Volume 23, Issue 4 (Winter 2016)                   J Birjand Univ Med Sci. 2016, 23(4): 286-297 | Back to browse issues page

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Ghorbani M, Doosti A. Isolation and cloning of Helicobacter Pylori ureE gene into pIRES2-DSRed expression vector to generate a gene vaccine. J Birjand Univ Med Sci.. 2016; 23 (4) :286-297
URL: http://journal.bums.ac.ir/article-1-2182-en.html
1- Department of Biology, Faculty of Basic Science, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran. , ghorbanim1003@gmail.com
2- Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
Abstract:   (9268 Views)

Background and Aim: As one of the factors of gastric ulcers and cancer, Helicobacter pylori can live in the acidic environment of stomach for many years due to having urease enzyme. This enzyme requires Ni2+ and a group of auxiliary proteins such as ureE for its catalytic activity. Urease is not only a requisite factor to colonize the Helicobacter pylori but it is also pathogenic with different mechanisms. Regarding the high prevalence of these bacteria finding a way to prevent infection with them  is necessary. The present research aimed at homogenizing . cloning of Helicobacter Pylori ureE gene into pIRES2-DS Red expression vector in order to create a DNA (gene) vaccine.

Materials and Methods: In this experimental study, the ureE gene fragment was amplified through PCR method and it was cloned using T/A cloning in the pTZ vector. Sub-cloning of the gene was done in pIRES2-DS Red vector using T4-ligase enzyme and it was transformed into E. coli TOP10F strain; and, then, amplified. The gene construct, which is a DNA vaccine candidate, was transferred to CHO cells using electroporation method to investigate the gene expression in eukaryotic systems. UreE gene expression was assessed in eukaryotic cells by means of SDS-PAGE.

Results: UreE gene cloning was confirmed in two vectors including pTZ as a replicative vector and pIRES2-DS Red expression vector by PCR, enzyme digestion, and sequencing methods. SDS-PAGE results confirmed the successful expression of the ureE gene in the eukaryotic system of CHO cells.

Conclusion: The recombinant pIRES2-DSRed-ureE construct is capable of successfully generating of polypeptides derived from Helicobacter Pylori ureE gene expression in bestial cells. Given that the protein product of the ureE gene is one of the most important proteins of the mentioned bacterium, . the created recombinant DNA in this research can be used as a DNA vaccine candidate against Helicobacter pylori in . future.

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Type of Study: Original Article | Subject: Medical Genetics
Received: 2016/10/4 | Accepted: 2016/11/23 | ePublished: 2017/01/9

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