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

本研究以水稻半胱氨酸蛋白酶抑制剂（Oryzacystatins：OCs）为重组蛋白，对其制备及性质进行了研究。本文还对大米和米糠中抗菌蛋白的筛选进行了初步研究。本研究的主要结果如下：1.将五种类型的重组OCs在大肠杆菌BL 21中表达为GST或His标签融合蛋白，并通过常规方法纯化。测定了OCs对几种半胱氨酸蛋白酶的抑制活性.为了更灵敏地测定抗菌活性，建立了浊度法、ATP化学发光法和DNA结合剂荧光法，并比较了它们的性能.在大米和米糠提取物中检测到牙龈卟啉单胞菌蛋白酶（Arg-和Lys-gingipains）的抑制蛋白.从米糠提取物中获得的分子量为1-16 kDa的蛋白质对牙龈卟啉单胞菌具有抗微生物活性。通过二维电泳纯化的蛋白质的化学结构进行了分析，MALDI-TOF-MS的纯化样品中的几个蛋白被确定为抗菌蛋白的候选者。

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.