布鲁氏菌Omp19基因的生物信息学分析、克隆表达和ELISA方法的初步建立

doi:10.16431/j.cnki.1671-7236.2020.10.005

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (10): 3078-3087.doi: 10.16431/j.cnki.1671-7236.2020.10.005

布鲁氏菌Omp19基因的生物信息学分析、克隆表达和ELISA方法的初步建立

杨宁宁¹, 王震¹, 杨亚军², 张倩³, 宋胜男¹, 张欢¹, 陈创夫^1,4, 刘明军^1,5

1. 石河子大学动物科技学院, 石河子 832000;
2. 石河子大学生命科学学院, 石河子 832000;
3. 新疆农垦科学院, 省部共建绵羊遗传改良与健康养殖国家重点实验室, 石河子 832000;
4. 西部地区高发人兽共患传染性疾病防治协同创新中心, 石河子 832000;
5. 新疆畜牧科学院生物技术研究所, 乌鲁木齐 830001

收稿日期:2020-01-17 出版日期:2020-10-20 发布日期:2020-10-17
通讯作者: 陈创夫, 刘明军 E-mail:ccf-xb@163.com;xjlmj2006@126.com
作者简介:杨宁宁(1991-),女,河南商丘人,博士生,研究方向:人畜共患病防控,E-mail:2450224604@qq.com;王震(1987-),男,山东诸城人,讲师,研究方向:人畜共患病防控,E-mail:530469380@qq.com
基金资助:
兵团重大科技项目（2017AA003）；石河子大学高层次人才科研启动项目（RCSX2018B13）

Bioinformatics Analysis,Cloning and Expression of Omp19 Gene of Brucella and Preliminary Establishment of ELISA Method

YANG Ningning¹, WANG Zhen¹, YANG Yajun², ZHANG Qian³, SONG Shengnan¹, ZHANG Huan¹, CHEN Chuangfu^1,4, LIU Mingjun^1,5

1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
2. College of Life Sciences, Shihezi University, Shihezi 832000, China;
3. State Key Laboratory for Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Science, Shihezi 832000, China;
4. Co-innovation Center for Zoontic Infectious Disesses in the Western Region, Shihezi 832000, China;
5. Institute of Biotechnology, Xinjiang Academy of Animal Husbandry, Urumqi 830001, China

Received:2020-01-17 Online:2020-10-20 Published:2020-10-17

摘要/Abstract

摘要： 为了进一步研究布鲁氏菌外膜蛋白19（outer membrane protein 19，OMP19）的结构与功能，并获得具有反应原性的OMP19重组蛋白，建立布鲁氏菌间接ELISA抗体检测方法，试验通过生物信息学软件对OMP19蛋白进行氨基酸序列分析，经PCR技术克隆Omp19基因，利用无缝克隆技术构建重组表达载体pET-32a-Omp19，将其转化大肠杆菌BL21（DE3）感受态细胞，诱导其表达并纯化，并通过Western blotting和ELISA分析方法分别检测OMP19蛋白的反应原性，以建立基于该蛋白的间接ELISA方法。结果显示，OMP19蛋白N端有19个信号肽序列，二级结构以无规则卷曲为主，且OMP19蛋白含有优势抗原表位，利用PCR技术和无缝克隆技术成功构建了Omp19基因的原核表达载体pET-32a-Omp19，成功诱导表达并纯化了OMP19融合蛋白，大小约为35 ku，与理论值相符，并通过Western blotting和ELISA方法鉴定OMP19的反应原性，结果表明该蛋白具有较好的反应原性，且包被浓度为10 μg/mL，一抗稀释比为1：50，二抗稀释比为1：8 000时，P/N值最大，为2.80。本研究结果为布鲁氏菌病快速诊断方法的建立和新型疫苗的研发奠定了理论基础。

关键词: 布鲁氏菌; Omp19基因; 生物信息学分析; 反应原性

Abstract: This study was aimed to further investigate the structure and function of Brucella outer membrane protein 19 (OMP19),obtain the recombinant protein of OMP19 with reactivity,and establish the new method for diagnosing brucellosis based on indirect ELISA.The amino acid sequence of OMP19 protein was analyzed by bioinformatics software,Omp19 gene was amplified by PCR,and the recombinant expression vector pET-32a-Omp19 was constructed by seamless cloning method,it was transformed into E.coli BL21 (DE3) competent cells,induced and purified OMP19 protein.The immunogenicity of OMP19 protein was detected by Western blotting and ELISA,respectively,to establish an indirect ELISA method based on the protein.The results showed that there were 19 signal peptide sequences in the N terminal of OMP19 protein,and the secondary structure was random coil,and OMP19 protein contained dominant antigenic epitopes.The prokaryotic expression vector pET-32a-Omp19 was successfully constructed by PCR and seamless cloning method,the OMP19 fusion protein was successfully induced,expressed and purified,the size of protein was about 35 ku,which was consistent with the expect size.The reactivity of OMP19 was identified by Western blotting and ELISA,the results showed that the fusion protein had good reactivity,and the coating concentration was 10 μg/mL.The dilution ratio of the first antibody was 1：50 and the second antibody dilution ratio was 1：8 000,the P/N value reach the highest at 2.80.The results laid a foundation for the establishment of rapid diagnosis of brucellosis and the development of new vaccine.

Key words: Brucella; Omp19 gene; bioinformatics analysis; reactivity

中图分类号:

杨宁宁, 王震, 杨亚军, 张倩, 宋胜男, 张欢, 陈创夫, 刘明军. 布鲁氏菌Omp19基因的生物信息学分析、克隆表达和ELISA方法的初步建立[J]. 中国畜牧兽医, 2020, 47(10): 3078-3087.

YANG Ningning, WANG Zhen, YANG Yajun, ZHANG Qian, SONG Shengnan, ZHANG Huan, CHEN Chuangfu, LIU Mingjun. Bioinformatics Analysis,Cloning and Expression of Omp19 Gene of Brucella and Preliminary Establishment of ELISA Method[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(10): 3078-3087.

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布鲁氏菌Omp19基因的生物信息学分析、克隆表达和ELISA方法的初步建立

Bioinformatics Analysis,Cloning and Expression of Omp19 Gene of Brucella and Preliminary Establishment of ELISA Method

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