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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-1847 |
| Date | 02 June 2009 |
| Creators | Abi Ghanem, Daad Ali |
| Contributors | Berghman, Luc R. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | electronic, application/pdf, born digital |
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