Preparation of recombinant rat interleukin-5 by baculovirus expression system and analysis of its biological activities
Preparation of recombinant rat interleukin-5 by baculovirus expression system and analysis of its biological activities
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DOI：
10.1016/s0167-4889(99)00090-7
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发表时间：
1999-08-12
影响因子：
5.1
通讯作者：
Ohuchi, K
中科院分区：
文献类型：
--
作者：
Ishihara, K;Satoh, I;Ohuchi, K
向免疫大鼠腹腔内注射猪蛔虫抗原溶液4小时后收集的腹膜细胞亚克隆大鼠白细胞介素5 (IL-5) cDNA。通过将大鼠IL-5 cDNA插入具有家蚕核多角体病毒的半胱氨酸蛋白酶基因缺失的CPd病毒中，构建半胱氨酸蛋白酶缺失(CPd)大鼠IL-5重组病毒。感染 CPd 大鼠 IL-5 重组病毒后，家蚕幼虫在血淋巴中产生二聚体形式的大鼠 IL-5。通过阴离子交换层析和疏水层析，重组大鼠IL-5的纯化率超过95.5%。纯化的重组大鼠IL-5以浓度依赖性方式促进T88-M细胞增殖，其作用被抗鼠IL-5中和多克隆抗体抑制。当来自正常大鼠的骨髓细胞与重组大鼠IL-5在含有甲基纤维素的培养基中孵育时，诱导嗜酸性细胞集落形成。此外，当大鼠腹膜嗜酸性粒细胞与重组大鼠IL-5一起孵育时，嗜酸性粒细胞活力的自发降低以时间和浓度依赖性方式受到抑制。此外，重组大鼠IL-5诱导的嗜酸性粒细胞存活被抗鼠IL-5中和多克隆抗体抑制。这些发现表明，大鼠 IL-5 充当 B 细胞生长因子 II (BCGF-II)、嗜酸性粒细胞分化因子 (EDF) 和嗜酸性粒细胞存活增强因子。 (C) 1999 Elsevier Science B.V. 保留所有权利。
Rat interleukin-5 (IL-5) cDNA was subcloned from peritoneal cells collected 4 h after intraperitoneal injection of Ascaris suum antigen solution into the immunized rats. Cysteine proteinase-deleted (CPd) rat IL-5 recombinant virus was constructed by inserting rat IL-5 cDNA into CPd virus having a deletion in the cysteine proteinase gene of the silkworm Bombyx mori nuclear polyhedrosis virus. On infection with the CPd rat IL-5 recombinant virus, the silkworm B. mori larvae produced rat IL-5 as a dimeric form in hemolymph. Recombinant rat IL-5 was purified more than 95.5% by anion-exchange chromatography and hydrophobic chromatography. The purified recombinant rat IL-5 promoted the proliferation of T88-M cells in a concentration-dependent manner, and its effect was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. When bone marrow cells from normal rats were incubated with recombinant rat IL-5 in medium containing methylcellulose, the colony formation by eosinophilic cells was induced. Furthermore, when rat peritoneal eosinophils were incubated with recombinant rat IL-5, the spontaneous decrease in the eosinophil viability was inhibited in time- and concentration-dependent manners. In addition, the recombinant rat IL-5-induced eosinophil survival was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. These findings suggest that rat IL-5 acts as B-cell growth factor II (BCGF-II), eosinophil differentiation factor (EDF), and eosinophil survival-enhancing factor. (C) 1999 Elsevier Science B.V. All rights reserved.