120 IDENTIFICATION OF THE RELATIVELY PREDOMINANT BUFFALO INTERFERON tau ISOFORM AND ITS EXPRESSION IN ESCHERICHIA COLI

Abstract

The maternal pregnancy recognition factor interferon tau (IFN-τ) is expressed in multiple isoforms in all pecoran ruminant species. Interferon-τ, as the first pregnancy signaling molecule, performs a significant role in implantation as well as establishment of pregnancy. Due to low reproductive efficiency of buffalo compared with bovine and IFN-τ being the key molecule of reproductive physiology in ruminants, the objective of our study was framed to identify the various IFN-τ transcripts in buffalo embryonic trophoblast cells, to know their relative abundance to identify the relatively predominant isoform, and lastly to clone and express it in a heterologous host. Following total cellular RNA extraction from primary trophectodermal cells, RT-PCR was performed using gene-specific primers designed against known bovine IFN-τ sequence. Cloning of the amplified product and screening of the recombinant colonies gave 13 distinct cDNA variants that encoded for 8 distinct buffalo IFN-τ isoforms. These buffalo IFN-τ isoforms have a greater nucleotide and amino acid homology with caprine IFN-τ (98–100% and 96–100%) than ovine (94–97% and 90–95%) and bovine (89.6–90.6% and 82–86%), respectively. The novel buffalo IFN-τ isoforms showed pronounced nucleotide and amino acid sequence identity with one another (99.1–99.8% and 98–99%) but only moderate identity with previously identified buffalo IFN-τ (90–92% and 82–86%). All the 13 transcript sequences were accepted in GenBank. Out of 8 isoforms, buffalo IFN-τ1 has been found to be the relatively predominant, which was subcloned into expression vector pET 22b without signal sequence from pJET cloning vector and expressed in competent BL21 (DE3) Escherichia coli strain. Expression of the recombinant protein in soluble form was induced by isopropyl β-D-1-thiogalactopyranoside (0.1 mM) at 30°C for 6 h. The recombinant BuIFN-τ obtained was confirmed by Western blot using anti-HIS antibody. A new 20-kDa protein was detected coinciding the molecular weight of IFN-τ reported earlier in literature. In conclusion, the current study revealed that there are 8 different isoforms of IFN-τ that are expressed in trophectodermal out-growths during early pregnancy of buffalo. Predominantly found isoform IFN-τ 1 was expressed in pET 22b vector, and recombinant soluble protein was confirmed by Western blot.