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首页> 外文期刊>Journal of Pharmacological and Toxicological Methods >Preparation of complement fragments C3b and C3a from purified rat complement component C3 by activated cobra venom factor.
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Preparation of complement fragments C3b and C3a from purified rat complement component C3 by activated cobra venom factor.

机译：通过活化的眼镜蛇毒因子从纯化的大鼠补体成分C3制备补体片段C3b和C3a。

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INTRODUCTION: Complement component C3 (C3) can be a target of pharmacological or toxicological agents. In the analysis of this, it is important to examine the involvement of fragments C3b and C3a since C3 function normally requires cleavage into these fragments. The present study describes a simple and efficient method for the preparation of rat complement C3b and C3a by using purified C3 and cobra venom factor (CVF) as a cleaving enzyme. METHODS: CVF was purified from lyophilized cobra venom (Naja naja kausia) by two-step chromatography and was activated by incubation with human factors B and D. C3 was cleaved by incubation with activated CVF (CVF,Bb), and C3b and C3a were isolated by anion- and cation-exchange chromatography, respectively. RESULTS: About 200 microg of CVF was purified from 100 mg of cobra venom. All the CVF was activated by incubation with factors B and D. The C3b and C3a obtained were pure as analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and no digestive by-products such as C3f were found. DISCUSSION: The advantage of the present method is that it is possible to prepare relatively large amounts of C3b by simple procedures without digestive by-products. C3a can be prepared from the flow through fraction of the C3b purification. C3b and C3a prepared by the present method would be useful for pharmacological or toxicological experiments involving receptor binding since their binding sites remain intact.
机译：简介：补体成分C3（C3）可以作为药理或毒理学目标。在对此进行分析时，重要的是检查片段C3b和C3a的参与，因为C3功能通常需要裂解成这些片段。本研究描述了一种简单有效的方法，通过使用纯化的C3和眼镜蛇毒因子（CVF）作为裂解酶来制备大鼠补体C3b和C3a。方法：通过两步色谱法从冻干的眼镜蛇毒（Naja naja kausia）中纯化CVF，并通过与人因子B和D的温育进行活化。通过与活化CVF（CVF，Bb）的温育裂解C3，C3b和C3a分别为分别通过阴离子交换色谱和阳离子交换色谱分离。结果：从100毫克眼镜蛇毒中纯化出约200微克CVF。通过与因子B和D一起温育来活化所有的CVF。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，获得的C3b和C3a是纯的，没有发现消化副产物如C3f。讨论：本方法的优点是可以通过简单的方法制备相对大量的C3b，而无需消化副产物。 C3a可以从C3b纯化的流通部分中制备。由于本发明方法制备的C3b和C3a的结合位点保持完整，因此可用于涉及受体结合的药理或毒理学实验。

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