【作者】 王小娟；

【导师】 傅中滇；

【作者基本信息】 华东师范大学 ， 植物学， 2004， 硕士

【摘要】 Luffin-B 是一种从丝瓜（Luffa cylindrica）籽中分离纯化出来的单链核糖体失活蛋白，是目前发现的活性最高的单链核糖体失活蛋白之一。但是以往纯化 Luffin-B的常规方法中的得率并不高，只有0.01％左右。所以本研究在前人纯化Luffin-B的方法基础上作了一些改进：1、由于Luffin-B的等电点在10左右，属于碱性蛋白质，所以本研究用冰醋酸将其粗提液的pH值调节到4.2，进行了一次酸性沉淀，以除去大部分酸性蛋白质，然后又采用50-85％的硫酸铵分级饱和沉淀法除去大部分杂蛋白，这样Luffin-B粗提液中的杂蛋白就少了很多，便于更好的纯化Luffin-B。2、在将Luffin-B的粗提液进行CM-52阳离子交换层析和Sephadex G-75凝胶过滤层析后，首次尝试采用Superdex 75 HR 10／30高效凝胶过滤层析进一步纯化Luffin-B，取得了很好的纯化效果，可以从100 g丝瓜籽中纯化到42.92g Luffin-B，得率接近于0.043％，比以往纯化方法的得率要高很多。这为以后开展Luffin-B的进一步深入研究提供了很好的基础。 用SDS-PAGE蛋白质变性电泳测定Luffin-B的分子量约为28KDa，这与文献中报道的一致。大鼠肝核糖体在Luffin-B和苯胺作用后产生明显的特征性的R-片断，表明Luffin-B属于典型的RNA N-糖苷酶型核糖体失活蛋白。它对兔网织红细胞裂解液的蛋白质生物合成有强烈的抑制作用，半抑制剂量IC₅₀约为2.1×10^-11M，其活力比天花粉蛋白的IC₅₀ (约为2.8×10^-10M)强很多，有望成为肿瘤导向药物的高效“弹头”。更多还原

【Abstract】 Luffin-B, which was purified from seeds of Luffa cylindrica, is one of the single -chain ribosome-inactivating proteins of the highest activity so far investigated. But the obtain rate of luffin-B purified by previous methods was no less than 0.01%. I changed some procedure based on previous methods:1. The crude extract of Luffin-B first was purified by ice acetum (pH4.2) precipitation in order to get rid of many acidity proteins , followed by protein precipitation with 50%- 85% saturation ammonium sulfate; 2. After the crude extract was chromatographed on a CM-52 ion exchange chromatography column and Sephadex G-75 gel filtration, Superdex 75 , the high performance gel filtration had been used to purify Luffin-B from the seed extract secondly. 42.92 mg Luffin-B can be obtained from 100 g seeds of Luffa cylindrical. The obtain rate is be close to 0.043% ,which is much higher than that of the luffin by previous methods. This has prepared much for the furthest studies about Luffin-B.The purified Luffin-B could be demonstrated as a single band on SDS-PAGE, estimated to be 28 KDa as was previously reported . It can depurinate RNA in rat ribosome and produce the diagnostic R-fragment after rRNA were treated with acid aniline, which proved that Luffin-B is a typical ribosome-inactivating protein of rRNA N-glycosidase activity. It inhibits the protein synthesis of rabbit reticulocyte lysate strongly with a concentration causing 50% inhibition (IC50) of 2.1×10-11 M, much lower than that of trichosanthin(TCS), which is 2.8×10-10 M.So, it is promising that Luffin-Bthan that of trichosanthin(TCS), which is 2.8×10-10 M. So, it is promising that Luffin-B may be used as an effect toxin moiety of immunotoxins.更多还原