Expression of Functional Antip24 scFv 183H125C in HEK293T and Jurkat T Cells

Purpose: More than half of the diagnostic and therapeutic recombinant protein production depends on mammalianbased 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 HIV1. Methods: The work was to test expression of a singlechain variable fragment (scFv) antibody against HIV1 Capsid p24 protein in a human mammalianbased expression system using HEK293T and Jurkat T cells as a model. Three expression plasmid vectors expressing scFv 183H125C 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 HIV1 replication and MAGI infectivity assay in Jurkat T cells. Results: Three plasmid vectors expressing scFv 183H125C was successfully engineered in this study. Recombinant antibodies scFv (~29 kDa) and scFvFc (~52 kDa) in the cytoplasm of HEK293T were effectively obtained by transfected the cells with engineered pCDNA3.3muIgGkscFv 183H125C and pCMX2.5scFv 183H125ChIgG1Fc plasmid vectors respectively. scFv and scFvFc are specifically bound recombinant p24, and HIV1 derived p24 (gag) evaluated by ELISA and Western blot. Jurkat T cells transfected by pCDNA3.3scFv 183H125C inhibit the replicationcompetent NL43 viral infectivity up to 60%. Conclusion: Antip24 scFv 183H125C antibody generated is suitable to be acted as intrabodies and may serve as a valuable tool for the development of antibodybased biotherapeutics against HIV1.