Expression of M2 Protein of Human Influenza Virus in Escherichia Coli
Background: Influenza A virus is an orthomyxovirus capable of infecting humans. The virus genome consists of eight negative single-strand RNA segments that encode structural and nonstructural viral proteins. M2, a disulfide-linked homotetramer and a membrane-bound protein, is conserved among all inf...
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doaj-8652b81e864540a4873171defa76b62c2020-11-25T01:09:10ZfasVesnu Publications مجله دانشکده پزشکی اصفهان1027-75951735-854X2013-02-0130216209121021218Expression of M2 Protein of Human Influenza Virus in Escherichia ColiMohammad Ali Alavi Esfahani0Fatemeh Fotouhi1Amir Ghaemi2Maryam Saleh3Siavash Chalabiani4Behrokh Farahmand5Masoumeh Tavasoti Kheiri6Department of Microbiology, School of Biological Sciences, Islamic Azad University, Qom Branch, Qom, IranAssistant Professor, Department of Virology, Influenza Research Laboratory, Pasteur Institute of Iran, Tehran, IranAssistant Professor, Department of Medical Virology and Immunology, School of Medicine, Golestan University of Medical Sciences, Gorgan, IranDepartment of Virology, Influenza Research Laboratory, Pasteur Institute of Iran, Tehran, IranDepartment of Microbiology, School of Biological Sciences, Islamic Azad University, Qom Branch, Qom, IranAssistant Professor, Department of Virology, Influenza Research Laboratory, Pasteur Institute of Iran, Tehran, IranAssistant Professor, Department of Virology, Influenza Research Laboratory, Pasteur Institute of Iran, Tehran, IranBackground: Influenza A virus is an orthomyxovirus capable of infecting humans. The virus genome consists of eight negative single-strand RNA segments that encode structural and nonstructural viral proteins. M2, a disulfide-linked homotetramer and a membrane-bound protein, is conserved among all influenza A viruses. Therefore, it is an appropriate target for the development of influenza vaccine with broad-spectrum protection. In this study, M2 protein of influenza virus A was expressed in a prokaryotic system. Methods: M2 gene of influenza virus A (New Caledonia/20/99, H1N1) was amplified by polymerase chain reaction (PCR) using specific primers. It was then digested with appropriate enzymes and cloned into the prokaryotic expression vector pQE30. The Escherichia coli (M15) competent cells were transformed with recombinant plasmid (pQE30-M2) and grown in LB broth media overnight after being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The media were supplemented with 50 mg/ml ampicillin and 50 mg/ml kanamycin. Expression of the M2 protein was approved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. Findings: The results of colony PCR, restriction enzyme digestion, and sequencing revealed that M2 gene was cloned in pQE30 properly in frame to histidine tag. Protein expression was confirmed in western blotting using specific monoclonal anti-M2 antibody. Conclusion: Experiments in animal models have shown M2-based vaccines to induce broad spectrum immunity against various influenza A viruses. Thus, the M2 protein prepared in this study will be a suitable vaccine candidate to be evaluated in further studies on animal models. Keywords: M2 protein, Vaccine, Protein expression, Influenza virus, Escherichia colihttp://jims.mui.ac.ir/index.php/jims/article/view/2381 |
collection |
DOAJ |
language |
fas |
format |
Article |
sources |
DOAJ |
author |
Mohammad Ali Alavi Esfahani Fatemeh Fotouhi Amir Ghaemi Maryam Saleh Siavash Chalabiani Behrokh Farahmand Masoumeh Tavasoti Kheiri |
spellingShingle |
Mohammad Ali Alavi Esfahani Fatemeh Fotouhi Amir Ghaemi Maryam Saleh Siavash Chalabiani Behrokh Farahmand Masoumeh Tavasoti Kheiri Expression of M2 Protein of Human Influenza Virus in Escherichia Coli مجله دانشکده پزشکی اصفهان |
author_facet |
Mohammad Ali Alavi Esfahani Fatemeh Fotouhi Amir Ghaemi Maryam Saleh Siavash Chalabiani Behrokh Farahmand Masoumeh Tavasoti Kheiri |
author_sort |
Mohammad Ali Alavi Esfahani |
title |
Expression of M2 Protein of Human Influenza Virus in Escherichia Coli |
title_short |
Expression of M2 Protein of Human Influenza Virus in Escherichia Coli |
title_full |
Expression of M2 Protein of Human Influenza Virus in Escherichia Coli |
title_fullStr |
Expression of M2 Protein of Human Influenza Virus in Escherichia Coli |
title_full_unstemmed |
Expression of M2 Protein of Human Influenza Virus in Escherichia Coli |
title_sort |
expression of m2 protein of human influenza virus in escherichia coli |
publisher |
Vesnu Publications |
series |
مجله دانشکده پزشکی اصفهان |
issn |
1027-7595 1735-854X |
publishDate |
2013-02-01 |
description |
Background: Influenza A virus is an orthomyxovirus capable of infecting humans. The virus genome consists of eight negative single-strand RNA segments that encode structural and nonstructural viral proteins. M2, a disulfide-linked homotetramer and a membrane-bound protein, is conserved among all influenza A viruses. Therefore, it is an appropriate target for the development of influenza vaccine with broad-spectrum protection. In this study, M2 protein of influenza virus A was expressed in a prokaryotic system.
Methods: M2 gene of influenza virus A (New Caledonia/20/99, H1N1) was amplified by polymerase chain reaction (PCR) using specific primers. It was then digested with appropriate enzymes and cloned into the prokaryotic expression vector pQE30. The Escherichia coli (M15) competent cells were transformed with recombinant plasmid (pQE30-M2) and grown in LB broth media overnight after being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The media were supplemented with 50 mg/ml ampicillin and 50 mg/ml kanamycin. Expression of the M2 protein was approved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis.
Findings: The results of colony PCR, restriction enzyme digestion, and sequencing revealed that M2 gene was cloned in pQE30 properly in frame to histidine tag. Protein expression was confirmed in western blotting using specific monoclonal anti-M2 antibody.
Conclusion: Experiments in animal models have shown M2-based vaccines to induce broad spectrum immunity against various influenza A viruses. Thus, the M2 protein prepared in this study will be a suitable vaccine candidate to be evaluated in further studies on animal models.
Keywords: M2 protein, Vaccine, Protein expression, Influenza virus, Escherichia coli |
url |
http://jims.mui.ac.ir/index.php/jims/article/view/2381 |
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