Production, gene cloning, and expression in maize of antibodies with inhibitory activity to corn stunt spiroplasma

• Main Author: Chen, Yangde
• Format: Dissertation
• Language: English
• Subjects: agriculture, plant pathology; biology, molecular
• ISBN: 0599141409; 9780599141407

In an attempt to develop an effective control method for corn stunt disease, we sought to express antibodies with inhibitory activity to corn stunt spiroplasm. (CSS, Spiroplasma kunkelii) in maize to influence the CSS infection. A panel of monoclonal antibodies specific to CSS were produced using standard hybridoma production technology. By screening these monoclonal antibodies using spiroplasma deformation, metabolism inhibition and growth inhibition tests, seven monoclonal antibodies with good inhibitory activity to CSS were selected. A protein of approximately 33 KD from CSS was recognized by the selected monoclonal antibodies in western blot.
To facilitate in vitro characterization and subsequent maize transformation, the variable heavy (VH) and light chain (V L) genes of the selected monoclonal antibodies were amplified by PCR, and the fragments of the variable genes were joined together as a single chain (scFv) through a DNA linker. The constructed scFv genes were expressed both as phage fusion proteins on the phage surface and soluble proteins in E. coli. CSS-positive scFv antibodies were screened by ELISA and their specificity was confirmed by competition ELISA. An E. coli-expressed E7-scFv antibody was shown to be more effective in inhibiting CSS compared to other positive scFv antibodies. DNA sequence analysis indicated that E7-scFv has longer CDR3 and CDR1 domains for heavy and light chains, respectively, as compared to several other positive scFv clones.
To determine whether the E7-scFv would be useful to induce resistance to CSS infection in plants, this E7-scFv gene was cloned into a plant expression vector under the control of maize ubiquitin gene 1 (Ubi1) promoter. The resulting construct was introduced into maize cells through particle bombardment. One transformant, E7-1, was genetically confirmed to express functional anti-CSS scFv antibody. Transgenic maize plants that regenerated from E7-1 had no influence on CSS infection when challenged with CSS in the greenhouse using infective corn leafhoppers, Dalbulus maidis. The failure of the transgenic maize plants in developing resistance to CSS infection might mainly ascribe to the low affinity of the scFv antibody and compartmentalization of the plant-expressed scFv antibody precluding antibody interference with pathogen.