oipA基因在携带小鼠IL-18基因的双顺反子载体中的设计、克隆和表达测定:对幽门螺杆菌疫苗研究的潜在意义

Mehran Nemattalab, M. Shenagari, A. Mojtahedi, M. Aghasadeghi, M. Pouriayevali, M. Taheri, Mahdieh Mondannizadeh
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引用次数: 2

摘要

引言:幽门螺杆菌(H.pylori)感染一直是一个全球性的健康问题。动物研究证实了幽门螺杆菌oipA基因在癌症发展中的作用。本研究的目的是在携带小鼠IL-18基因的双顺反子载体中克隆和表达幽门螺杆菌oipA基因。材料与方法:利用聚合酶链式反应从共优化克隆中扩增出编码oipA的靶基因,然后用限制性内切酶进行双酶切。pIRESIgk/mIL18/Fc质粒同时被BstXI/NotI酶消化以引发eGFP片段。使用T4连接酶将oipA的PCR产物插入pIRES-Igk/mIL18/Fc质粒中。转化为DH5α菌株。通过PCR、酶切和测序证实了克隆。通过TaqMan实时PCR评估oipA和IL-18mRNA的表达。结果:PCR产物电泳、酶切和测序结果表明,成功地将幽门螺杆菌oipA基因克隆到pIRES-Igk/mIL18/Fc中,构建了mIL-18-pIRES-2-oipA质粒。实时PCR结果证实了oipA和IL-18在小鼠巨噬细胞系中的成功表达。结论:考虑到oipA在幽门螺杆菌发病机制中的作用以及IL-18作为分子佐剂的强大活性,本研究结果表明密码子优化的oipA基因在包括小鼠IL-18在内的双顺反子载体中的表达是成功的。因此,在未来的研究中,它可以被认为是一种合适的幽门螺杆菌基因候选疫苗。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Design, cloning and expression assay of oipA gene in a bicistronic vector harboring mice IL-18 gene: potential implications for Helicobacter pylori vaccine investigations
Introduction: Helicobacter pylori (H. pylori) infection has remained as a global health problem. Animal studies demonstrated the role of H. pylori oipA gene in the development of gastric cancer. The aim of this study was the cloning and expression of Helicobacter pylori oipA gene in a bicistronic vector harboring mice IL-18 gene. Materials and methods: The target gene encoding oipA was amplified from a codonoptimized clone by PCR, and then double-digested by restriction enzymes. The pIRESIgk/ mIL18/Fc plasmid was simultaneously digested by BstXI/NotI enzymes to elicit the eGFP segment. PCR product of oipA was inserted into pIRES-Igk/mIL18/Fc plasmid using T4 ligase. Transformation into DH5α strain was done. Cloning was confirmed by PCR, enzymatic digestion and sequencing. Expression of the oipA and IL-18 mRNA was assessed by means of TaqMan Real-time PCR. Results: Electrophoresis of PCR product, enzymatic digestion and sequencing showed that the H. pylori oipA gene was successfully cloned into pIRES-Igk/mIL18/Fc to generate mIL- 18-pIRES2-oipA plasmid. The results of Real-time PCR confirmed the successful expression of both oipA and IL-18 in mouse macrophage cell line. Conclusion: Considering the role of oipA in pathogenesis of H. pylori and potent activity of IL-18 as a molecular adjuvant, the results of the present study showed that the expression of codon-optimized oipA gene in bicistronic vector including mouse IL-18 is successful. So, it could be considered as an appropriate genetic vaccine candidate for H. pylori in future investigations.
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