A single-chain variable region immunoglobulin library from the abomasal lymph node of sheep infected with the gastrointestinal nematode parasite Haemonchus contortus

Sheep immunoglobulin (Ig) heavy-chain (V(H)DJ(H)) and lambda light-chain va riable region (V(lambda)J(lambda)) nucleotide coding sequence was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) from abomasal l ymph node (ALN) B cells of immune sheep challenged with the gastrointestina l nematode parasite Haemonchus contortus. Single-chain antibodies (scFv) we re then constructed with the purified V(H)DJ(H) and V(lambda)J(lambda) Ig g ene region DNA using oligonucleotides to PCR and join the variable regions to a central [Gly(4)Ser](3)-linker. In a similar fashion 5'-SfiI and 3'-Not I restriction endonuclease sites were added for cloning into a phagemid exp ression vector. Expression of sheep scFv from pHFA phagemid in an amber-sup presser strain of Escherichia coli, after infection with filamentous phage, resulted in 10(9) sheep scFv antibodies displayed as a library on phagemid particles. Western blot analysis demonstrated sheep scFv gene expression i n E. coli cell lysate and on purified library phage. In addition, four roun ds of scFv-library selection against H. contortus surface antigen resulted in a 300-fold increase in the elution titre of phage recovered from parasit e surface antigen. Nearly 1000 of the selected and eluted scFvs were expres sed in an attempt to identify monoclonal sheep scFv against parasite antige n. Only low affinity clones were isolated during screening of this sheep sc Fv-library, suggesting different strategies will be needed for isolation of specific high affinity recombinant antibody in future studies. (C) 2001 El sevier Science B.V. All rights reserved.