ENGINEERING OF A SINGLE-CHAIN VARIABLE-FRAGMENT (SCFV) ANTIBODY SPECIFIC FOR THE STOLBUR PHYTOPLASMA (MOLLICUTE) AND ITS EXPRESSION IN ESCHERICHIA-COLI AND TOBACCO PLANTS

From a hybridoma cell line (2A10) producing an immunoglobulin G1 direc ted against the major membrane protein of the stolbur phytoplasma, we have engineered scFv (single-chain variable fragment) antibodies from the variable heavy (VH) and light (VL) domains of the immunoglobulin. The scFv gene was cloned and expressed in Escherichia coli. The expres sed protein of 30 kDa could be recovered from the periplasmic fraction of the bacterial cells and was shown to be fully functional toward it s phytoplasmal antigen, since enzyme-linked immunosorbent assay or imm unofluorescence (IF) detection of the stolbur phytoplasma antigen by t he scFv was identical to that of the native immunoglobulin. The scFv g ene was then cloned in plasmid pBG-dAb-BIN of Agrobacterium tumefacien s to transform tobacco plants. The transformed plants were screened by PCR and Northern blotting for the presence and expression of the tran sgene, respectively, and by IP for expression of the scFv. One transge nic tobacco line, 1A6, was selected for challenge inoculation with the stolbur phytoplasma. When grafted on a stolbur phytoplasma-infected t obacco rootstock, the transgenic tobacco shoots grew free of symptoms and flowered after 2 months, while normal tobacco shoots showed severe stolbur symptoms during the same period and eventually died.