Trapman J, van Heuvel M, de Jonge P, Bosveld I J, Klaassen P, Zwarthoff E C
Department of Pathology, Erasmus University, Rotterdam, The Netherlands.
J Gen Virol. 1988 Jan;69 ( Pt 1):67-75. doi: 10.1099/0022-1317-69-1-67.
A mouse interferon alpha gene (MuIFN-alpha 10) was isolated from a BALB/c cosmid genomic library. The gene was located on a 1.8 kb HindIII fragment and a 5.1 kb EcoRI fragment. The coding region and parts of the 5' and 3' non-coding regions were sequenced. The results showed that the MuIFN-alpha 10 gene encoded a protein of 167 amino acids. Like most other MuIFN-alpha species it contained a putative N-glycosylation site at amino acid positions 78 to 80. It also possessed cysteine residues at positions 1, 29, 86, 99 and 129. In the signal peptide, in addition to cysteine 21, which is present in all MuIFN-alpha species sequenced so far, a cysteine was found at position 22. At the amino acid level MuIFN-alpha 10 showed strong homology to MuIFN-alpha 1 (only 15 out of 167 amino acids were different). The MuIFN-alpha 10 gene was transiently expressed in monkey COS cells under the direction of the simian virus 40 early promoter. The protein product secreted by COS cells was equally active on mouse (L929) and hamster (CHO) cells. However, as compared to MuIFN-alpha 1 and MuIFN-alpha 4 the specific activity on mouse cells of the protein was 10- to 100-fold lower. To find out which region of its structure was responsible for this low activity, hybrids of the genes encoding MuIFN-alpha 10 and MuIFN-alpha 1 were constructed using the two common XmmI sites which correspond to positions between amino acids 67 and 68 and 123 and 124, respectively. The data showed that hybrid constructs which were MuIFN-alpha 1-like from amino acid 68 or MuIFN-alpha 10-like from position 124 to the C terminus possessed high antiviral activity. Other hybrid constructs were hardly active at all. This implied that the amino acid 68 to 123 region was mainly responsible for the low antiviral activity of MuIFN-alpha 10. In this part of the molecule MuIFN-alpha 1 and MuIFN-alpha 10 differed in only five amino acids. A serine at position 110 and a valine at 85 were unique to MuIFN-alpha 10 as compared to all known MuIFN-alpha and human IFN-alpha subspecies.
从BALB/c黏粒基因组文库中分离出一个小鼠干扰素α基因（MuIFN-α10）。该基因位于一个1.8 kb的HindIII片段和一个5.1 kb的EcoRI片段上。对编码区以及5'和3'非编码区的部分进行了测序。结果表明，MuIFN-α10基因编码一种由167个氨基酸组成的蛋白质。与大多数其他小鼠干扰素α种类一样，它在氨基酸位置78至80处含有一个推定的N-糖基化位点。它在位置1、29、86、99和129处也有半胱氨酸残基。在信号肽中，除了迄今测序的所有小鼠干扰素α种类中都存在的半胱氨酸21外，在位置22处还发现了一个半胱氨酸。在氨基酸水平上，MuIFN-α10与MuIFN-α1具有很强的同源性（167个氨基酸中只有15个不同）。MuIFN-α10基因在猴COS细胞中由猿猴病毒40早期启动子指导进行瞬时表达。COS细胞分泌的蛋白质产物对小鼠（L929）和仓鼠（CHO）细胞具有同等活性。然而，与MuIFN-α1和MuIFN-α4相比，该蛋白质对小鼠细胞的比活性低10至100倍。为了找出其结构的哪个区域导致了这种低活性，利用两个常见的XmmI位点构建了编码MuIFN-α10和MuIFN-α1的基因的杂种，这两个位点分别对应于氨基酸67和68以及1,23和124之间的位置。数据表明，从氨基酸68起类似MuIFN-α1或从位置124到C末端类似MuIFN-α10的杂种构建体具有高抗病毒活性。其他杂种构建体几乎没有活性。这表明氨基酸68至123区域主要是MuIFN-α10抗病毒活性低的原因。在分子的这一部分，MuIFN-α1和MuIFN-α10仅在五个氨基酸上不同。与所有已知的小鼠干扰素α和人干扰素α亚种类相比，位置110处的丝氨酸和85处的缬氨酸是MuIFN-α10特有的。
