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Construction And Characterization Of Recombinant Adenovirus Expressing Single Chain Antibody Against DEC205 Receptor Fused To Carcino Embryonic Antigen For Preliminary Application In Immunotherapy Of Cancer In Mice
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Nazila Hajiahmadi   , Seyed Mohammad Moazzeni , Taravat Bamdad *  |
| Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. |
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Abstract: (159 Views) |
Background and Aims: Selecting appropriate antigens, suitable delivery vehicles and proper immune adjuvants are important items in vaccine designing. According to the previous studies, Carcinoembryonic antigen (CEA) and Adenoviral vector are considered as proper antigen and reliable delivery vehicle for cancer vaccines, respectively. Directing antigens to DCs with antibody single-chain fragment variable (scFv) against to DEC-205 (scDEC) can play the role of adjuvant. The aim of this study was to produce a construct encoding CEA targeted to DCs by scDEC in Adenovirus vector platform.
Materials and Methods: scDEC were cloned into p-track-CEA-GFP. Linearized p-track-scDEC-CEA-GFP and pAdenoVator ∆E1/E3 were co-transformed into BJ5183 cells. Resulting pAd scDEC-CEA-GFP was transfected into HEK293 cells to produce Adenoviruses (Ad scDEC-CEA-GFP). The presence of scDEC-CEA gene in the recombinant virus and expression of CEA protein was detected by PCR and electrochemiluminescence tests, respectively. Flow cytometry tests were used to confirm the attachment of scDEC-CEA protein to DCs. Adenovirus infectivity in cells other than HEK293 was confirmed by transduction of Ad scDEC-CEA-GFP into L929 cells.
Results: PCR test showed the presence of scDEC-CEA gene in adenovirus genome. Electrochemilu-minescence and flow cytometry analysis confirmed expression of CEA protein and attachment of scDEC-CEA protein to DCs, respectively. GFP expression in L929 cells indicated Ad scDEC-CEA-GFP could effectively infect cells other than HEK293.
Conclusion: As construction of a suitable construct for immunotherapy is a major goal of many research groups, the Ad scDEC-CEA-GFP engineered in this study, which properly targets DCs, can be applied in future in vivo cancer immunotherapy investigations in mice. |
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| Keywords: Cancer immunotherapy, CEA, DEC205, Adenoviral vector |
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Full-Text [PDF 540 kb]
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Type of Study: Original article |
Subject:
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Received: 2022/11/28 | Accepted: 2023/08/28 | Published: 2022/12/19
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