208 CLONING AND CHARACTERIZATION OF BUFFALO INTERFERON-TAU AND EFFICACY OF RECOMBINANT BUFFALO INTERFERON-TAU FOR IN VITRO EMBRYO DEVELOPMENT

Abstract

Interferon tau (IFN-tau) is known as maternal pregnancy recognition factor in ruminants. IFN-tau not only acts as a signalling molecule of pregnancy recognition but also performs various functions for successful implantation and pregnancy establishment. The aim of the present study was to produce recombinant buffalo interferon-tau (BuIFN-Tau) and observe if it has any effect on in vitro embryo development. The BuIFN-Tau gene was obtained through polymerase chain reaction (PCR) from hatched buffalo blastocysts and was cloned into pJET cloning vector. Screening of the recombinant colonies gave 8 distinct buffalo IFN-tau isoforms, out of which the predominant buffalo IFN-t tau1 isoform (gene bank accession number JX481984), was subcloned into expression vector pET22b without signal sequence. The recombinant plasmid was induced to express the recombinant protein by isopropyl b-D-1-thiogalactopyranoside. Analysis of the products of recombinant BuIFN-tau without signal sequence by SDS–PAGE revealed a new 20-kDa protein coinciding with the molecular weight of IFN-tau as reported earlier in literature. The purified recombinant BuIFN-tau was confirmed by Western blot using anti-HIS antibody and was subjected to three steps of large-scale purification using HIS affinity chromatography, anion exchange chromatography, and gel filtration chromatography. Finally, a relatively pure histidine-tagged recombinant protein, which had a purity of at least 90%, was generated as confirmed through SDS. The concentration of recombinant BuIFN-tau was 1 mg mL^–1 by Bradford assay. The purified recombinant BuIFN-tau was used as supplement of the culture medium for IVF early buffalo embryos at the following concentrations: control, 1, 2, and 4 µg mL^–1. Sixty oocytes each in 4 groups (with 20 oocytes/drop in three replicates for each group) were used for in vitro maturation. After 24 h, the matured oocytes were incubated with in vitro capacitated sperm cells for 18 h; thereafter, the presumptive zygotes were cultured in IVC medium supplemented with 0, 1, 2, or 4 µg mL^–1 of the purified recombinant BuIFN-tau. The experiment was repeated 3 times. The data were analysed using SYSTAT 7.0 (SPSS Inc., Chicago, IL, USA) after arcsin transformation of percentage values. The differences were analysed by one-way ANOVA followed by Fisher's least significant difference test. Out of 3 concentrations of recombinant BuIFN-tau, the 2 µg mL^–1 concentration significantly promoted the rate of blastocyst development, 45.55% against 31.1% (control; P < 0.01). Blastocyst development rate for low and high concentrations was 29.97% and 10.18% respectively. It is concluded that the addition of 2 µg mL^–1 of recombinant BuIFN-tau enhances the blastocyst development rate in buffalo, and hence there is some evidence that BuIFN-tau has not only a role in maternal recognition of pregnancy but also in embryonic development.