人IL-24基因的克隆、 表达、 纯化及体外诱导肿瘤细胞凋亡的研究

目的:克隆人白细胞介素-24(IL-24)基因,在大肠杆菌中融合表达,纯化复性,研究此融合蛋白的抗肿瘤活性。方法:分离人外周血单个核细胞,ConA刺激培养,提取细胞总RNA,RT-PCR技术克隆人IL-24基因。将IL-24插入到pET32a( )中,构建重组表达载体IL-24/pET32a,IPTG诱导在E.coli表达。Edman法N端测序,将鉴定正确的蛋白亲合层析纯化。MTT比色法体外分析杀伤肿瘤细胞的活性。结果:获得人IL-24基因,序列分析与GenBank公布一致。表达载体用双酶切和PCR鉴定正确。重组工程菌经IPTG诱导后表达相对分子质量(Mr)约为35000的融合蛋白。N端测序正确,鉴定该蛋白以包涵体形式表达。包涵体经洗涤、溶解后,亲合纯化得到纯度大于95%的融合蛋白。复性后表达产物能显著诱导MCF-7乳腺癌细胞凋亡(P<0.05)。结论:IL-24融合蛋白在原核细胞中高效表达,并获得高纯度、在体外明显诱导乳腺癌细胞凋亡的重组蛋白,为后续的研究提供实验基础。

Cloning, expression and purification of human interleukin 24 gene and study on its activity of inducing tumor cells apoptosis in vitro

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The fusion protein can be highly expressed in E.coli and the obtained protein of high purity can induce tumor cells apoptosis.