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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (11): 21-27    DOI: 10.13523/j.cb.2007040
研究报告     
幽门螺杆菌致病岛CagL重组抗原的可溶性表达及其多克隆抗体的制备和分析*
何萌1,张国林2,李元1,韩学波1,刘宏鹏1,李欣1,钱玲玲1,刘昆梅3**(),郭乐1**()
1 宁夏医科大学临床医学院 宁夏临床病原微生物重点实验室 银川 750021
2 苏州市药品检验检测研究中心 苏州 215000
3 宁夏医科大学颅脑疾病国家重点实验室培育基地 银川 750021
Soluble Expression of Recombinant Antigen CagL from Helicobacter pylori Pathogenicity Island and Preparation and Analysis of Anti-CagA Polyclonal Antibody
HE Meng1,ZHANG Guo-lin2,LI Yan1,HAN Xue-bo1,LIU Hong-peng1,LI Xin1,QIAN Ling-ling1,LIU Kun-mei3**(),GUO Le1**()
1 Provincial Key Laboratory of Clinical Pathogenic Microbiology, School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China
2 Suzhou Pharmaceutical Testing and Research Center, Suzhou 215000, China
3 Breeding Base of State Key Laboratory for Craniocerebral Diseases, Ningxia Medical University, Yinchuan 750021, China
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摘要：

目的:利用原核表达和蛋白质纯化技术获得高纯度的幽门螺杆菌致病岛CagL重组抗原(rCagL),利用其制备anti-CagL多克隆抗体,并分析抗体的特异性。方法:通过生物信息学软件分析rCagL的抗原结构;利用PCR长片段DNA合成技术合成不含有信号肽序列的幽门螺杆菌致病岛CagL基因,将其插入表达质粒pCzn1中,构建重组质粒pCzn1-rCagL。然后,将pCzn1-rCagL转入大肠杆菌Arctic Express中,经IPTG诱导表达后,通过Ni-IDA镍离子亲和层析纯化重组抗原rCagL,利用Western blot鉴定rCagL与His标签抗体和Anti-H. pylori抗体的免疫反应性;最后,通过rCagL辅以弗氏佐剂免疫BALB/c小鼠,制备anti-CagL多克隆抗血清,通过ELISA方法分析抗血清的特异性。结果:生物信息学软件表明重组抗原rCagL具有较好的抗原性质;重组质粒pCzn1-rCagL经双酶切和基因测序等技术鉴定,证实rCagL核苷酸序列与理论序列完全一致;基因工程菌株pCzn1-rCagL/Arctic Express在低温11℃条件经IPTG诱导表达。 SDS-PAGE实验结果证实:rCagL可实现相对高效地可溶性蛋白表达,可溶性蛋白约占包涵体的62.07%。经Ni-IDA亲和层析柱纯化,可获得高纯度rCagL,纯度约为96.6%。Western blot结果证实:重组抗原rCagL可特异性与His标签抗体和Anti-H. pylori抗体结合。ELISA结果证实:经rCagL免疫小鼠制备的多克隆抗体anti-CagL可特异性识别rCagL和H. pylori裂解物,具有较高的抗体特异性。结论:重组抗原rCagL在低温条件下可实现可溶性表达,经纯化可获得高纯度抗原蛋白;rCagL具有较好的抗原性,制备的多克隆抗体具有较好的免疫特异性,为发展H. pylori相关诊断试剂奠定了实验基础。
关键词： 幽门螺杆菌;  致病岛;  CagL蛋白;  特异性抗体    
Abstract:

Objective: After prokaryotic expression of recombinant antigen CagL (rCagL) from Helicobacter pylori pathogenic island, recombinant antigen rCagL with high purity was obtained by protein purification technology. Anti-CagL polyclonal antibody was prepared and its specificity was also analyzed. Methods:The antigen structure of rCagL was analyzed by bioinformatics software. CagL gene without signal peptide was synthetized by PAS (PCR-based accurate synthesis), and then inserted into expression plasmid pCzn1 to construct recombinant plasmid pCzn1-rCagL. After that, the pCzn1-rCagL plasmids were transferred into E. coli Arctic Express. After expression with IPTG induction, the rCagL protein was purified by Ni-IDA affinity chromatography. The immunoreactivity of rCagL reaction with His label antibodies and anti-H. pylori antibodies was identified by Western blot. Lastly, anti-CagL polyclonal antibodies were prepared by immunizing BALB/c mice with rCagL plus Freund’s adjuvant, and the specificity of anti-CagL polyclonal antibodies was also analyzed by ELISA. Results: Bioinformatics software showed that recombinant antigen rCagL has good antigenic properties. The recombinant plasmid pCzn1-rCagL was identified by double enzyme digestion and gene sequencing, and the nucleotide sequence of rCagL was completely consistent with the theoretical sequence. After induction with IPTG at low temperature of 11℃, the recombinant genetically engineered strains pCzn1-rCagL/Arctic Express can express soluble rCagL protein efficiently by SDS-PAGE analysis. And the soluble rCagL protein accounted for 62.07% of inclusion bodies. The purity of the protein was about 96.6% after purification by Ni-IDA affinity chromatography. Western blot results confirmed that recombinant antigen rCagL could bind specifically to His label antibodies and anti-H. pylori antibodies. ELISA results showed that the prepared anti-CagL polyclonal antibodies could specifically recognize rCagL and H. pylori lysates, indicating anti-CagL polyclonal antibodies had high antibody specificity. Conclusion: The recombinant antigen rCagL can be expressed at low temperature, and the rCagL protein with high purity was obtained after purification. The recombinant antigen rCagL had good antigenicity, and anti-CagL polyclonal antibodies had good immune specificity, which laid a foundation for the development of H. pylori-related diagnostic reagents.
Key words: Helicobacter pylori    Pathogenicity island    CagL    Specific antibody
收稿日期: 2020-07-24 出版日期: 2020-12-11
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(32070930);国家自然科学基金(81760359);宁夏重点研发计划(2020BFG02012);宁夏自然科学基金(2019AAC03079);宁夏自然科学基金(2020AAC03152);江苏省市场监督管理局科技计划(KJ207561)
通讯作者: 刘昆梅,郭乐     E-mail: lkm198507@126.com;guoletian1982@163.com
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引用本文:

何萌,张国林,李元,韩学波,刘宏鹏,李欣,钱玲玲,刘昆梅,郭乐. 幽门螺杆菌致病岛CagL重组抗原的可溶性表达及其多克隆抗体的制备和分析*[J]. 中国生物工程杂志, 2020, 40(11): 21-27.

HE Meng,ZHANG Guo-lin,LI Yan,HAN Xue-bo,LIU Hong-peng,LI Xin,QIAN Ling-ling,LIU Kun-mei,GUO Le. Soluble Expression of Recombinant Antigen CagL from Helicobacter pylori Pathogenicity Island and Preparation and Analysis of Anti-CagA Polyclonal Antibody. China Biotechnology, 2020, 40(11): 21-27.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2007040    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I11/21

图1  rCagL的抗原结构组成及其生物信息学分析
图2  重组质粒pCzn1-rCagL的构建与鉴定
图3  重组抗原rCagL的表达及纯化
图4  Western blot鉴定重组抗原rCagL的免疫反应性
图5  Anti-CagL多克隆抗体的特异性检测
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