Niu P D, Lefevre F, Mege D, La Bonnardiere C
Unité de Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
Eur J Biochem. 1995 May 15;230(1):200-6. doi: 10.1111/j.1432-1033.1995.tb20551.x.
A recombinant baculovirus was designed to express short porcine type I interferon (spI interferon), a novel and atypical type I interferon that was recently described as the product of a gene transcribed in pig trophoblast at the time of implantation in the uterus [Lefèvre, F. & Boulay, V.C. (1993) J. Biol. Chem. 268, 19,760-19,768]. The recombinant protein, secreted into the culture medium of Sf9 cells at 3 days post infection (60,000 IU/ml), was purified by ion-exchange and reverse-phase HPLC. N-terminal sequencing confirmed the predicted signal peptide cleavage site and therefore the size of the mature protein (149 amino acids), the shortest of all reported type I interferons. Purified spI interferon, with a specific antiviral activity using Madin-Darby bovine kidney cells of 3.7 x 10(7) IU/mg, is an N-glycosylated monomer of 19 kDa that possesses several physicochemical characteristics of interferons: (a) disulfide bonds are necessary for bioactivity; spI interferon is thermolabile, stable at pH 2, and able to renature after complete denaturation (1% 2-mercaptoethanol, 1% SDS, and 5 M urea); (b) the carbohydrate chain is not essential for bioactivity since no loss of antiviral activity is observed following complete deglycosylation. In this study, antiviral and anti-proliferation activities of spI interferon in cell culture were compared with those of other interferons, especially with porcine type 1 interferon-alpha. A major difference with porcine type 1 interferon-alpha was that spI interferon was not active on human cells in either test, and it was relatively more active on pig cells compared to bovine cells than porcine type 1 interferon-alpha. Serological cross-neutralization results obtained with anti-(spI interferon) serum confirmed that several members of interferon families are not antigenically related to spI interferon, in agreement with previous observations; this provides further evidence that spI interferon could represent a new family of type I interferon.
一种重组杆状病毒被设计用于表达短猪I型干扰素（spI干扰素），这是一种新型且非典型的I型干扰素，最近被描述为猪滋养层细胞在子宫着床时转录的一个基因的产物[勒费弗尔，F. & 布莱，V.C.（1993年）《生物化学杂志》268卷，第19760 - 19768页]。感染后3天，重组蛋白分泌到Sf9细胞的培养基中（60,000国际单位/毫升），通过离子交换和反相高效液相色谱法进行纯化。N端测序证实了预测的信号肽切割位点，从而确定了成熟蛋白的大小（149个氨基酸），它是所有已报道的I型干扰素中最短的。纯化后的spI干扰素，使用马迪 - 达比牛肾细胞测定其比抗病毒活性为3.7×10⁷国际单位/毫克，是一种19 kDa的N - 糖基化单体，具有干扰素的几个物理化学特性：（a）二硫键对于生物活性是必需的；spI干扰素对热不稳定，在pH 2时稳定，并且在完全变性（1% 2 - 巯基乙醇、1% SDS和5 M尿素）后能够复性；（b）糖链对于生物活性不是必需的，因为在完全去糖基化后未观察到抗病毒活性的丧失。在本研究中，将spI干扰素在细胞培养中的抗病毒和抗增殖活性与其他干扰素，特别是猪I型α干扰素的活性进行了比较。与猪I型α干扰素的一个主要区别在于，在任何一项测试中spI干扰素对人细胞均无活性，并且与猪I型α干扰素相比，它对猪细胞的活性相对高于对牛细胞的活性。用抗 - （spI干扰素）血清获得的血清学交叉中和结果证实，干扰素家族的几个成员与spI干扰素无抗原相关性，这与先前的观察结果一致；这进一步证明spI干扰素可能代表一种新的I型干扰素家族。
