year 5, Issue 3 (Fall 2011)
Iran J Med Microbiol 2011, 5(3): 18-28 |
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Sadat Shandiz A, Yazdanian M, Aghasadeghi M, Khanahmad H, Memarnejadian A, Motevali F, et al . Designing and construction of recombinant BCG secreting HCV polytope as a candidate vaccine. Iran J Med Microbiol 2011; 5 (3) :18-28
URL: http://ijmm.ir/article-1-195-en.html
URL: http://ijmm.ir/article-1-195-en.html
Attaollah Sadat Shandiz1 
, Maryam Yazdanian2 , Mohammadreza Aghasadeghi2 , Hossein Khanahmad3 , Arash Memarnejadian2 , Fatemeh Motevali4 , Tayebeh Sohrabi3 , Farzin Roohvand 5
, Maryam Yazdanian2 , Mohammadreza Aghasadeghi2 , Hossein Khanahmad3 , Arash Memarnejadian2 , Fatemeh Motevali4 , Tayebeh Sohrabi3 , Farzin Roohvand 5
1- Biology Department, Khatam Institute of Higher Education, Tehran, Iran
2- Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran )NRGB Lab, Pasteur Institute of Iran, Tehran
3- BCG Department ,Research and Production Complex ,Pasteur Institute of Iran
4- NRGB Lab, Pasteur Institute of Iran, Tehran
5- Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran )NRGB Lab, Pasteur Institute of Iran, Tehran , rfarzin@pasteur.ac.ir
2- Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran )NRGB Lab, Pasteur Institute of Iran, Tehran
3- BCG Department ,Research and Production Complex ,Pasteur Institute of Iran
4- NRGB Lab, Pasteur Institute of Iran, Tehran
5- Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran )NRGB Lab, Pasteur Institute of Iran, Tehran , rfarzin@pasteur.ac.ir
Abstract: (10726 Views)
Background and objectives: Liver diseases associated with chronic hepatitis c virus
(HCV) infection are serious public health problems worldwide. There is neither an
efficient therapy nor a vaccine available against this virus. Cellular immune responses,
specially the cytotoxic T-lymphocytes (CTLs) play a key role towards the HCV
clearance. Considering that the Bacillus Calmette-Guerin (BCG) as a tuberculosis
vaccine has the ability to stimulate the cellular immunity, the final aim of this study was construction of a recombinant BCG (rBCG) as an HCV vaccine capable of secreting a
polytope sequence composed of the immunodaminant HCV epitopes for the effective
induction of HCV-specific CTLs.
Material and Method: Hepatitis B surface antigen (HBsAg)-fused polytope sequence composed of 5 HCV epitopes derived from core132-142 , E2405-414 , E2614-622 , NS31406-1415 and core39-48 antigens made by SOEing PCR was further fused to the HSP60 promoter and BCG-alpha-antigen signal sequences. The produced HSP-S-HPOL fragment was further cloned into the kanamycin-encoding pVN2 shuttle vector and the final pHSPS-HPOL recombinant plasmid was transformed into the BCG cells by electroporation technique. The transformants were screened on the kanamycin containing media.
Results: The pHSP-S-HPOL recombinant BCG-plasmid was successfully constructed and confirmed by restriction analyses and sequencing reactions. Transformation of BCG cells was efficiently achieved following intensive optimizations of electroporation parameters. The kanamycin-resistant transformants were screened in kanamycin-media in four weeks.
Conclusion: Appearance of kanamycin-resistant BCG colonies indicated the possibility of expression of pHSP-S-HPOL plasmid and its successful transformation, while motivated us to analyse the secretion of HCV-polytope and perform the in vivo immunogenicity assays.
Material and Method: Hepatitis B surface antigen (HBsAg)-fused polytope sequence composed of 5 HCV epitopes derived from core132-142 , E2405-414 , E2614-622 , NS31406-1415 and core39-48 antigens made by SOEing PCR was further fused to the HSP60 promoter and BCG-alpha-antigen signal sequences. The produced HSP-S-HPOL fragment was further cloned into the kanamycin-encoding pVN2 shuttle vector and the final pHSPS-HPOL recombinant plasmid was transformed into the BCG cells by electroporation technique. The transformants were screened on the kanamycin containing media.
Results: The pHSP-S-HPOL recombinant BCG-plasmid was successfully constructed and confirmed by restriction analyses and sequencing reactions. Transformation of BCG cells was efficiently achieved following intensive optimizations of electroporation parameters. The kanamycin-resistant transformants were screened in kanamycin-media in four weeks.
Conclusion: Appearance of kanamycin-resistant BCG colonies indicated the possibility of expression of pHSP-S-HPOL plasmid and its successful transformation, while motivated us to analyse the secretion of HCV-polytope and perform the in vivo immunogenicity assays.
Type of Study: Original Research Article |
Subject:
Medical Virology
Received: 2013/12/28 | Accepted: 2013/12/28 | ePublished: 2013/12/28
Received: 2013/12/28 | Accepted: 2013/12/28 | ePublished: 2013/12/28
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