重组人干扰素-λ1的纯化与鉴定

Purification and identification of recombinant human interferon-lamda 1

背景:2003年研究发现一个全新的III型干扰素家族,即IFN-λs,包括λ1,2,3三种亚型。目的:对在毕赤酵母中高效表达重组人干扰素-λ1(rhIFN-λ1)进行产率纯度分析,理化性质、受体表达鉴定以及抗肿瘤活性初步探讨。设计、时间及地点:单一样本观察,于2006-09/2008-07在中国医学科学院基础所生物化学与分子生物学系完成。材料:含rhIFNλ1表达质粒的酵母菌种pAO815-4αF-IFNλ1/GS115,人宫颈癌细胞系Hela细胞和HCT116细胞,为中国医学科学院基础所医学分子生物学国家重点实验室保存。方法:诱导含rhIFN-λ1表达质粒的甲醇营酵母Pichia pastoris进行表达,表达产物经阳离子交换层析及凝胶层析纯化。主要观察指标:通过HPLC,质谱,毛细管电泳等鉴定其纯度及理化性质,反转录-聚合酶链反应及real-time荧光定量聚合酶链反应鉴定其在肿瘤细胞中特异受体的表达,应用四甲基偶氮唑盐比色法初步评价其抗肿瘤活性。结果:毕赤酵母表达的rhIFN-1表达水平约9.23mg/L,纯化后的纯度近90%。rhIFN-λ1等电点为8.01,相对分子质量为20960。在Hela和HCT116中均检测到rhIFN-λ1受体小亚基IL-10R2和特异大亚基IL-28RmRNA的表达,但U937细胞中未检测到IL-28RmRNA的表达。rhIFN-λ1具有与IFN-α2a相似的降低细胞增殖的作用,但在经检测无或极少表达IL-28R的细胞中此作用不明显。结论:rhIFN-λ1可以在毕赤酵母中实现高效表达,表达产物的理化性质与天然IFN-λ1一致,具有与IFN-α2a相似的抗肿瘤活性,但对其特异受体分布少的骨髓造血系统降低增殖作用相对较弱。

OBJECTIVE： To analyze the recombinant human interferon- λ 1 （rhIFN-λ1） expressed in Pichia pastoris, further more, to identify its physical and chemical characteristics, expression of receptors, and anti-tumor activities. DESIGN, TIME AND SETTING： The single sample observation was performed at the Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences from September 2006 to July 2008. MATERIALS： The barms pAO815-4α F-IFNλ1/GS115 contained rhIFN λ 1 expressive plasmids, human cervical carcinoma cell Hela and HCT116 cells were preserved in the state key lab for Molecular Biology from Institute of Basic Medical Sciences Chinese Academy of Medical Sciences METHODS： The Pichia pastoris contained rhIFN- λ 1 expressive plasmids was cultured and induced to express IFN-λ 1. The positive ion exchange chromatography and gel chromatography were used to purify the expression product. MAIN OUTCOME MEASURES： The physico-chemical characters of the purified product were tested. The product＇s IL-10R2 and IL-28R were investigated by RT-PCR and real-time quantitative PCR and its antitumor activity was tested by MTT analysis. RESULTS： The expressed product rhIFN-λ1 yield reached to 9.23 mg/L, and its purity was near 90%. The isoelectric point of rhlFN- 1 was 8.01, with molecular weight of 20 960. The expressions of IL-10R2 and IL-28R were detected in U937 cell. rhlFN-λ1 could decrease the cell proliferation just like IFN-α 2a, but this function was unclearly in the IL-28R cell. CONCLUSION： rhIFN- λ1 can be highly expressed in Pichia pastoris, and its product has the physical and chemical characteristics consistent with natural IFN-λ1, and the anti-tumor activity is similar to IFN-α 2a, however, it is weak in decreasing the proliferation of relatively lowerintensity of receptor distribution in marrow-hematopoiesis system.