CLONING OF ANTIFUNGAL ANTIBODY GENES
V MANATUNGA1, H SANATI2 and PA O'BRIEN1
1School of Biological Sciences and Biotechnology, and WA State Agricultural Biotechnology Centre, Murdoch University, Murdoch, WA 6150, Australia; 2National Research Centre for Genetic Engineering and Biotechnology, PO Box 14155-6343, Tehran, Iran
Background and objectives
Genes for antibodies active against fungal enzymes can potentially be used to engineer resistance in the same way as pectinase inhibitors. Antibody genes can now be cloned and expressed in a variety of organisms including plants. To test this we are investigating the placement of genes for antipectinase antibodies into plants as a means of generating resistance to R. solani.
Materials and methods
Results and conclusions
Amplification of first-strand spleen cDNA produced correct size bands of immunoglobulin variable heavy (VH) and variable light (VL) chain genes in two separate reactions. Those VH and VL genes were joined together with a linker molecule to make a single chain variable fragment (ScFv), and a major band of the appropriate size for an assembled ScFv gene (750 bp) was cloned into the pCANTAB 5E phagemid vector. Phage displaying antipectinase ScFv antibodies were isolated by biopanning against immobilized pectinases. 192 colonies were isolated by this procedure. All of these gave positive results in ELISA tests against fungal enzymes. These clones are being used for production of soluble antibodies in E. coli.