Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco
To explore the feasibility of employing antibodies to obtain disease resistance against plant root pathogens, we have studied the expression of genes encoding antibodies in roots of transgenic plants. A model monoclonal antibody was used that binds to a fungal cutinase. Heavy and light chain cDNAs were amplified by PCR, fused to a signal sequence for secretion and cloned behind CaMV 35S and TR2′ promoters in a single T-DNA. The chimeric genes were cloned both in tandem and in a divergent orientation. The roots of tobacco plants transformed with these constructs produced antibodies that were able to bind antigen in an ELISA. Immunoblotting showed assembly to a full-size antibody. In addition, a F(ab′)2-like fragment was observed, which is probably formed by proteolytic processing. Both antibody species were properly targeted to the apoplast, but the full-size antibody was partially retained by the wall of suspension cells. The construct with divergent promoters showed a better performance than the construct with promoters in tandem. It directed the accumulation of functional antibodies to a maximum of 1.1% of total soluble protein, with half of the plants having levels higher than 0.35%. The high efficiency of this construct probably results from coordinated and balanced expression of light and heavy chain genes, as evidenced by RNA blot hybridization.
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article/Letter to editor biblioteca |
| Language: | English |
| Subjects: | Antibody, coordinated expression, genetic engineering, protein assembly, root, secretion, |
| Online Access: | https://research.wur.nl/en/publications/coordinate-expression-of-antibody-subunit-genes-yields-high-level |
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| Summary: | To explore the feasibility of employing antibodies to obtain disease resistance against plant root pathogens, we have studied the expression of genes encoding antibodies in roots of transgenic plants. A model monoclonal antibody was used that binds to a fungal cutinase. Heavy and light chain cDNAs were amplified by PCR, fused to a signal sequence for secretion and cloned behind CaMV 35S and TR2′ promoters in a single T-DNA. The chimeric genes were cloned both in tandem and in a divergent orientation. The roots of tobacco plants transformed with these constructs produced antibodies that were able to bind antigen in an ELISA. Immunoblotting showed assembly to a full-size antibody. In addition, a F(ab′)2-like fragment was observed, which is probably formed by proteolytic processing. Both antibody species were properly targeted to the apoplast, but the full-size antibody was partially retained by the wall of suspension cells. The construct with divergent promoters showed a better performance than the construct with promoters in tandem. It directed the accumulation of functional antibodies to a maximum of 1.1% of total soluble protein, with half of the plants having levels higher than 0.35%. The high efficiency of this construct probably results from coordinated and balanced expression of light and heavy chain genes, as evidenced by RNA blot hybridization. |
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