Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution
Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution
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DOI：
10.1016/0305-0491(96)00025-9
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发表时间：
1996-07-01
影响因子：
2.2
通讯作者：
Abrahamson, M
中科院分区：
文献类型：
--
作者：
Hakansson, K;Huh, C;Abrahamson, M
通过在大肠杆菌中表达、分离和功能表征来产生重组小鼠（Mus musculus）和大鼠（Rattusnorvegicus）胱抑素 C。小鼠和大鼠抑制剂在木瓜蛋白酶滴定中均具有完全活性。测定其与目标蛋白酶组织蛋白酶 B 的复合物的解离平衡常数 (K-1)，所得值与人半胱氨酸蛋白酶抑制剂 C (K-1 0.07-0.13 nM) 的值没有太大差异。生产了针对小鼠和大鼠胱抑素 C 的兔抗血清，并用于改进重组抑制剂的亲和纯化。从抗小鼠半胱氨酸蛋白酶抑制剂 C 的抗血清中分离出的亲和纯化的免疫球蛋白用于构建灵敏的酶联免疫吸附测定。该测定用于证明小鼠和大鼠胱抑素 C 之间的高度免疫交叉反应性，并可用于小鼠和大鼠组织匀浆中胱抑素 C 的定量。所有分析的组织均含有胱抑素C，其相对含量与人体组织非常相似。对于所有物种，脑组织中胱抑素 C 含量最高，肝脏最低，而肾脏、脾脏和肌肉组织含量居中。在小鼠中，观察到腮腺组织中胱抑素 C 含量显着升高。小鼠、大鼠和人类半胱氨酸蛋白酶抑制剂 C 的分布模式和功能特性高度相似，表明小鼠模型应该与人类疾病、遗传性半胱氨酸蛋白酶抑制剂 C 淀粉样血管病的研究相关。
Recombinant mouse (Mus musculus) and rat (Rattus norvegicus) cystatin C were produced by expression in Escherichia coli, isolated and functionally characterized. The mouse and rat inhibitors were both fully active in titrations of papain. Determination of equilibrium constants for dissociation (K-1) for their complexes with the target proteinase, cathepsin B, produced values not largely different from that for human cystatin C (K-1 0.07-0.13 nM). Rabbit antisera against mouse and rat cystatin C were produced and used for improved affinity purification of the recombinant inhibitors. Affinity purified immunoglobulins isolated from the antiserum against mouse cystatin C were used for construction of a sensitive enzyme-linked immunosorbent assay. The assay was used to demonstrate a high degree of immunological cross-reactivity between mouse and rat cystatin C and could be used for cystatin C quantification in mouse and rat tissue homogenates. All tissues analyzed contained cystatin C, with a relative content very similar to that of human tissues. For all species, brain tissue contained the highest cystatin C amounts and liver the lowest, whereas kidney, spleen and muscle tissues were intermediate in content. In the mouse, a notable high cystatin C content in parotid gland tissue was observed. The high degree of similarity in distribution pattern and functional properties for mouse, rat and human cystatin C indicates that a murine model should be relevant for studies of the human disease, hereditary cystatin C amyloid angiopathy.