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:: Volume 17, Number 1 (spring 2017) ::
J Ardabil Univ Med Sci 2017, 17(1): 46-57 Back to browse issues page
Cloning and Expression Study of the hcpD Gene of Helicobacter pylori
Eslami, Abbas Doosti
Abstract:   (1199 Views)

Background & Objectives: hcpD gene in Helicobacter pylori is a member of cysteine-rich proteins family which triggers the host's immune system and antibody production. H. pylori is found in human's stomach and causes digestive diseases such as duodenal ulcer, chronic gastritis and stomach cancer. The objectives of this study were to isolate, amplify and clone H. pylori's hcpD gene in pcDNA3.1 (-) vector and to study its expression in eukaryotic system.

Methods: H. pylori genomic DNA was isolated by extraction kit. The hcpD gene was amplified using PCR reaction and then purified from gel, followed by pTZ cloning. Subcloning of hcpD was performed in pcDNA3.1 (-) eukaryotic expression vector. The accuracy of cloning steps was investigated through PCR, enzymatic digestion by BamHI and EcoRV enzymes, and sequencing, respectively. Transfer of expression construct into CHO cells was done by electroporation. The gene expression in these cells was analyzed using RT-PCR and SDS-PAGE.

Results: PCR results showed amplification of a 933bp segment related to hcpD gene. Successful cloning of the gene in pTZ vector and construction of pTZ-hcpD recombinant vector were achieved. Enzymatic digestion and sequencing confirmed the correctness of subcloning and creation of pcDNA3.1 (-)-hcpD construct. hcpD was expressed in eukaryotic system, and its protein product was observed on SDS-PAGE gel.

Conclusion: pTZ-hcpD construct can be used as a source of H. pylori's hcpD gene for future research, like production of recombinant protein and vaccine in different systems. Furthermore, successful expression of the gene using pcDNA3.1 (-)-hcpD in CHO animal cells shows the potential of vector as a gene vaccine against H. pylori.

Keywords: hcpD Gene, Helicobacter pylori, Electroporation, Gene Expression
Full-Text [PDF 274 kb]   (523 Downloads)    
Type of Study: article | Subject: General
Received: 2016/07/22 | Accepted: 2017/02/18 | Published: 2017/03/30
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Eslami, Doosti A. Cloning and Expression Study of the hcpD Gene of Helicobacter pylori . J Ardabil Univ Med Sci. 2017; 17 (1) :46-57
URL: http://jarums.arums.ac.ir/article-1-1314-en.html
Volume 17, Number 1 (spring 2017) Back to browse issues page
مجله دانشگاه علوم پزشکی اردبیل Journal of Ardabil University of Medical Sciences
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