title: Molecular characterization of IFN-T expressed in buffalo embryonic trophoblasts and expression of recombinant BuIFN-T1a2 and BuIFN-T8 isoforms in E. coli.
abstract: Interferon tau (IFN-T) acts as a signaling molecule for maternal recognition of pregnancy (MRP) in ruminants. Aim of the present study was to identify various Buffalo Interferon tau (BuIFN-T) transcripts in buffalo trophoblast, phylogenetic comparison of these sequences with known mRNA sequences of buffalo, bovine, caprine and ovine and to express and purify the recombinant BuIFN-T (rBuIFN-T) isoforms. Following RNA extraction from trophectodermal cells, RT-PCR was performed using Ifn-t gene specific primers. 13 distinct cDNA variants encoding eight different BuIFN-T proteins were identified. BuIFN-T1a2 and BuIFN-T8 were expressed in prokaryotic expression system at 37 °C, 25 °C and 16 °C with 1 mM IPTG for 12 h and the recombinant proteins expressed at 16 °C were partially purified by Immobilised Metal Affinity Chromatography (IMAC). BuIFN-T isoforms have greater nucleotide and amino acid homology with caprine (98-100%, 96-100%), ovine (94-97%, 90-95%) and bovine (89.6-90.6%, 82-86%). These novel BuIFN-T isoforms contained pronounced nucleotide and amino acid sequence identity with one another (99.1-99.8%, 98-99%) but moderate sequence identity with previously identified buffalo IFN-T (90-92%, 82-86%). Solubility of expressed recombinant isoforms (rBuIFN-T1a2 and rBuIFN-T8) was highest at 16 °C. In conclusion, 13 distinct Ifn-t gene variants exist in trophectoderm of in vitro developed buffalo blastocysts that encode eight different proteins. rBuIFN-T1a2 and rBuIFN-T8 were successfully expressed in soluble form in Escherichia coli expression system at 16 °C with 1 mM IPTG and the resulting recombinant proteins were partially purified by IMAC.
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India.
authors.forename: Shrabani
authors.initials: S
authors.lastname: Saugandhika
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India.
authors.forename: Vishal
authors.initials: V
authors.lastname: Sharma
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India.
authors.forename: Hrudananda
authors.initials: H
authors.lastname: Malik
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India.
authors.forename: Sushil Kumar
authors.initials: SK
authors.lastname: Mohapatra
authors.affiliation: National Institute of Virology, Pune, India.
authors.forename: Vijay P
authors.initials: VP
authors.lastname: Bondre
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India.
authors.forename: Sudarshan
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India.
authors.forename: Ashok Kumar
authors.initials: AK
authors.lastname: Mohanty
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India. Electronic address: dhrubamalakar@gmail.com.
authors.forename: Dhruba
authors.initials: D
authors.lastname: Malakar
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism; D002020:Buffaloes / Q000196:embryology / Q000235:genetics*; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006041:Goats; D007370:Interferon Type I / Q000235:genetics*; D010802:Phylogeny; D011257:Pregnancy Proteins / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D012756:Sheep; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D007370:Interferon Type I; D011257:Pregnancy Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; C020947:interferon tau
keywords: Buffalo; Cloning; Interferon tau; Isoform; Sequencing; Trophectodermal cells