Buffalo (Bubalus bubalis) interleukin-12: analysis of expression profiles and functional cross-reactivity with bovine system.
Interleukin-12, a heterodimeric pro-inflammatory cytokine, from water buffaloes (Bubalus bubalis) was analyzed for its for its tissue specific expression and functionality. Concanavalin A stimulated splenocytes displayed an up-regulation of the IL-12 p40 subunit 8-24h post-stimulation, whereas the p35 subunit did not show any quantitative variation at different time intervals. Basal level expressions of both the subunits were observed by RT-PCR in spleen. In addition p40 transcripts could be detected in liver and p35 in brain and muscle tissues as well in very low levels. Functional recombinant buffalo IL-12 was expressed in HEK 293T cells as a heterodimer using foot-and-mouth disease virus 2A polypeptide as a linker. Culture supernatants from transfected cells contained a hetero-dimeric p70 subunit as revealed in western blot of the proteins separated by native polyacrylamide gel electrophoresis (PAGE) using a monoclonal antibody against bovine IL-12 p40. IL-12 containing culture supernatant induced production of nitric oxide in cultured splenocytes of both buffalo and bovine origin. Our study reveals that buffalo IL-12, which shares a high-level sequence identity with bovine IL-12, also has functional cross-reactivity with the bovine immune cells.
Premraj
Avinash
A
Animal Biotechnology Laboratory, Rajiv Gandhi Center for Biotechnology (RGCB), Jagathy, Thycaud PO, Trivandrum 695014, Kerala, India.
Sreekumar
E
E
Jain
Mamta
M
Rasool
T J
TJ
D000818:Animals; D002020:Buffaloes / Q000276:immunology*; D002417:Cattle; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D019281:Dimerization; D020869:Gene Expression Profiling; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D053762:Interleukin-12 Subunit p40; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D021122:Protein Subunits / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014018:Tissue Distribution; D014158:Transcription, Genetic; D019076:Transgenes; D015854:Up-Regulation
D053762:Interleukin-12 Subunit p40; D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003208:Concanavalin A; D018664:Interleukin-12; D009569:Nitric Oxide