幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究

doi:10.13523/j.cb.20141204

中国生物工程杂志

2014, Vol. 34

Issue (12): 23-29 DOI: 10.13523/j.cb.20141204

研究报告

幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究

秦玉红¹, 刘昆梅³, 廖国玲¹, 杨华¹, 徐广贤¹, 李秀萍^1,2, 郭乐¹

1. 宁夏医科大学检验学院宁夏临床病原微生物重点实验室银川 750021;
2. 宁夏医科大学宁夏医科大学总医院银川 750021;
3. 宁夏医科大学颅脑疾病重点实验室银川 750021

Purification and Immunologic Study of the Recombinant Urease B subunit from Helicobacter pylori

QIN Yu-hong¹, LIU Kun-mei³, LIAO Guo-ling¹, YANG Hua¹, XU Guang-xian¹, LI Xiu-ping^1,2, GUO Le¹

1. Ningxia Key Laboratory of Clinical Pathogens, School of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, China;
2. General Hospital of Ningxia Medical University, Ningxia Medical University, Yinchuan 750004, China;
3. Ningxia Key Laboratory of Cerebrocranial Diseases, Ningxia Medical University, Yinchuan 750004, China

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摘要：

目的:幽门螺旋杆菌(Hp)尿素酶是Hp重要的定制因子和致病因子,Hp尿素酶活性位点位于Hp尿素酶B亚基(UreB),研发基于UreB的Hp疫苗是一种很有前景的防治Hp感染的策略。方法:主要利用基因克隆技术从幽门螺旋杆菌标准菌株SS1(Hp SS1)获得Hp尿素酶B亚基基因,并构建含有重组Hp尿素酶B亚基(rUreB)基因的重组表达载体pET-rUreB及其重组菌株;重组菌株经蛋白表达和优化后,利用Ni-NTP镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化重组尿素酶B亚基(rUreB),并进一步通过腹腔注射免疫BALB/c小鼠,研究rUreB的免疫学性质。结果:通过基因克隆技术成功获得了Hp尿素酶B亚基基因,并成功构建了重组表达载体pET-rUreB及其重组菌株BL21(DE3)/pET-rUreB,经蛋白表达优化及纯化,可获得高纯度(96.5%)的重组蛋白rUreB。重组蛋白rUreB辅以弗氏佐剂腹腔注射免疫BALB/c小鼠,经间接ELISA鉴定小鼠能够产生针对天然Hp尿素酶和UreB的高滴度特异性抗体,且能够显著性抑制Hp尿素酶的活性。结论:重组Hp尿素酶B亚基能够在大肠杆菌表达系统中获得较高水平的表达,具有较高的免疫学特异性,其抗体能够有效抑制Hp尿素酶活性。为研究基于尿素酶的防治Hp感染的Hp疫苗奠定了一定的实验基础。

关键词： 幽门螺旋杆菌; 尿素酶; 尿素酶B亚基

Abstract:

Objective: Helicobacter pylori (Hp) urease is an important colonization factor as well as a pathogenic factor, and the enzymatic activity sites of Hp urease locate in the Hp urease B subunit (UreB). Research and development of Hp vaccine based on urease is a promising strategy for the prevention of Hp infection. The aims are to obtain recombinant Hp urease with high purity, and study its immunological properties. Methods:The UreB gene was obtained from Helicobacter pylori standard strain SS1 (Hp SS1), and the expression vector pET-rUreB and the recombinant strain BL21(DE3)/pET-rUreB were also constructed by gene cloning technology. After protein expression and optimization, the recombinant protein rUreB was purified by Ni²⁺-charged column chromatography and anion-exchange chromatography using DEAE Sepharose FF. Then, the immunological properties of rUreB protein was investigated by intraperitoneal immunization experiments in BALB/c mice with Freund's adjuvant. Results:Hp UreB gene was obtained successfully from Hp SS1 through PCR, and the expression vector pET-rUreB and recombinant strain BL21(DE3)/pET-rUreB were constructed successfully by gene cloning technology. After protein expression optimization and purification, about 69 mg of pure target protein was obtained from 1 L of fermentation broth and the purity of rUreB was 96.5%. Mice immunized with rUreB using Freund's adjuvant could induce high level of antibodies specific for both natural Hp urease and UreB by ELISA, which can significantly inhibit the activity of Hp urease. Conclusion:The recombinant UreB was expressed at a medium level in E. coli and had good immunological specificity. Besides, the antibodies induced by rUreB can effectively inhibit the activity of Hp urease. This will provide an experimental basis for the development of Hp vaccine based on urease.

Key words: Helicobacter pylori Urease Urease B subunit

收稿日期: 2014-09-17 出版日期: 2014-12-25

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通讯作者: 李秀萍, 郭乐 E-mail: guoletian1982@163.com;lixp7777@sina.com

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秦玉红, 刘昆梅, 廖国玲, 杨华, 徐广贤, 李秀萍, 郭乐. 幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究[J]. 中国生物工程杂志, 2014, 34(12): 23-29.

QIN Yu-hong, LIU Kun-mei, LIAO Guo-ling, YANG Hua, XU Guang-xian, LI Xiu-ping, GUO Le. Purification and Immunologic Study of the Recombinant Urease B subunit from Helicobacter pylori. China Biotechnology, 2014, 34(12): 23-29.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20141204 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I12/23

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