR and TcfSR in the Pathogenesis of Brucella" (31572491)

摘要/Abstract

摘要： 【目的】对布鲁氏菌GMD蛋白进行生物信息学预测分析和免疫原性研究,并在小鼠模型上评价其免疫效果。【方法】对GMD蛋白进行生物信息学分析,分析预测其结构及功能,采用PCR技术对布鲁氏菌gmd基因进行特异性扩增,与pET-28a表达载体连接后转化至E.coli BL21(DE3)中,通过SDS- PAGE和Western Blot检测目的蛋白的表达及其反应原性,并通过小鼠免疫和攻毒实验评价其免疫原性以及免疫保护力。【结果】生物信息学分析显示,该蛋白无跨膜区结构,有15个抗原决定簇,不存在信号肽,二级结构以α-螺旋为主。并利用Phyre2服务器成功构建了该蛋白的三维模型。SDS-PAGE分析表明获得了约43.7 ku的融合蛋白,Western Blot结果显示该融合蛋白与羊抗布鲁氏菌阳性血清发生特异性反应。间接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。免疫保护力测定结果显示小鼠脾指数和脾脏CFU均低于PBS组。【结论】GMD重组蛋白具有良好的反应原性,其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;攻毒后能发挥一定免疫保护作用。

关键词: 布鲁氏菌; GMD; 重组蛋白; 免疫原性; 保护力

Abstract: 【Objective】 To predict and analyze bioinformatics function for the GMD protein of Brucella, and to identify its immunogenicity and then the immune effect was evaluated on mouse model.【Method】The structure and function of ManB protein was analyzed by bioinformatics tools. The GMD gene of Brucella was amplified by PCR and the recombinant plasmid was transformed into E.coli BL21 (DE3) after being connected with pET-28a expression vector. The expression of target protein was detected by SDS-PAGE and its reaction was detected by Western Blot. Then the immunogenicity and immunoprotection were evaluated by mouse immunoassay and challenge experiments.【Result】Bioinformatics analysis showed that the protein had no transmembrane domain structure and no signal peptide. There were 15 antigenic determinants and its secondary structure was mainly α-helix. And the three-dimensional structure of the protein was constructed by Phyre2 Server. SDS-PAGE analysis showed that the fusion protein was about 43.7 ku. Western Blot showed that the protein could react with sheep serum against Brucella, indicating it possessed reactogenicity. Indirect ELISA results showed that the mice in the experimental group produced antibodies at 7th, 14th and 21st after immunization and the antibody level increased with time. The results of immune protection test showed that the spleen index and CFU of the spleen were lower than those in the PBS group. 【Conclusion】The GMD recombinant protein has good immunogenicity and can induce high level of specific antibodies after immunization in mice. After immunization, it can play a certain role in immune protection.

Key words: brucella abortus; GMD; fusion protein; immunogenicity; protection

中图分类号:

徐锦凤, 王震, 易德武, 易继海, 马旭升, 姬祥, 张辉, 陈创夫. 布鲁氏菌GMD重组蛋白表达及对昆明系小鼠的免疫效果研究[J]. 新疆农业科学, 2017, 54(12): 2304-2313.

XU Jin-feng, WANG Zhen, YI De-wu, YI Ji-hai, MA Xu-sheng, JI Xiang, ZHANG Hui, CHEN Chuang-fu. Expression of GMD Recombinant Protein of Brucella and Its Immune Effect on Kunming Mice[J]. Xinjiang Agricultural Sciences, 2017, 54(12): 2304-2313.

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布鲁氏菌GMD重组蛋白表达及对昆明系小鼠的免疫效果研究

Expression of GMD Recombinant Protein of Brucella and Its Immune Effect on Kunming Mice

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