Devos R, Cheroutre H, Taya Y, Degrave W, Van Heuverswyn H, Fiers W
Nucleic Acids Res. 1982 Apr 24;10(8):2487-501. doi: 10.1093/nar/10.8.2487.
Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.
从源自葡萄球菌肠毒素A诱导的人脾细胞的mRNA开始，合成双链DNA并将其插入真核表达载体pSV529的独特BamHI位点（1）。通过将结合在硝酸纤维素滤膜上的插入质粒DNA与源自SEA诱导的脾细胞的mRNA杂交、在非洲爪蟾卵母细胞中对洗脱的RNA进行翻译并检测IFN活性，鉴定出含有人类免疫干扰素（IFN-γ）cDNA的重组质粒。通过菌落杂交鉴定出含有整个人IFN-γ cDNA序列的质粒并进行测序。鉴定出一个独特的编码区，预测其编码一种166个氨基酸的蛋白质，其N端的20个氨基酸可能代表信号肽。用从其中一个重组克隆（pHIIF-SV-γ 1）分离的质粒DNA转染猴细胞后，IFN分泌到培养基中。通过血清学标准或细胞靶物种特异性，这种IFN与人类IFN-γ没有区别。
