year 5, Issue 1 And 2 (6-2011)
Iran J Med Microbiol 2011, 5(1 And 2): 43-52 |
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Saffar S, Golkar M, Amminian M, Mirshahabi H, Rafatpanah H, Golmohammadi T et al . Production of recombinant HTLV1 Tax protein in Rosetta(DE3) bacterial host. Iran J Med Microbiol 2011; 5 (1 and 2) :43-52
URL: http://ijmm.ir/article-1-180-en.html
URL: http://ijmm.ir/article-1-180-en.html
Samaneh Saffar1 
, Majid Golkar2 , Mahdi Amminian3 , Hesam Mirshahabi4 , Houshang Rafatpanah5 , Taghi Golmohammadi6 , Keihan Azadmanesh 7
, Majid Golkar2 , Mahdi Amminian3 , Hesam Mirshahabi4 , Houshang Rafatpanah5 , Taghi Golmohammadi6 , Keihan Azadmanesh 7
1- Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Science.
2- Department of Parasitology, Pasteure Institute of Iran, Tehran, Iran
3- Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Science
4- Department of Virology, School of Medicine, Tarbiat Modares University, Tehran, Iran.
5- Department of Immunology, Mashhad University of Medical Science.
6- Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Science
7- Department of Virology, Pasteure Institute of Iran, Tehran, Iran , azadmanesh@pasteur.ac.ir
2- Department of Parasitology, Pasteure Institute of Iran, Tehran, Iran
3- Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Science
4- Department of Virology, School of Medicine, Tarbiat Modares University, Tehran, Iran.
5- Department of Immunology, Mashhad University of Medical Science.
6- Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Science
7- Department of Virology, Pasteure Institute of Iran, Tehran, Iran , azadmanesh@pasteur.ac.ir
Abstract: (14839 Views)
Background and objectives: HTLV-1 is the first declared Retrovirus that cause disease in
human. physio pathology of HTLV-1 related diseases ,was linked with tax protein
characteristic.Use of mutant Tax proteins accompanied by immune regulator drugs could help
treating HTLV1 Associated Myelopathy patients as a modulator of potent immune response
against this viral protein.Since Tax protein is not commercially available, produce of
recombinant Tax protein was targeted for this study.
Matherial and methods: Coding sequence of Tax protein (contain R222K mutant) in the pcDNA3.1(+) digested with BamHI and XhoI restriction enzymes then removed and then inserted into the expression vector pET32a(+) within the same cutting site s and cloned into Ecoli DH5α. Recombinant vector was confirmed with enzyme digestive processes,colony PCR and sequencing of cloned gene and then expression vector. E-coli Rosetta (DE3) was transformed with the recombinant plasmid and the expression was induced. The expression of protein was assayed with SDS-PAGE and Western blot. Using monoclonal antibodies against Tax and 5His epitope. Finally antigenic characteristic of the recombinant protein was evaluated by wester n blotting against patient serums.
Results: presence of Tax protein band in the recording of SDS-PAGE and Westernblot was confirmed.western blotting the recombinant protein with patient sera showed the band related to Tax protein.
Conclusion: The expression recombinant protein is well produced and could be detected by patients' sera,making it eligible to be used as a recombinant viral antigen for future purposes.
Matherial and methods: Coding sequence of Tax protein (contain R222K mutant) in the pcDNA3.1(+) digested with BamHI and XhoI restriction enzymes then removed and then inserted into the expression vector pET32a(+) within the same cutting site s and cloned into Ecoli DH5α. Recombinant vector was confirmed with enzyme digestive processes,colony PCR and sequencing of cloned gene and then expression vector. E-coli Rosetta (DE3) was transformed with the recombinant plasmid and the expression was induced. The expression of protein was assayed with SDS-PAGE and Western blot. Using monoclonal antibodies against Tax and 5His epitope. Finally antigenic characteristic of the recombinant protein was evaluated by wester n blotting against patient serums.
Results: presence of Tax protein band in the recording of SDS-PAGE and Westernblot was confirmed.western blotting the recombinant protein with patient sera showed the band related to Tax protein.
Conclusion: The expression recombinant protein is well produced and could be detected by patients' sera,making it eligible to be used as a recombinant viral antigen for future purposes.
Type of Study: Original Research Article |
Subject:
Medical Virology
Received: 2013/11/30 | Accepted: 2013/11/30 | ePublished: 2013/11/30
Received: 2013/11/30 | Accepted: 2013/11/30 | ePublished: 2013/11/30
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