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Construction of recombinant yeast expressing EpEX as a suitable candidate in cancer diagnosis and therapy
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Mozhdeh Zamani   , Atieh Hashemi  , Najmeh Zarei , Hoda Jahandar |
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Abstract: (3155 Views) |
| Introduction: Epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that is overexpressed on the majority of tumor cells of epithelial origin and thereby can be used as a target of immunodetection and immunotherapy of cancer. So, it is important to produce this protein in its native form. Interestingly, during the last two decades, the yeast Pichia pastoris (P. pastoris) has become a popular host for the production of recombinant proteins because it combines the advantages of both mammalian and prokaryotic expression systems. In this study, the Pichia expressing EpCAM extracellular domain (EpEX) was constructed.
Materials and Methods: The codon optimized gene encoding EpEX protein was cloned in the XhoI and XbaI restriction sites of the pPICZαB vector. The constructed plasmid was integrated into GS115 strain by electroporation. Positive clones were evaluated by PCR using AOX1 primers.
Results: Sequencing as well as restriction enzyme analysis utilizing XhoI and XbaI (3506, 843 bp bands), as well as BamHI (3651, 698 bp bands) confirmed construction of recombinant EpEX pPICZαB. PCR based screening results of integrants showed two bands (2200 and 1345 bp), when AOX1 primer set was used.
Conclusion: These findings imply that the engineered strain was constructed. The constructed strain can be used in EpEX recombinant protein production for diagnostic and therapeutic purposes. |
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| Keywords: Pichia pastoris, Molecular Cloning, EpEX, Epithelial Cell Adhesion Molecule, Neoplasms |
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Full-Text [PDF 2718 kb]
(1041 Downloads)
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Type of Study: Research |
Subject:
General
Received: 2019/02/18 | Accepted: 2019/05/4 | Published: 2019/10/29
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