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 *

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。

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

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.