Generation of monoclonal antibodies to porcine interleukin 6 (PIL-6) using the recombinant PIL-6 expressed in Escherichia coli.
Porcine interleukin-6 (PIL-6) protein without signal peptide was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The fusion protein was expressed in an insoluble fraction, however, it was solubilized by refolding procedure using urea. From the solubilized protein, the recombinant PIL-6 (rPIL-6) was purified by a batch method using glutathione sepharose 4B and PreScission protease cleavage. By the B3B1 hybridoma cell proliferation assay, biological activity of the purified rPIL-6 was confirmed. Three monoclonal antibodies (MAbs) named 2B-1, 5A-8 and 4C-3 were generated by using the rPIL-6 as an immunogen. Immunoglobulin isotypes of the MAbs were IgG2a (4C-3) and IgG2b (2B-1 and 5A-8). For the epitope analysis, additive enzyme-linked immunosorbent assay and immunoblot analysis using deletion mutants of PIL-6 were performed. These experiments revealed that the two MAbs (2B-1 and 5A-8) recognize an overlapped epitope and the other (4C-3) recognizes a distinct epitope, and all epitopes reside in the region of aa26-64 of PIL-6.
Watanabe
Rie
R
Department of Veterinary Hygiene, Yamaguchi University, Yoshida, Japan.
Hasegawa
Atsuhiko
A
Miyazawa
Takayuki
T
Kato
Hirotomo
H
Iwata
Hiroyuki
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D049109:Cell Proliferation; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000378:metabolism*; D005982:Glutathione Transferase; D015151:Immunoblotting; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013552:Swine / Q000276:immunology*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase