:: Volume 4, Issue 3 (6-2017) ::
JBRMS 2017, 4(3): 1-7 Back to browse issues page
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, Mohammad Shenagari, Ali Mojtahedi, Mohammad Reza Aghasadeghi, Mohammad Hassan Pouriayevali, Mojtaba Taheri, Mahdieh Mondannizadeh
Department of Microbiology, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran , shenagari@gmail.com
Abstract:   (5636 Views)

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 codon-optimized clone by PCR, and then double-digested by restriction enzymes. The pIRES-Igk/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.

Keywords: Cloning, Codon-optimization, oipA gene, Mouse IL-18, Bicistronic vector
Full-Text [PDF 706 kb]   (2008 Downloads)    
Type of Study: Research | Subject: Biotechnology
Received: 2016/05/24 | Accepted: 2016/08/8 | Published: 2016/11/1

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