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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| Format: | Article |
| Language: | English |
| Published: |
Tabriz University of Medical Sciences
2017
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| Subjects: | |
| 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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| Summary: | 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. |
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