Fiers W, Remaut E, Devos R, Cheroutre H, Contreras R, Gheysen D, Degrave W, Stanssens P, Tavernier J, Taya Y, Content J
Philos Trans R Soc Lond B Biol Sci. 1982 Sep 24;299(1094):29-38. doi: 10.1098/rstb.1982.0103.
The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.
编码人成纤维细胞干扰素（IFN-β）的遗传信息已被克隆为DNA拷贝（cDNA）和基因组克隆。人IFN-β以前体形式产生，由一个21个氨基酸残基长的信号序列和其后166个氨基酸长的成熟蛋白组成。存在一个糖基化位点。人IFN-β基因不含内含子。用含有在SV40晚期启动子控制下的人IFN-β cDNA克隆的嵌合SV40衍生物转染猴细胞，可导致高水平IFN-β的分泌。当在同一载体中使用基因组克隆时，通过用聚（rI）·聚（rC）处理，IFN-β的合成可进一步增强达30倍；这表明该克隆中存在一个顺式活性控制元件。基于含有噬菌体λ的PL启动子的质粒，构建了一个在大肠杆菌中的高效表达系统，该启动子由温度敏感阻遏物调控。该启动子后面是一段来自噬菌体MS2的片段，其中包含复制酶基因的核糖体结合位点。然而，后者被人IFN-β基因取代。诱导后，细菌合成高水平（约5×10⁹ IU 1⁻¹）的IFN-β；这相当于细菌总蛋白的约2%。人免疫（II型）干扰素（IFN-γ）基因也同样被克隆。以用葡萄球菌肠毒素A诱导的人脾细胞来源的部分纯化mRNA为起始材料。对一个全长cDNA克隆进行了测序。cDNA总序列约1150个核苷酸长；它包含一个编码166个氨基酸的单一开放阅读框，其中前20个氨基酸构成跨膜信号。有两个糖基化位点。氨基酸序列与IFN-α或IFN-β的序列有很大不同，尽管可以注意到一些相似之处。未翻译的3'末端区域约550个核苷酸长。IFN-γ基因也通过使用SV40衍生载体在猴细胞中表达，分泌产物被鉴定为真正的人IFN-γ。还获得了噬菌体λ衍生物形式的基因组克隆。IFN-γ基因至少延伸5千碱基，并且包含至少两个内含子。
