幽门螺杆菌多价表位疫苗CWAE的表达及其免疫学性质的研究 <sup>*</sup>

doi:10.13523/j.cb.20191201

中国生物工程杂志

2019, Vol. 39

Issue (12): 1-8 DOI: 10.13523/j.cb.20191201

研究报告

幽门螺杆菌多价表位疫苗CWAE的表达及其免疫学性质的研究 ^*

郭乐^1,³,王淑娥³,何萌³,张帆³,刘宏鹏^3,^**(

),刘昆梅^2,^3,^**(

)

1 宁夏医科大学总医院临床病原微生物省级重点实验室银川 750021
2 宁夏医科大学颅脑疾病国家重点实验室培育基地银川 750021; 宁夏医科大学临床医学院银川 750021

Expression and Immunological Properties of Multivalent Epitope Vaccine CWAE Against Helicobacter pylori

GUO Le^1,³,WANG Shu-e³,HE Meng³,ZHANG Fan³,LIU Hong-peng^3,^**(

),LIU Kun-mei^2,^3,^**(

)

1 Provincial Key Laboratory of Clinical Pathogenic Microbiology, General Hospital of Ningxia Medical University, Yinchuan 750004, China
2 Breeding Base of State Key Laboratory for Craniocerebral Diseases, Ningxia Medical University, Yinchuan 750021, China

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摘要：

目的利用生物信息学软件评价幽门螺杆菌(Helicobacter pylori)多价表位疫苗CWAE的抗原结构,经原核表达获得高纯度CWAE蛋白,进而鉴定多价表位疫苗CWAE的免疫学性质。方法通过生物信息学软件分析H. pylori多价表位疫苗CWAE的抗原结构;用人工合成的H. pylori多价表位肽融合基因WAE替换重组质粒pET28a-CUE中的UE基因,构建重组质粒pET28a-CWAE。然后,将pET28a-CWAE转入大肠杆菌BL21(DE3)中,经IPTG诱导表达,并通过Ni-NTP镍离子亲和层析纯化抗原蛋白CWAE;利用GM1-ELISA鉴定CWAE中CTB组分的黏膜免疫佐剂活性。最后,通过ELISA和小鼠脾脏淋巴细胞增殖实验检测CWAE激发BALB/c小鼠产生抗H. pylori抗体体液免疫和淋巴细胞免疫应答的能力。结果通过生物信息学软件证实H. pylori多价表位疫苗CWAE具有科学合理的结构;重组表达质粒pET28a-CWAE经PCR、双酶切和基因测序鉴定,融合基因CWAE与设计序列完全一致;重组基因工程菌株pET28a-CWAE/BL21(DE3)经IPTG诱导表达,抗原蛋白CWAE主要以包涵体形式存在,经Ni-NTP镍离子亲和层析纯化,纯度约达93.2%;GM1-ELISA实验证实,CWAE中CTB组分依旧保持有较好的黏膜佐剂活性;ELISA结果证实CWAE能够激发BALB/c小鼠产生H. pylori特异性抗体,而小鼠脾脏淋巴细胞增殖实验进一步证实CWAE能够激发针对H. pylori多种致病因子的淋巴细胞免疫反应。结论 H. pylori多价表位疫苗CWAE具有科学合理的抗原结构,经原核表达可获得高纯度抗原蛋白,能够激发BALB/c小鼠产生H. pylori特异性抗体体液免疫和淋巴细胞免疫应答。为研发防治H. pylori感染的多价表位疫苗奠定实验基础。

关键词： 幽门螺杆菌; 多价表位疫苗; 特异性抗体

Abstract:

Objective: To evaluate antigenic structure of multivalent epitope vaccine CWAE against Helicobacter pylori (H. pylori) by bioinformatics softs, obtain the purified CWAE protein after prokaryotic expression, and further identify the immunological properties of CWAE.Methods: The antigenic structure of CWAE was analyzed by bioinformatics softs. The recombinant plasmid pET28a-CWAE was obtained by using a synthetical WAE gene to replace the UE gene in pET28a-CUE plasmid. Then, the recombinant plasmid pET28a-CWAE was transformed into E. coli BL21 (DE3). After induction by IPTG, the antigen protein CWAE was purified by Ni-NTP nickel ion affinity chromatography. The adjuvant activity of CTB components in CWAE was identified by GM1-ELISA. Finally, the ability of CWAE to induce antibodies and lymphocyte immune response against H. pylori was detected by ELISA and spleen lymphocyte proliferation test.Methods: The multivalent epitope vaccine CWAE had a scientific and reasonable structure. The CWAE gene in recombinant plasmid pET28a-CWAE was consistent with the design sequence. After induction with IPTG, CWAE protein mainly exists as inclusion body. The purified CWAE was about 93.2% after purification.Results: from GM1-ELISA confirmed that CTB components in CWAE had good mucosal adjuvant activity. ELISA results confirmed that CWAE could stimulate BALB/c mice to produce anti-H. pylori antibodies. Moreover, CWAE vaccine could stimulate lymphocyte responses against various pathogenic factors from H. pylori.Conclusion: H. pylori multivalent epitope vaccine CWAE has scientific and reasonable antigen structure, and CWAE with high purity can be obtained by prokaryotic expression. Furthermore, the CWAE vaccine can stimulate specific antibodies and lymphocyte response against H. pylori. Experimental evidences for the development of multivalent epitope vaccine against H. pylori will be provided.

Key words: Helicobacter pylori Multivalent epitope vaccine Specific antibody

收稿日期: 2019-05-27 出版日期: 2020-01-15

ZTFLH:

Q93

基金资助: * 宁夏自然科学基金(2019AAC03079)

通讯作者: 刘宏鹏,刘昆梅 E-mail: 70576980@qq.com;lkm198507@126.com

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引用本文:

郭乐,王淑娥,何萌,张帆,刘宏鹏,刘昆梅. 幽门螺杆菌多价表位疫苗CWAE的表达及其免疫学性质的研究 ^*[J]. 中国生物工程杂志, 2019, 39(12): 1-8.

GUO Le,WANG Shu-e,HE Meng,ZHANG Fan,LIU Hong-peng,LIU Kun-mei. Expression and Immunological Properties of Multivalent Epitope Vaccine CWAE Against Helicobacter pylori. China Biotechnology, 2019, 39(12): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191201 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I12/1

图1 CWAE疫苗的抗原结构和DNAstar分析

图2 重组质粒pET28a-CWAE的构建与鉴定

图3 CWAE抗原蛋白的表达和纯化

图4 GM1-ELISA鉴定CWAE中CTB组分的黏膜佐剂活性

图5 CWAE疫苗免疫后小鼠血清中抗H. pylori IgG和IgA抗体的检测

图6 CWAE疫苗免疫后小鼠脾脏淋巴细胞增殖反应的检测

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