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 variable region (V(lambda)J(lambda)) nucleotide coding sequence was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) from abomasal lymph node (ALN) B cells of immune sheep challenged with the gastrointestinal nematode parasite Haemonchus contortus. Single-chain antibodies (scFv) were then constructed with the purified V(H)DJ(H) and V(lambda)J(lambda) Ig gene 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'-NotI restriction endonuclease sites were added for cloning into a phagemid expression vector. Expression of sheep scFv from pHFA phagemid in an amber-suppresser 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 in E. coli cell lysate and on purified library phage. In addition, four rounds of scFv-library selection against H. contortus surface antigen resulted in a 300-fold increase in the elution titre of phage recovered from parasite surface antigen. Nearly 1000 of the selected and eluted scFvs were expressed in an attempt to identify monoclonal sheep scFv against parasite antigen. Only low affinity clones were isolated during screening of this sheep scFv-library, suggesting different strategies will be needed for isolation of specific high affinity recombinant antibody in future studies.
White
G P
GP
The Centre for Animal Biotechnology, School of Veterinary Science, The University of Melbourne, Parkville, 3052, Vic., Australia. gregoryw@ichr.uwa.edu.au
Meeusen
E N
EN
Newton
S E
SE
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D016384:Consensus Sequence; D018509:DNA, Helminth / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D015723:Gene Library; D006188:Haemonchiasis / Q000276:immunology / Q000662:veterinary*; D006189:Haemonchus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D007146:Immunoglobulin lambda-Chains / Q000235:genetics / Q000276:immunology; D008198:Lymph Nodes / Q000276:immunology; D008969:Molecular Sequence Data; D018510:RNA, Helminth / Q000737:chemistry / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D012757:Sheep Diseases / Q000276:immunology* / Q000469:parasitology
D000909:Antibodies, Helminth; D018509:DNA, Helminth; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007146:Immunoglobulin lambda-Chains; D018510:RNA, Helminth