title: Antibody against biologically active p40 subunit of porcine interleukin-12 expressed in Escherichia coli.
abstract: A truncated p40 subunit of porcine interleukin-12 (pIL-12) gene without the N-terminal signal peptide sequence was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1. The resulting recombinant plasmid pGEX-IL12-40 was transformed into host cells BL21(DE3)pLysS, and the expression of the p40 subunit was induced using isopropyl β-D-thiogalactoside (IPTG). An anti-p40 polyclonal antibody was generated by immunizing a rabbit with the purified protein. Immunoreactivities of the p40 protein and the antibody were confirmed by immunoblotting. At the same time, a recombinant plasmid expressing the entire pIL-12 consisting of p35 and p40 genes was constructed by splicing by overlap extension (SOE)-PCR and transiently transfected into BHK-21 cells. Expression of p40 subunit on the surface of the transfected cells was identified using the anti-p40 antibody. The p40 protein and the specific antibody are biologically active and can be used as detecting reagents.
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Xiangfang District, Harbin, China.
authors.forename: Xunliang
authors.initials: X
authors.lastname: Li
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Ren
mesh_terms: D000818:Animals; D000917:Antibody Formation / Q000276:immunology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D019084:Fluorescent Antibody Technique, Indirect; D053762:Interleukin-12 Subunit p40 / Q000276:immunology* / Q000302:isolation & purification*; D007544:Isopropyl Thiogalactoside; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011817:Rabbits; D013552:Swine; D014162:Transfection
chemical_list: D017931:DNA Primers; D053762:Interleukin-12 Subunit p40; D007544:Isopropyl Thiogalactoside