IL18-IL2融合基因的克隆及其在大肠埃氏菌(E.coli)中的表达

Cloning and Expression of a Fusion Protein IL18-IL2 in Escherichia Coli

构建IL-18和IL-2成熟区的融合基因IL18-IL2,并实现其在大肠埃氏菌中的大量表达.抽提经PHA刺激增殖的人淋巴细胞总RNA,RT-PCR法获得IL-18和IL-2基因的成熟区序列,并通过一段Linker相连得到融合基因IL18-IL2;将IL18-IL2克隆至原核表达载体pBV220,并转化至大肠埃氏菌BL21(DE3);阳性克隆经42℃温度诱导表达后用SDS-PAGE和Western blot分析重组蛋白的表达情况及正确性.经SDS-PAGE分析,融合基因IL18-IL2在宿主菌中能够表达,其相对表观分子质量约为34.5 kD;Western blot进一步证明重组蛋白正确.实验结果表明,已成功构建了表达人IL18-IL2的菌株,为进一步研究融合蛋白IL18-IL2体内外抗肿瘤活性提供了基础.

We report here the cloning and expressing of an fusion protein IL18-IL2 containing the mature hu-man interleukin-18 peptide and interleukin-2 peptide in Escherichia Coli.The mature human IL-18 and IL-2cDNAs are cloned by RT-PCR with the total RNAs,extracted from human PBMC stimulated by PHA,as tem-plates.The fusion gene IL18-IL2 connected by a flexible linker is inserted into the pBV220 expression vector and sequenced.Then the recombinant plasmid pBV-IL18-IL2 is transformed into BL21（DE3） and induced at 42 ℃.SDS-PAGE analysis indicates that the relative molecular weight of IL18-IL2 is about 34.5 ku and Western blot further confirms its correctness.The fusion protein IL18-IL2 is successfully cloned and expressed in E.coli.This work has settled the foundation for further biological research on IL18-IL2.