|YUAN, QING - China Medical University|
|BRLANSKY, RONALD - University Of Florida|
|MINENKOVA, OLGA - National Council Of Agricultural Research (CRA)|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/16/2010
Publication Date: 6/1/2010
Citation: Yuan, Q., Jordan, R.L., Brlansky, R.H., Minenkova, O., Hartung, J.S. 2010. Selection of single chain variable fragments (scFv) against Xylella fastidiosa subsp. pauca by phage display. Phytopathology 100(6):S43.
Technical Abstract: Xylella fastidiosa is a gram-negative member of the gamma proteobacteria. Xylella fastidiosa subsp pauca causes citrus variegated chlorosis in Brazil and enjoys ‘select agent’ status in the United States. Antibody based detection assays are commercially available for Xylella fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against Xylella fastidiosa subsp pauca strain 9a5c (citrus) by using phage display technology. BALB/c mice were immunized with 9a5c bacteria at a concentration of 108 cfu/100 ul buffer. mRNA from the spleens of the immunized mice was purified and converted into cDNA. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (VH) and 21 primers for the light chain variable region (VL). The VH and VL were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×107 independent clones with full-length scFv inserts. In each of 3 cycles of affinity-selection with 9a5c, about 1.0×1012 phage were used for panning with 4.1×106, 7.1×106, 2.1×107 phage recovered after the first, second and third cycles respectively. 66% of clones from the final library bound Xylella fastidiosa 9a5c. Some of these phage that expressed scFv recognized strain 9a5c but did not recognize Xylella fastidiosa strains that cause Pierce’s disease of grapevine.