Lieschke G J, Rao P K, Gately M K, Mulligan R C
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Nat Biotechnol. 1997 Jan;15(1):35-40. doi: 10.1038/nbt0197-35.
Interleukin-12 (IL-12) is unique amongst cytokines in being a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40). We expressed single chain IL-12 proteins from retroviral constructs in which the two IL-12 subunits were linked by a 6-15 amino acid polypeptide linker, with deletion of the 22 amino acid leader sequence of the trailing subunit. The murine fusion protein IL-12.p40.L.delta p35 containing a (Gly4Ser)3 linker was stably expressed, bioactive in vitro, and had an apparent specific activity comparable to that of native and recombinant IL-12. Western blotting confirmed that murine IL-12.p40.L.delta p35 retained the linking polypeptide sequences. The analogous human IL-12.p40.L.delta p35 fusion protein containing a Gly6Ser linker was bioactive with an apparent specific activity comparable to recombinant human IL-12. In a preexisting CMS-5 tumor model, CMS-5 cells secreting either native or fusion protein forms of IL-12 prolonged survival and led to complete tumor regression.
白细胞介素-12（IL-12）在细胞因子中独具特色，它是由两个分别编码的亚基（p35和p40）通过二硫键连接而成的异源二聚体。我们从逆转录病毒构建体中表达了单链IL-12蛋白，其中两个IL-12亚基由一个6 - 15个氨基酸的多肽接头连接，并缺失了尾随亚基的22个氨基酸前导序列。含有(Gly4Ser)3接头的小鼠融合蛋白IL-12.p40.L.delta p35得以稳定表达，在体外具有生物活性，其表观比活性与天然和重组IL-12相当。蛋白质印迹法证实小鼠IL-12.p40.L.delta p35保留了连接多肽序列。含有Gly6Ser接头的类似人类IL-12.p40.L.delta p35融合蛋白具有生物活性，其表观比活性与重组人IL-12相当。在一个预先存在的CMS - 5肿瘤模型中，分泌天然或融合蛋白形式IL-12的CMS - 5细胞延长了生存期，并导致肿瘤完全消退。
