DNA versus protein immunisation for production of monoclonal antibodies against Choristoneura fumiferana ecdysone receptor (CfEcR).
The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S-transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native CfEcR-B protein induced by 20E in CF-203 insect cells. DNA immunisation was shown to overcome the solubility problem of the bacterial expressed EcR and created a more direct route for monoclonal antibody production for this receptor protein obviating the need for EcR expression and purification to generate the antigen.
Yang
Dan-Hui
DH
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Makhmoudova
Amina
A
Arif
Basil M
BM
Feng
Qili
Q
Retnakaran
Arthur
A
Palli
Subba Reddy
SR
Krell
Peter J
PJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D009036:Moths / Q000276:immunology*; D011987:Receptors, Steroid / Q000235:genetics / Q000276:immunology*; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000276:immunology*; D022223:Vaccines, Subunit / Q000008:administration & dosage / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000276:immunology
D000911:Antibodies, Monoclonal; D011987:Receptors, Steroid; D019444:Vaccines, DNA; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; C055469:ecdysone receptor