Yang Jun, Cai Shao-xi, Zou Quan-ming, Zhang Wei-jun, Yang Qin
Key Laboratory of Biomechanics and Tissue Engineering, Chongqing University, Chongqing 400044, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2005 Nov;21(6):693-6.
To clone and express IL-24 gene in E.coli and to study its antitumor activity to induce apoptosis of tumor cells after purification and refolding.
The total RNA was extracted from peripheral blood mononuclear cells (PBMC) and induced by ConA. The human IL-24 gene was amplified by RT-PCR before subcloned into expression vector pET32a(+) and expressed in E. coli. The fusion protein was identified by Edman and purified in chelating sepharose fast flow chromatography and refolding. The activity of inducing tumor cells apoptosis was observed by MTT colorimetry and morphological study in vitro.
The obtained human IL-24 gene was identical with that included in GenBank. The recombinant IL-24 could be expressed in E. coli by IPTG induction as inclusion body (35 kD) with purity over 95%. The apoptosis of MCF-7 cells was induced by Trx-IL-24 protein.
The fusion protein can be highly expressed in E. coli and the obtained protein with high purity can induce tumor cells apoptosis.
在大肠杆菌中克隆并表达白细胞介素-24（IL-24）基因，纯化复性后研究其诱导肿瘤细胞凋亡的抗肿瘤活性。
从外周血单个核细胞（PBMC）中提取总RNA，经刀豆蛋白A（ConA）诱导。通过逆转录聚合酶链反应（RT-PCR）扩增人IL-24基因，随后亚克隆至表达载体pET32a(+)中并在大肠杆菌中表达。采用埃德曼法鉴定融合蛋白，通过螯合琼脂糖快速流动层析法进行纯化及复性。体外通过MTT比色法和形态学研究观察诱导肿瘤细胞凋亡的活性。
获得的人IL-24基因与GenBank中收录的序列一致。重组IL-24经异丙基-β-D-硫代半乳糖苷（IPTG）诱导可在大肠杆菌中以包涵体形式表达（35 kD），纯度超过95%。Trx-IL-24蛋白可诱导MCF-7细胞凋亡。
融合蛋白能在大肠杆菌中高效表达，获得的高纯度蛋白可诱导肿瘤细胞凋亡。
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