具有野生型活性和新活性的小鼠白介素12融合基因的构建与表达

天然白介素(IL)12是一种异二聚体分子,其工程产品只能在真核细胞内表达,因而产量低,造价高,限制了临床应用。根据融合蛋白的原理,结合IL-12本身的结构特点进行推断,若将IL-12的二亚基(p35/p40)适当改造和融合,其产物可能同样地表达野生型IL-12的活性,这将为在原核表达系统内表达融合蛋白提供先决条件。将p40亚基cDNA中编码Ig-样区域的序列缺失,残余部分与p35亚基的cDNA3’端通过连接序列连接,构成融合IL-12 cDNA(fmIL-12C)。当此融合基因克隆人真核表达载体pMT/EP后在CHO细胞内表达,其产物具有野生型IL-12的活性,即刺激活化淋巴母细胞增殖。与此同时,融合IL-12的cDNA的表达使CHO细胞发生十分明显的形态变化,提示融合IL-12可能具有一些新活性。对此融合基因进一步改造及在原核系统内表达的可能性也进行了讨论。

Construction and expression of a murine IL-12 fused gene showing activity of wild type (wt-) IL-12 and novel activities

As a heterodimeric cytokine, wild type interleukin 12 can only be produced in eukaryotic cells, leading to low production and high manufacturing cost, thus greatly limits its clinical usage. On the knowledge of fusion proteins, we deduced that when modified and fused adequately, p35 and p40 may give some properties of wild type IL-12. If so, it would be possible to express the fusion protein in prokaryotic expression system. The sequence coding for the Ig-like domain in p40 subunit cDNA was deleted and the residual part was linked to 3'-end of p35 subunit cDNA, resulting in a fused IL-12 cDNA (fmIL-12C). When the fusion gene was cloned into eukaryotic expression vector pMT/EP and transfected into CHO cells, the culture supernatant showed IL-12-like activity, namely to stimulate activated lym-phoblasts to proliferate. At the same time, fmIL-12C expression altered CHO cellular morphology significantly. The possibility of modification and expression of fmIL-12C in prokaryotic system was discussed.