Saugandhika Shrabani, Sharma Vishal, Malik Hrudananda, Mohapatra Sushil Kumar, Bondre Vijay P, Kumar Sudarshan, Mohanty Ashok Kumar, Malakar Dhruba
Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India.
National Institute of Virology, Pune, India.
Protein Expr Purif. 2016 Jun;122:8-14. doi: 10.1016/j.pep.2016.02.005. Epub 2016 Feb 11.
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.
干扰素τ（IFN-T）作为反刍动物母体识别妊娠（MRP）的信号分子。本研究的目的是鉴定水牛滋养层细胞中的各种水牛干扰素τ（BuIFN-T）转录本，将这些序列与水牛、牛、山羊和绵羊的已知mRNA序列进行系统发育比较，并表达和纯化重组BuIFN-T（rBuIFN-T）亚型。从滋养外胚层细胞中提取RNA后，使用Ifn-t基因特异性引物进行RT-PCR。鉴定出13种不同的cDNA变体，它们编码8种不同的BuIFN-T蛋白。将BuIFN-T1a2和BuIFN-T8在原核表达系统中于37°C、25°C和16°C下用1 mM IPTG表达12小时，在16°C下表达的重组蛋白通过固定化金属亲和色谱（IMAC）进行部分纯化。BuIFN-T亚型与山羊（98 - 100%，96 - 100%）、绵羊（94 - 97%，90 - 95%）和牛（89.6 - 90.6%，82 - 86%）具有更高的核苷酸和氨基酸同源性。这些新的BuIFN-T亚型彼此之间具有明显的核苷酸和氨基酸序列同一性（99.1 - 99.8%，98 - 99%），但与先前鉴定的水牛IFN-T具有中等序列同一性（90 - 92%，82 - 86%）。表达的重组亚型（rBuIFN-T1a2和rBuIFN-T8）在16°C时溶解度最高。总之，体外发育的水牛囊胚的滋养外胚层中存在13种不同的Ifn-t基因变体，它们编码8种不同的蛋白质。rBuIFN-T1a2和rBuIFN-T8在16°C下用1 mM IPTG在大肠杆菌表达系统中成功以可溶形式表达，所得重组蛋白通过IMAC进行部分纯化。
