Expression of functional recombinant antibody molecules in insect cell expression systems

Area of Science:

• Molecular biology
• Biotechnology
• Plant virology

Background:

• African cassava mosaic virus (ACMV) poses a significant threat to cassava production.
• Development of rapid and sensitive diagnostic tools for ACMV is crucial for disease management.
• Recombinant antibody fragments offer potential for improved diagnostic assays.

Purpose of the Study:

• To construct and express recombinant single-chain variable fragment (scFv) molecules targeting ACMV.
• To investigate the efficacy of different expression systems for scFv production and secretion.
• To develop a diagnostic assay for ACMV detection in plant tissues using secreted scFv.

Main Methods:

• Construction of scFv molecules from a monoclonal antibody against ACMV.
• Expression of scFv in Escherichia coli, insect cell lines (using baculovirus), and Drosophila DS2 cells.
• Genetic manipulation to enhance scFv secretion, including the use of a honeybee melittin leader sequence and Ckappa domain fusion.
• Detection of ACMV using enzyme-linked immunosorbent assay (ELISA) with secreted scFv.

Main Results:

• High levels of scFv were produced in insect cell lines, but not secreted.
• Stable recombinant Drosophila DS2 cell lines were established, secreting functional scFv into the culture medium.
• Secreted scFv from Drosophila cells were directly used in ELISA for ACMV detection in plant tissues.
• Fusion of the scFv with a human IgG Ckappa domain enhanced secretion and ELISA activity.

Conclusions:

• Drosophila DS2 cells provide a suitable system for the secretion of functional scFv antibodies.
• Secreted scFv antibodies can be directly applied in ELISA for efficient ACMV detection.
• The Ckappa domain fusion strategy enhances the utility of scFv for diagnostic applications.