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.