title: Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferon-tau for in vitro embryo development.
abstract: The aim of our study was to optimize growth and induction parameters, for expression and large scale purification of functionally active buffalo interferon tau, and to study its possible impact on in vitro blastocyst development. The buffalo interferon-tau gene (BuIFN-T1) bearing gene bank accession No. JX481984, with signal sequence, was obtained through polymerase chain reaction (PCR) from bovine early embryos and was cloned into pJET vector. After being verified, the fragments without signal sequence, were inserted into the expression vector pET-22b and the recombinant plasmid was induced to express the recombinant protein in a prokaryotic expression system. The recombinant BuIFN-T was confirmed by SDS-PAGE and Western blot and subjected to three steps of large scale purification using His Affinity chromatography, Anion Exchange chromatography and Gel Filtration chromatography. The purified recombinant BuIFN-T protein was validated by mass spectroscopy analysis. To examine the effect of recombinant BuIFN-T protein on developmental competency of buffalo embryos, purified recombinant BuIFN-T protein was added to in vitro embryo culture medium (at concentration of 0, 1μg/ml, 2μg/ml, 4μg/ml) for 9days. Addition of recombinant BuIFN-T (2μg/ml) significantly improved the rate of blastocyst production, 45.55% against 31.1% control (p<0.01). Here we conclude that the recombinant BuIFN-T was successfully purified to homogeneity from a prokaryotic expression system and it significantly increased the blastocyst production rate in buffalo. These findings suggest a potential impact of IFN-T in promoting embryonic growth and development.
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: Sikander
authors.initials: S
authors.lastname: Saini
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India.
authors.forename: Sudam
authors.initials: S
authors.lastname: Bag
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, GADVASU, Ludhiana, Punjab, India.
authors.forename: Niraj Kumar
authors.initials: NK
authors.lastname: Singh
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 / Q000737:chemistry; D001755:Blastocyst / Q000378:metabolism; D002020:Buffaloes / Q000196:embryology*; D002417:Cattle; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D047108:Embryonic Development; D004664:Encephalitis Virus, Japanese / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D007370:Interferon Type I / Q000096:biosynthesis / Q000737:chemistry*; D008297:Male; D013058:Mass Spectrometry; D009865:Oocytes / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011257:Pregnancy Proteins / Q000096:biosynthesis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013094:Spermatozoa / Q000378:metabolism; D013696:Temperature; D014709:Vero Cells
chemical_list: D000998:Antiviral Agents; D007370:Interferon Type I; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; C020947:interferon tau
keywords: Blastocyst; Buffalo; Interferon tau; Isoform; Trophectodermal cells