Volume 23, Issue 2 (Iranian South Medical Journal 2020)
Iran South Med J 2020, 23(2): 143-152 |
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1- Department of microbiology, School of Agriculture and Basic Sciences, Shiraz Branch, Islamic Azad University, Shiraz, Iran
2- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran , mfooladvand39@yahoo.com
3- Cellular and Molecular Biology Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran , mfooladvand39@yahoo.com
3- Cellular and Molecular Biology Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract: (2853 Views)
Background: Molecular methods are nowadays used to diagnose diseases, produce vaccines, drugs and recombinant proteins. Therefore, one of the important steps in these procedures is selecting the appropriate vector for cloning and expression of the target genes. Because the expression of synthetic polygenes or so-called chimeric antigens with high molecular weight requires special conditions, the aim of this study was to evaluate the importance of vector type for expression of such antigens.
Materials and Methods: The 1250bp sequence consisting of epitopes from eight important antigens of Leishmania infantum parasite was designed and synthesized by Biomatic Company (Cambridge, Canada). The sequence was cloned separately in two expression vectors pET25b and pET32a and then transformed into E.coli BL21 (DE3) and expressed under similar appropriate conditions. Bacterial lysates were analyzed by SDS PAGE and evaluated by Western blotting.
Results: of SDS- PAGE and Western blot analysis showed that intact recombinant protein production by pET25b vector was not successful. However, the recombinant protein resulted from the expression of the aforementioned poly-epitope in pET32a vector was successfully produced and confirmed.
Conclusion: According to the successful expression confirmation of the poly-epitopic sequence within the pET32a vector and furthermore the failure to obtain protein in the pET25b vector showed that in the case of some specific mosaic sequences expression, protein isolation will be difficult because of low solubility. So, the expression vector choose should be made more carefully.
Materials and Methods: The 1250bp sequence consisting of epitopes from eight important antigens of Leishmania infantum parasite was designed and synthesized by Biomatic Company (Cambridge, Canada). The sequence was cloned separately in two expression vectors pET25b and pET32a and then transformed into E.coli BL21 (DE3) and expressed under similar appropriate conditions. Bacterial lysates were analyzed by SDS PAGE and evaluated by Western blotting.
Results: of SDS- PAGE and Western blot analysis showed that intact recombinant protein production by pET25b vector was not successful. However, the recombinant protein resulted from the expression of the aforementioned poly-epitope in pET32a vector was successfully produced and confirmed.
Conclusion: According to the successful expression confirmation of the poly-epitopic sequence within the pET32a vector and furthermore the failure to obtain protein in the pET25b vector showed that in the case of some specific mosaic sequences expression, protein isolation will be difficult because of low solubility. So, the expression vector choose should be made more carefully.
Keywords: Poly-epitopic structure, Leishmania infantum, Expression level, pET25b vector, pET32a vector
Type of Study: Original |
Subject:
Parasitology
Received: 2019/08/11 | Accepted: 2020/05/15 | Published: 2020/07/1
Received: 2019/08/11 | Accepted: 2020/05/15 | Published: 2020/07/1
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