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

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          Abstract

          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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          Most cited references35

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          Fusion protein linkers: property, design and functionality.

          Xiaoying Chen, Jennica Zaro, Wei-Chiang Shen (2013)
          As an indispensable component of recombinant fusion proteins, linkers have shown increasing importance in the construction of stable, bioactive fusion proteins. This review covers the current knowledge of fusion protein linkers and summarizes examples for their design and application. The general properties of linkers derived from naturally-occurring multi-domain proteins can be considered as the foundation in linker design. Empirical linkers designed by researchers are generally classified into 3 categories according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers. Besides the basic role in linking the functional domains together (as in flexible and rigid linkers) or releasing the free functional domain in vivo (as in in vivo cleavable linkers), linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles. Copyright © 2012 Elsevier B.V. All rights reserved.
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            Characteristics of a human cell line transformed by DNA from human adenovirus type 5.

            F Graham, J. Smiley, W Russell … (1977)
            Human embryonic kidney cells have been transformed by exposing cells to sheared fragments of adenovirus type 5 DNA. The transformed cells (designated 293 cells) exhibited many of the characteristics of transformation including the elaboration of a virus-specific tumour antigen. Analysis of the polypeptides synthesized in the 293 cells by labelling with 35S-methionine and SDS PAGE showed a variable pattern of synthesis, different in a number of respects from that seen in otheruman cells. On labelling the surface of cells by lactoperoxidase catalysed radio-iodination, the absence of a labelled polypeptide analogous to the 250 K (LETS) glycoprotein was noted. Hybridization of labelled cellular RNA with restriction fragments of adenovirus type 5 DNA indicated transcription of a portion of the adenovirus genome at the conventional left hand end.
            Article 0 comments Cited 396 times – based on 0 reviews     Review now
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              HIV-1 Dynamics in Vivo: Virion Clearance Rate, Infected Cell Life-Span, and Viral Generation Time

              A S Perelson, A Neumann, Steven Markowitz … (1996)
              A new mathematical model was used to analyze a detailed set of human immunodeficiency virus-type 1 (HIV-1) viral load data collected from five infected individuals after the administration of a potent inhibitor of HIV-1 protease. Productively infected cells were estimated to have, on average, a life-span of 2.2 days (half-life t 1/2 = 1.6 days), and plasma virions were estimated to have a mean life-span of 0.3 days (t 1/2 = 0.24 days). The estimated average total HIV-1 production was 10.3 x 10(9) virions per day, which is substantially greater than previous minimum estimates. The results also suggest that the minimum duration of the HIV-1 life cycle in vivo is 1.2 days on average, and that the average HIV-1 generation time--defined as the time from release of a virion until it infects another cell and causes the release of a new generation of viral particles--is 2.6 days. These findings on viral dynamics provide not only a kinetic picture of HIV-1 pathogenesis, but also theoretical principles to guide the development of treatment strategies.
              Article 0 comments Cited 325 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Adv Pharm Bull
                Journal ID (iso-abbrev): Adv Pharm Bull
                Journal ID (publisher-id): Adv Pharm Bull
                Journal ID (pmc): APB
                Journal ID (publisher-id): TBZMED
                Title: Advanced Pharmaceutical Bulletin
                Publisher: Tabriz University of Medical Sciences
                ISSN (Print): 2228-5881
                ISSN (Electronic): 2251-7308
                Publication date (Print): June 2017
                Publication date (Electronic): 30 June 2017
                Volume: 7
                Issue: 2
                Pages: 299-312
                Affiliations
                1Cluster of Oncology and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200 Kepala Batas, Pulau Pinang, Malaysia.
                2Biology Program, School of Distance Education, Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia.
                Author notes
                [* ] Corresponding author: Mohammad Tasyriq Che Omar, Tel: 604-6535930, Fax: 604-6576000, mtasyriq@ 123456usm.my
                Article
                DOI: 10.15171/apb.2017.036
                PMC ID: 5527245
                SO-VID: dc81b316-8e2f-484b-a539-1e8b4d8968c0
                Copyright © ©2017 The Authors.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers.

                History
                Date received : 20 April 2017
                Date revision received : 06 June 2017
                Date accepted : 08 June 2017
                Page count
                Figures: 7, Tables: 3, References: 41, Pages: 14
                Categories
                Subject: Research Article

                Keywords: single-chain variable fragment (scfv),scfv-fc,hiv-1 capsid p24 protein,mammalian-based expression,intrabody
                Data availability:
                Keywords: single-chain variable fragment (scfv), scfv-fc, hiv-1 capsid p24 protein, mammalian-based expression, intrabody

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