Genetic and proteomic analysis of novel antimicrobial proteins from rice and their application to harmful microorganisms

本研究对半胱氨酸蛋白酶抑制剂(oryzacystatins：OCS)重组蛋白的制备及其性质进行了研究。并对大米和米皮中抗牙龈卟啉单胞菌的抗菌蛋白进行了筛选。本研究取得的研究结果总结如下：1.以GST或His-Tag融合蛋白的形式在大肠杆菌BL21中表达了5种类型的重组OCS，并用常规方法纯化。结果：1.测定了OCS对几种半胱氨酸蛋白酶的抑制活性。为了灵敏地检测抗菌活性，建立了浊度法、三磷酸腺苷化学发光法和DNA结合试剂荧光法，并对它们的性能进行了比较。在大米和米饭的提取液中检测到牙龈假单胞菌对精氨酸和赖氨酸蛋白水解酶的抑制蛋白。从米糠提取液中提取的分子量为1-16 kDa的蛋白质对牙周炎假单胞菌具有一定的抑菌活性。用MALDI-TOF-MS分析了二维电热纯化的蛋白质的化学结构。纯化的样品中的几个蛋白质被鉴定为抗菌蛋白质的候选。

In this study, the preparation of cysteine protease inhibitors (oryzacystatins: Ocs) as a recombinant protein and their properties were investigated. The screening of antimicrobial proteins from rice and rice bran against Porphyromonas gingivalis was also studied. The results obtained in this study were summarized as follows.1. Five types of recombinant-OCs were expressed in Escherichia coli BL21 as the GST or His-tag fusion protein and purified by the conventional method. The inhibitory activities of OCs for several cysteine proteases were estimated.2. To measure sensitively antimicrobial activity, turbidity assay, ATP-chemical luminescence assay and fluorescence assay using DNA-binding reagent were developed and their performances were compared.3. The proteins inhibitory to proteases (Arg- and Lys-gingipains) from P. gingivalis were detected in the extracts of rice and rice bran.4. The proteins of MW 1-16 kDa obtained from the extract of rice bran have antimicrobial activity against P. gingivalis. The chemical structure of the proteins purified by two-dimensional electrohoresis was analyzed by MALDI-TOF-MS. The several proteins in the purified samples were identified as candidates of antimicrobial proteins.