Design, cloning and expression assay of oipA gene in a bicistronic vector harboring mice IL-18 gene: potential implications for Helicobacter pylori vaccine investigations
Mehran Nemattalab, M. Shenagari, A. Mojtahedi, M. Aghasadeghi, M. Pouriayevali, M. Taheri, Mahdieh Mondannizadeh
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引用次数: 2
Abstract
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.