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Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells

Purpose: More than half of the diagnostic and therapeutic recombinant protein production depends on mammalian-based expression system. However, the generation of recombinant antibodies remains a challenge in mammalian cells due to the disulfide bond formation and reducing cytoplasm. Therefore, th...

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Main Author: Che Omar, Mohammad Tasyriq
Format: Article
Language:English
Published: Tabriz University of Medical Sciences 2017
Subjects:
LC5800-5808 Distance education.
RK1-715 Dentistry
Online Access:http://eprints.usm.my/36956/1/%28Expression_of_Functional%29_APB-7-299.pdf
http://eprints.usm.my/36956/
https://www.ncbi.nlm.nih.gov/pubmed/28761833
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spelling my.usm.eprints.36956 http://eprints.usm.my/36956/ Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells Che Omar, Mohammad Tasyriq LC5800-5808 Distance education. RK1-715 Dentistry Purpose: More than half of the diagnostic and therapeutic recombinant protein production depends on mammalian-based expression system. However, the generation of recombinant antibodies remains a challenge in mammalian cells due to the disulfide bond formation and reducing cytoplasm. Therefore, the production of functional recombinant antibodies in target cell line is necessary to be evaluated before used in therapeutic application such intrabodies against HIV-1. Methods: The work was to test expression of a single-chain variable fragment (scFv) antibody against HIV-1 Capsid p24 protein in a human mammalian-based expression system using HEK293T and Jurkat T cells as a model. Three expression plasmid vectors expressing scFv 183-H12-5C were generated and introduced into HEK293T. Expression of the scFv was analyzed, while ELISA and immunoblotting analysis verified its binding. The evaluation of the recombinant antibody was confirmed by HIV-1 replication and MAGI infectivity assay in Jurkat T cells. Results: Three plasmid vectors expressing scFv 183-H12-5C was successfully engineered in this study. Recombinant antibodies scFv (~29 kDa) and scFv-Fc (~52 kDa) in the cytoplasm of HEK293T were effectively obtained by transfected the cells with engineered pCDNA3.3-mu-IgGk-scFv 183-H12-5C and pCMX2.5-scFv 183-H12-5C-hIgG1-Fc plasmid vectors respectively. scFv and scFv-Fc are specifically bound recombinant p24, and HIV-1 derived p24 (gag) evaluated by ELISA and Western blot. Jurkat T cells transfected by pCDNA3.3-scFv 183-H12-5C inhibit the replication-competent NL4-3 viral infectivity up to 60%. Conclusion: Anti-p24 scFv 183-H12-5C antibody generated is suitable to be acted as intrabodies and may serve as a valuable tool for the development of antibody-based biotherapeutics against HIV-1. Tabriz University of Medical Sciences 2017 Article PeerReviewed application/pdf en http://eprints.usm.my/36956/1/%28Expression_of_Functional%29_APB-7-299.pdf Che Omar, Mohammad Tasyriq (2017) Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells. Advanced Pharmaceutical Bulletin, 7 (2). pp. 299-312. ISSN 2251-7308 https://www.ncbi.nlm.nih.gov/pubmed/28761833
institution Universiti Sains Malaysia
building Hamzah Sendut Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Sains Malaysia
content_source USM Institutional Repository
url_provider http://eprints.usm.my/
language English
topic LC5800-5808 Distance education.
RK1-715 Dentistry
spellingShingle LC5800-5808 Distance education.
RK1-715 Dentistry
Che Omar, Mohammad Tasyriq
Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells
description Purpose: More than half of the diagnostic and therapeutic recombinant protein production depends on mammalian-based expression system. However, the generation of recombinant antibodies remains a challenge in mammalian cells due to the disulfide bond formation and reducing cytoplasm. Therefore, the production of functional recombinant antibodies in target cell line is necessary to be evaluated before used in therapeutic application such intrabodies against HIV-1. Methods: The work was to test expression of a single-chain variable fragment (scFv) antibody against HIV-1 Capsid p24 protein in a human mammalian-based expression system using HEK293T and Jurkat T cells as a model. Three expression plasmid vectors expressing scFv 183-H12-5C were generated and introduced into HEK293T. Expression of the scFv was analyzed, while ELISA and immunoblotting analysis verified its binding. The evaluation of the recombinant antibody was confirmed by HIV-1 replication and MAGI infectivity assay in Jurkat T cells. Results: Three plasmid vectors expressing scFv 183-H12-5C was successfully engineered in this study. Recombinant antibodies scFv (~29 kDa) and scFv-Fc (~52 kDa) in the cytoplasm of HEK293T were effectively obtained by transfected the cells with engineered pCDNA3.3-mu-IgGk-scFv 183-H12-5C and pCMX2.5-scFv 183-H12-5C-hIgG1-Fc plasmid vectors respectively. scFv and scFv-Fc are specifically bound recombinant p24, and HIV-1 derived p24 (gag) evaluated by ELISA and Western blot. Jurkat T cells transfected by pCDNA3.3-scFv 183-H12-5C inhibit the replication-competent NL4-3 viral infectivity up to 60%. Conclusion: Anti-p24 scFv 183-H12-5C antibody generated is suitable to be acted as intrabodies and may serve as a valuable tool for the development of antibody-based biotherapeutics against HIV-1.
format Article
author Che Omar, Mohammad Tasyriq
author_facet Che Omar, Mohammad Tasyriq
author_sort Che Omar, Mohammad Tasyriq
title Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells
title_short Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells
title_full Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells
title_fullStr Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells
title_full_unstemmed Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells
title_sort expression of functional anti-p24 scfv 183-h12-5c in hek293t and jurkat t cells
publisher Tabriz University of Medical Sciences
publishDate 2017
url http://eprints.usm.my/36956/1/%28Expression_of_Functional%29_APB-7-299.pdf
http://eprints.usm.my/36956/
https://www.ncbi.nlm.nih.gov/pubmed/28761833
_version_ 1643708932104912896
score 13.211869

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