Cloning of porcine interleukin (IL)-12 receptor beta2 (IL-12Rbeta2) gene and its application to a rapid biological assay for human/porcine IL-12.
Porcine IL-12Rbeta2 gene was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs), and its complete nucleotide sequence was determined. To confirm the biological function, the entire open reading frame (ORF) was re-cloned into a mammalian expression vector, pcDNA3.1/Zeo(+), at the downstream of CMV promoter, and introduced to a Th1-like human lymphoma cell line, Jurkat E6-1. Antibiotic-resistant cells retaining the expression construct were selected then, isolated by the limiting dilution method. An established clone (10B10) constitutively expressed chimeric IL-12Rs composed of intrinsic (human) beta1 and extrinsic (porcine) beta2 subunits, and produced interferon (IFN)-gamma in response to IL-12 of both species with optimal PHA/PMA stimulation. The production of IFN-gamma was observed as early as 42 h after culture and appeared to be dose-dependent within the range between 20 and 2000 pg/ml. Thus, this clone not only reacts with IL-12 of both species but also provides a useful tool for quick and sensitive detection of IL-12 bioactivity.
Kokuho
Takehiro
T
Laboratory of Comparative Immunology, Department of Immunology, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. takehiro@afrc.go.jp
Inumaru
Shigeki
S
Watanabe
Satoko
S
Kubota
Takayuki
T
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D018664:Interleukin-12 / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018123:Receptors, Interleukin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D053707:Receptors, Interleukin-12; D017386:Sequence Homology, Amino Acid; D013552:Swine / Q000235:genetics*
C508438:IL12RB1 protein, human; C508439:IL12RB2 protein, human; D012333:RNA, Messenger; D018123:Receptors, Interleukin; D053707:Receptors, Interleukin-12; D018664:Interleukin-12