权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Structure and Functions of an IL-1 inhibitor found at inflammatory site in rabbit.

兔炎症部位发现的 IL-1 抑制剂的结构和功能。

基本信息

批准号：
03454172
负责人：
YOSHINAGA Masaru
金额：
$ 4.1万
依托单位：
KUMAMOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454172/
关键词：
IL-1 IL-1 receptor antagonist Inflammation Inflammatory exudate cells neutrophils Anti-inflammatory agent インターロイキン1レセプターアンダゴニスト Interleukin 1 Interlukin 1 receptor antagonigt 炎症抑制物質

项目摘要

A rabbit interleukin-1 (IL-1) inhibitor in inflammatory peritoneal exudate cells was purified to apparent homogeneity. This inhibitor was extracted from exudate cells of the 24-hr stage of peritoneal inflammation and purified using isoelectrofocusing (IEF), gel filtration, followed in this order by high-performance liquid chromatography steps with hydroxylapatite and anionic ion exchanger. The purified factor showed a single band on silver-stained SDS-PAGE. This molecule of MW 19 kD and pI 5.5 inhibited the binding of both IL-1alpha and beta to receptors on a thymoma cell line, EL-4 and a B-cell line, 70Z/3. We determined its primary structure by a combination of peptide chemistry and molecular cloning. The inhibitor was synthesized as a precursor composed of 177 amino acids and was processed to a mature molecule of 143 amino acids. The N-terminal amino acid of the mature inhibitor was N-acetyl-methionine residue. The deduced amino acid sequence of the inhibitor showed a 77% homology t … More o the human IL-1 receptor antagonist (IL-1ra) and essentially the same mode of action as seen with human IL-1ra. We consider the this inhibitor is a rabbit counterpart of human IL-1ra, although there are differences with respect to the molecular structure; the N-terminus of the mature rabbit IL-1ra at a position of nine amino acids downstream from that of human IL-1ra.Further, we made a recombinant rabbit IL-1ra (rrIL-1ra) expressed in E. coli. Using the rrIL-1ra, we also made a sheep anti-serum and developed an ELISA system for the rabbit IL-1ra. The rrIL-1ra was purified using DEAE-cellulose and Sephadex G-75, in a conventional chromatography system, followed by a linear NaCl gradient-elution of DEAE-cellulose in an FPLC system. Its specific biologic activity was almost the same as the authentic natural product. Using the above described tools, we examined the generation of IL-1ra during the course of LPS-arthritis in rabbits. Production of IL-1ra peaked at 9 hr (196.7 ng/joint) and the amount was 180-200 fold molar excess of IL-1 found at the lesion, and a large amount of IL-1ra was sustained for one week. The LPS-induced arthritis was strongly suppressed by intra-articular injection of 10 mug of IL-1ra when the severity of arthritis was monitored by the infiltration of leukocytes and destruction of articular cartilage. Then, we concluded that IL-1ra play an regulatory role in inflammation and may become a potent anti-inflammatory agent. Less

兔炎症性腹膜渗出细胞中的白细胞介素-1 （IL-1）抑制剂被纯化到明显的均匀性。该抑制剂从腹膜炎症24小时期的渗出细胞中提取，采用等电聚焦（IEF）、凝胶过滤纯化，然后用羟基磷灰石和阴离子交换剂进行高效液相色谱纯化。纯化后的因子在SDS-PAGE银染色上呈单条带。在胸腺瘤细胞系EL-4和b细胞系70Z/3上，mw19kd和pi5.5分子抑制il -1 α和β与受体的结合。我们通过多肽化学和分子克隆相结合的方法确定了其一级结构。该抑制剂是由177个氨基酸组成的前体合成的，经过加工得到143个氨基酸的成熟分子。成熟抑制剂的n端氨基酸为n -乙酰蛋氨酸残基。该抑制剂的氨基酸序列与人IL-1受体拮抗剂（IL-1ra）有77%的同源性，其作用方式与人IL-1ra基本相同。我们认为这种抑制剂是人类IL-1ra的兔子对应物，尽管在分子结构方面存在差异；成熟兔IL-1ra的n端位于人类IL-1ra下游9个氨基酸的位置。进一步，我们制作了在大肠杆菌中表达的重组兔IL-1ra （rrIL-1ra）。我们还利用ril -1ra制备了绵羊抗血清，并建立了兔IL-1ra的ELISA系统。在常规色谱系统中使用deae -纤维素和Sephadex G-75纯化rrIL-1ra，然后在FPLC系统中使用deae -纤维素线性NaCl梯度洗脱。其比生物活性与正品天然产物基本一致。使用上述工具，我们检测了兔lps关节炎过程中IL-1ra的产生。IL-1ra的产生在9小时达到峰值（196.7 ng/关节），病变处IL-1的量为180-200倍摩尔过量，大量IL-1ra持续一周。通过白细胞浸润和关节软骨破坏监测关节炎的严重程度，关节内注射10杯IL-1ra可明显抑制lps诱导的关节炎。因此，我们得出结论，IL-1ra在炎症中起调节作用，可能成为一种有效的抗炎剂。少