Abi Ghanem, Daad Ali (2006-08). Phage display selection of recombinant antibodies derived from a chicken immune library against cryopreserved Eimeria tenella sporozoites. Doctoral Dissertation.
Thesis
An antibody library against Eimeria tenella sporozoites was constructed by phage display. Total RNA was isolated from the spleen, bone marrow, and ceca of immune chickens, and was used to reverse-transcribe cDNA. Heavy and light antibody variable genes were amplified from cDNA by the Polymerase Chain Reaction (PCR), using primer pairs that contain complementary sequences encoding a short linker sequence. The single-chain antibody fragment (scFv) was obtained by a secondary overlap PCR with primers that incorporate SfiI restriction sites, thus allowing for subsequent cloning into the phagemid vector pComb3X. Vector and scFv insert were digested with SfiI, ligated, and transformed into competent XL1-Blue Escherichia coli cells by electroporation, yielding a library with 7.4 x 107 total transformants. The culture was grown under carbenicillin selective pressure, rescued with helper phage, and the antibody-displaying phage was precipitated by PEG/NaCl, and subsequently used for panning. Five panning rounds were performed using cryopreserved E. tenella sporozoites, with a gradual increase of washing stringency to select for specific, highaffinity binders. A 1000-fold increase in phage output was obtained after 3 rounds of panning. There was clear enrichment of the positive clones over the panning rounds, with the 3rd round resulting in a 3,000-fold enrichment over the first one, as the binding clones became the dominant population in the library. Selected antibodies from the last round of panning were sequenced and characterized by immunoblotting. Soluble antibody fragments were produced in a non-suppressor E. coli strain, and recognized a 66-KDa sporozoite antigen on a Western blot. Primary cultures of chicken enterocytes were prepared in the hope of serving for invasion assays with E. tenella sporozoites. The isolation procedure, however, proved to be cumbersome and time-consuming. Future investigations will focus on purification and further characterization of antibodies selected from the constructed library. Such antibodies can be tested, alone or in combination, for their ability to block in vitro the invasion mechanism of E. tenella.
An antibody library against Eimeria tenella sporozoites was constructed by phage display. Total RNA was isolated from the spleen, bone marrow, and ceca of immune chickens, and was used to reverse-transcribe cDNA. Heavy and light antibody variable genes were amplified from cDNA by the Polymerase Chain Reaction (PCR), using primer pairs that contain complementary sequences encoding a short linker sequence. The single-chain antibody fragment (scFv) was obtained by a secondary overlap PCR with primers that incorporate SfiI restriction sites, thus allowing for subsequent cloning into the phagemid vector pComb3X. Vector and scFv insert were digested with SfiI, ligated, and transformed into competent XL1-Blue Escherichia coli cells by electroporation, yielding a library with 7.4 x 107 total transformants. The culture was grown under carbenicillin selective pressure, rescued with helper phage, and the antibody-displaying phage was precipitated by PEG/NaCl, and subsequently used for panning. Five panning rounds were performed using cryopreserved E. tenella sporozoites, with a gradual increase of washing stringency to select for specific, highaffinity binders. A 1000-fold increase in phage output was obtained after 3 rounds of panning. There was clear enrichment of the positive clones over the panning rounds, with the 3rd round resulting in a 3,000-fold enrichment over the first one, as the binding clones became the dominant population in the library. Selected antibodies from the last round of panning were sequenced and characterized by immunoblotting. Soluble antibody fragments were produced in a non-suppressor E. coli strain, and recognized a 66-KDa sporozoite antigen on a Western blot. Primary cultures of chicken enterocytes were prepared in the hope of serving for invasion assays with E. tenella sporozoites. The isolation procedure, however, proved to be cumbersome and time-consuming. Future investigations will focus on purification and further characterization of antibodies selected from the constructed library. Such antibodies can be tested, alone or in combination, for their ability to block in vitro the invasion mechanism of E. tenella.
