isopropylβ-D-thio galactosidase (IPTG) and contains a human
Cκdomain downstream of the cloned antibody fragment that
facilitates immunodetection and purification of the recombi-
nant protein, and also improves its solubility.2.7 Oligonucleotides Degenerate nucleotide code (M¼A/C, R¼A/G, W¼A/T,
S¼G/C, Y¼C/T, K¼G/T).
2.7.1 Routine PCR
Reactions and Sequencing
- AH18 REV: 5^0 -AAA TAC CTA TTG CCT ACG GCA GCC
GCT GG-3^0. - GIII FOR: 5^0 -GAA TTT TCT GTA TGA GGT TTT GC-3^0.
- HuCκforward: 5^0 -GAA GAT GAA GAC AGA TGG TGC-3^0.
2.7.2 Sheep Single Chain
Fv Phage Display Library
Construction
- Ovine heavy chain constant region 3^0 primer.
(a) OvCHFOR: 5^0 -GAC TTT CGG GGC TGT GGT GGA
GGC-3^0. - Ovine kappa chain constant region 3^0 primer.
(a) OvCKFOR: 5^0 -GA TGG TTT GAA GAG GGA GAC
GGA TGG CTG AGC-3^0. - Ovine lambda chain constant region 3^0 primer.
(a) OvCLFOR: 5^0 -A CAG GGT GAC CGA GGG TGC GGA
CTT GG-3^0. - Ovine heavy chain variable region 5^0 primers.
(a) OvVH1BACK: 5^0 -CAG GTK CRR CTG CAG GRG
TCG GG-3^0.
(b) OvVH2BACK: 5^0 -CAG GTK CAG YTK CAG GAG TCG
GG-3^0.
(c) MuVH1BACK: 5^0 -SAG GTS MAR CTG CAG SAG TCW
GG-3^0.
(d) Hu4aBACK: 5^0 -CAG GTG CAG CTG CAG GAG TCG
GG-3^0. - Ovine heavy chain variable region 3^0 primers.
(a) OvJH1LINKFOR: 5^0 -CTC AGA AGG CGC GCC AGA
AGA TTT ACC TTC TGA GGA GAC GGT GAC CAG
GAG TCC-3^0.
(b) OvJH2LINKFOR: 5^0 -CTC AGA AGG CGC GCC AGA
AGA TTT ACC TTC TGA GGA GRC GGW GAY YAG
KAG TCC-3^0.
(c) OvJH3LINKFOR: 5^0 -CTC AGA AGG CGC GCC AGA
AGA TTT ACC TTC TGA GGA GAY RGT RAS CAG
GAS TCC-3^0.
(d) OvJH4LINKFOR: 5^0 -CTC AGA AGG CGC GCC AGA
AGA TTT ACC TTC TGA AAG AAC GCT GAT CAG
GAG-3^0.
Monoclonal Antibodies Inactivating AHL Signal Molecules 331