Saugandhika Shrabani, Sharma Vishal, Malik Hrudananda, Saini Sikander, Bag Sudam, Kumar Sudarshan, Singh Niraj Kumar, Mohanty Ashok Kumar, Malakar Dhruba
Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India.
Animal Biotechnology Centre, GADVASU, Ludhiana, Punjab, India.
Cytokine. 2015 Sep;75(1):186-96. doi: 10.1016/j.cyto.2015.03.012. Epub 2015 Apr 15.
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.
我们研究的目的是优化生长和诱导参数，以实现功能活性水牛干扰素τ的表达和大规模纯化，并研究其对体外囊胚发育的可能影响。通过聚合酶链反应（PCR）从牛早期胚胎中获得带有信号序列、基因库登录号为JX481984的水牛干扰素τ基因（BuIFN-T1），并将其克隆到pJET载体中。经验证后，将无信号序列的片段插入表达载体pET-22b中，并在原核表达系统中诱导重组质粒表达重组蛋白。通过SDS-PAGE和Western blot对重组BuIFN-T进行确认，并使用His亲和层析、阴离子交换层析和凝胶过滤层析进行三步大规模纯化。通过质谱分析对纯化的重组BuIFN-T蛋白进行验证。为了检测重组BuIFN-T蛋白对水牛胚胎发育能力的影响，将纯化的重组BuIFN-T蛋白添加到体外胚胎培养基中（浓度分别为0、1μg/ml、2μg/ml、4μg/ml），培养9天。添加重组BuIFN-T（2μg/ml）显著提高了囊胚产生率，分别为45.55%和31.1%（对照组，p<0.01）。我们得出结论，重组BuIFN-T已成功从原核表达系统中纯化至同质，并且它显著提高了水牛的囊胚产生率。这些发现表明干扰素τ在促进胚胎生长发育方面具有潜在影响。
