应用蛋白质组学方法研究盐角草（Salicornia europaea L.）的抗盐性-盐角草双向电泳体系的建立

盐角草（Salicornia europaea L.）是世界上最抗盐的高等植物之一。应用蛋白质组学方法对其抗盐性进行研究，对于我们理解植物抗盐机理和改进作物耐盐性都有重要意义。双向电泳是蛋白质组学的核心技术，样品制备是双向电泳的关键步骤。盐角草是一种聚盐的真盐生高等植物，其体内除含有多种次生代谢物质外还含有大量盐分，而盐离子的存在严重干扰等电聚焦的进行，其蛋白质提取较其他植物更为困难。因此，在进行蛋白质组学研究之前，有必要对其蛋白质提取方法进行摸索，建立一个高效的双向电泳体系。 　　比较了三氯乙酸/丙酮沉淀法（TCA）、三氯乙酸沉淀法（E-TCA）和酚抽法（Phe）三种方法对盐角草蛋白的提取效果。提取700mM处理2d的盐角草幼苗蛋白时，三种方法分别得到579，343和535个蛋白点;TCA和E-TCA法所得图谱均存在严重的横向纹理，Phe法所得图谱则背景干净，基本上没有纹理。说明Phe法不仅能很好地提取盐角草蛋白，而且能有效去除样品中的盐分。对200mM处理90d的盐角草蛋白的提取也证实了这一点。比较了不同沉淀剂对Phe法蛋白提取效果的影响，结果表明，甲醇不能代替乙酸铵甲醇溶液。对Phe法的提取液进行了改进，所得图谱背景更加清晰，蛋白点数增加。此外，还对聚焦时间，上样量和染色方法进行了改进优化，建立了盐角草双向电泳体系。 　　本研究表明，Phe法适合于盐角草双向电泳样品的制备，这为其他盐生植物以及嗜盐微生物蛋白质的提取提供了重要参考。 　　

Identification and characterization of a novel envelope protein in Rana grylio virus

Viral envelope proteins have been proposed to play significant roles in virus infection and assembly. In this study, an envelope protein gene, 53R, was cloned and characterized from Rana grylio virus (RGV), a member of the family Iridoviridae. Database searches found its homologues in all sequenced iricloviruses, and sequence alignment revealed several conserved structural features shared by virus capsid or envelope proteins: a myristoylation site, two predicted transmembrane domains and two invariant cysteine residues. Subsequently, RT-PCR and Western blot detection revealed that the transcripts encoding RGV 53R and the protein itself appeared late during infection of fathead minnow cells and that their appearance was blocked by viral DNA replication inhibitor, indicating that RGV 53R is a late expression gene. Moreover, immunofluorescence localization found an association of 53R with virus factories in RGV-infected cells, and this association was further confirmed by expressing a 53R-GFP fusion protein in pEGFP-N3/53R-transfected cells. Furthermore, detergent extraction and Western blot detection confirmed that RGV 53R was associated with virion membrane. Therefore, the current data suggest that RGV 53R is a novel viral envelope protein and that it may play an important role in virus assembly. This is thought to be the first report on a viral envelope protein that is conserved in all sequenced iridoviruses.

Electrochemiluminescent detection based on solid-phase extraction at tris(2,2 '-bipyridyl)ruthenium(II)-modified ceramic carbon electrode

A sensitive electrochemiluminescent detection scheme by solid-phase extraction at Ru(bpy)(3)(2+)-modified ceramic carbon electrodes (CCEs) was developed. The as-prepared Ru(bpy)(3)(2+)-modified CCEs show much better long-term stability than other Nafion-based Ru(bpy)(3)(2+)-modified electrodes and enjoy the inherent advantages of CCEs. The log-log calibration plot for dioxopromethazine is linear from 1.0 x 10(-9) to 1.0 x 10(-4) mol L-1 using the new detection scheme. The detection limit is 6.6 x 10(-10) mol L-1 at a signal-to-noise ratio of 3. The new scheme improves the sensitivity by similar to 3 orders of magnitude, which is the most sensitive Ru(bpy)(3)(2+) ECL method. The scheme allows the detection of dioxopromethazine in a urine sample within 3 min. Since Ru(bpy)(3)(2+) ECL is a powerful technique for determination of numerous amine-containing substances, the new detection scheme holds great promise in measurement of free concentrations, investigation of protein-drug interactions and DNA-drug interactions, pharmaceutical analysis, and so on.