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:: Volume 20, Issue 3 (Iranian South Medical Journal 2017) ::
Iran South Med J 2017, 20(3): 245-256 Back to browse issues page
Cloning and Study of Expression of Helicobacter Pylori FlaAGene in Eukaryotic System
Motaram Sadeghi 1, Abbas Doosti * 2
1- Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
2- Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran , abbasdoosti@yahoo.com
Abstract:   (1346 Views)

Background: Helicobacter pylori is the most common bacterium causing chronic infections worldwide. Expression of flagella and bacterial motility are essential in colonization and virulence. FlaA gene is one of the flagellin-encoding genes that play the key role in the colonization and bacterial motility, and it has a significant impact on immunization. This study aimed to design, construct and evaluate the Helicobacter pylori flaA gene expression in eukaryotic cells.
Material and Methods: In this experimental study, genomic DNA was purified from the Helicobacter pylori standard strain, and flaA gene was amplified and isolated by PCR method with using the specific primers. Then, this gene was cloned into pTZ vector by T/A cloning technique. To express flaA gene and generate the final construct, the flaA gene was removed from pTZ plasmid and subcloned into the pcDNA3.1 (-) expression vector. The pcDNA3.1 (-)-flaA construct was transformed into CHO cells by electroporation, and flaA eukaryotic gene expression was studied using the SDS-PAGE method.
Results: The results showed that flaA gene PCR product was cloned into pTZ vector and amplified in Escherichia coli TOP10F strain. Also, the enzymatic digestion and sequencing showed that the pcDNA3.1 (-)-flaA was performed. Finally, the evaluation of the Helicobacter pyloriflaA gene expression in CHO cells showed that the generated gene construct could express the flaA product in a eukaryotic system, successfully.
Conclusion: Given that the pcDNA3.1 (-)-flaA as a final construct can express the flaA protein of the Helicobacter pylori in animal cells. Flagellin protein is one of the essential antigens of the bacterium So we can appropriately say that gene pcDNA3.1(-)-flaA construct is a suitable candidate for usage in the field of gene vaccines against Helicobacter pylori and it can be used to check the immunization in the laboratory animals in the future research.

Keywords: Helicobacter pylori, flaA gene, Cloning, Electroporation
Full-Text [PDF 791 kb]   (329 Downloads)    
Type of Study: Original | Subject: Biochemistry. Cell Biology and Genetics
Received: 2017/07/5 | Accepted: 2017/07/5 | Published: 2017/07/5
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Sadeghi M, Doosti A. Cloning and Study of Expression of Helicobacter Pylori FlaAGene in Eukaryotic System . Iran South Med J. 2017; 20 (3) :245-256
URL: http://ismj.bpums.ac.ir/article-1-876-en.html

Volume 20, Issue 3 (Iranian South Medical Journal 2017) Back to browse issues page
دانشگاه علوم پزشکی بوشهر، طب جنوب ISMJ

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