Purification and Immunologic Study of the Recombinant Urease B subunit from <em>Helicobacter pylori</em>

doi:10.13523/j.cb.20141204

China Biotechnology

2014, Vol. 34

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

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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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

Received: 17 September 2014 Published: 25 December 2014

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This work was supported by a Grant from Ningnia High School Scientific Research Program(NGY2013065)

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

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