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非融合rIL-24蛋白的制备及活性鉴定

Preparation and characterization of non-fusion recombinant IL-24 in Escherichia coli
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摘要 目的制备非融合的rIL-24蛋白,鉴定其体外诱导肿瘤细胞凋亡活性。方法为制备非融合IL-24蛋白,将IL-24基因插入到pET32a(+)上KpnⅠ与BamHⅠ之间,构建表达载体IL-24/pET32a,IPTG诱导在E.coliBL21(DE3)中表达。洗涤后的包涵体溶解在2 mol/L尿素(pH9.0)中,采用镍离子亲和色谱和凝胶分子筛的二步法纯化,肠激酶酶切(肠激酶与蛋白1∶400,pH7.5处理36 h),再用亲和柱色谱纯化制备非融合的rIL-24。MTT法和形态学观察分析rIL-24体外诱导肿瘤细胞凋亡的活性。结果重组工程菌以包涵体形式表达出相对分子质量约为35 000的融合蛋白。包涵体经洗涤、溶解及二步纯化得到纯度大于95%的Trx-IL-24。融合蛋白用肠激酶酶切之后,经亲和色谱制备得到纯度大于98%的非融合蛋白rIL-24。rIL-24显著诱导MCF-7乳腺癌细胞凋亡(P<0.05),而对正常人肺成纤维细胞NHLF没有影响。结论IL-24基因在E.coli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非融合蛋白rIL-24,预示其具有肿瘤治疗应用的前景。 Purpose To prepare non-fusion recombinant IL-24 and to analyze its biological activities in vitro. Methods The confirmed IL-24 gene was inserted between the sites of Kpn Ⅰane Bam H Ⅰ in pET32a and expressed in E. coli BL21 (DE3). The inclusion bodies were disrupted, washed, and isolated in 2 mol/L urea at pH 9.0. After nickel ion metal affinity chromatography and gel filtration chromatography, the refolded fusion protein was digested by enterokinase(EK) to both Trx and rIL-24 fragments which then were separated by affinity chromatography. The activity of inducing tumor cells apoptosis was confirmed by MTT assay and morphologies. Results The Trx-IL-24 was expressed in form of inclusion bodies as 35 000(Mr). By two-step chromatography Trx-IL-24 with a purity of 〉 90% was obtained, then it was digested and purified by affinity chro- matography. Cell proliferation experiments proved that the non-fusion rIL-24 had a purity of more than 95 % retains apoptosis inducing of MCF-7. Conclusion This result suggested that the non-fusion rIL-24 may have cancer therapeutic applications.
出处 《中国生化药物杂志》 CAS CSCD 北大核心 2009年第5期289-293,共5页 Chinese Journal of Biochemical Pharmaceutics
基金 国家惯技术863计划资助课题(No.2001AA21516)
关键词 rIL-24 非融合蛋白 纯化 诱导肿瘤细胞凋亡 rIL-24 non-fusion protein purification inducing tumor cells apoptosis
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参考文献8

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