Crustacean Molecular Biology and Genomics Division, Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Science Block 6th floor, Burma Colony, Karaikudi 630004, Tamil Nadu, India.
Crustacean Molecular Biology and Genomics Division, Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Science Block 6th floor, Burma Colony, Karaikudi 630004, Tamil Nadu, India.
Int J Biol Macromol. 2018 Jul 15;114:864-873. doi: 10.1016/j.ijbiomac.2018.03.159. Epub 2018 Mar 27.
In this study, we purified β-GBP from hemolymph of Scylla serrata crabs using affinity chromatography. The purified S. serrata β-GBP (Ss-β-GBP) had 100kDa molecular mass in the SDS-PAGE. MALDI-TOF/TOF analysis was conducted, revealing that the purified 100kDa protein had 96% similarity with β-GBP of Astacus leptodactylus. Ss-β-GBP was characterized using high-performance liquid chromatography (HPLC), X-ray diffraction (XRD) analysis, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, which confirmed the structure of the Ss-β-GBP. The purified Ss-β-GBP was functionally analyzed by yeast agglutination and phagocytic reaction assays. Moreover, the PO enhancing ability of Ss-β-GBP was evidenced through PO activity. Specifically, the antibacterial activity of the Ss-β-GBP against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria was evaluated by determining its minimum inhibitory concentration (MIC)<60μg/ml for all tested species. Furthermore, the antibiofilm efficacy of Ss-β-GBP at 50 and 100μg/ml was outlined using light microscopy and confocal laser scanning microscopy (CLSM). Bacterial viability assays also outlined the dose-dependent activity of Ss-β-GBP based on the ratio of live/dead bacterial cells. The results of this study revealed that crab-borne Ss-β-GBP might be widely used to suppress the growth of pathogenic bacteria.
在这项研究中，我们使用亲和层析从锯缘青蟹的血淋巴中纯化β-GBP。纯化的锯缘青蟹β-GBP（Ss-β-GBP）在 SDS-PAGE 中具有 100kDa 的分子量。进行 MALDI-TOF/TOF 分析，结果表明，纯化的 100kDa 蛋白与Astacus leptodactylus的β-GBP具有 96%的相似性。使用高效液相色谱（HPLC）、X 射线衍射（XRD）分析、圆二色性（CD）和傅里叶变换红外（FTIR）光谱对 Ss-β-GBP 进行了表征，证实了 Ss-β-GBP 的结构。通过酵母凝集和吞噬反应试验对纯化的 Ss-β-GBP 进行了功能分析。此外，通过 PO 活性证实了 Ss-β-GBP 的 PO 增强能力。具体而言，通过测定其对革兰氏阳性（粪肠球菌和金黄色葡萄球菌）和革兰氏阴性（大肠杆菌和铜绿假单胞菌）细菌的最小抑菌浓度（MIC）<60μg/ml，评估了 Ss-β-GBP 对所有测试物种的抗菌活性。此外，使用明场显微镜和共聚焦激光扫描显微镜（CLSM）概述了 Ss-β-GBP 在 50 和 100μg/ml 时的抗生物膜功效。细菌活力测定也根据活/死细菌细胞的比例概述了 Ss-β-GBP 的剂量依赖性活性。这项研究的结果表明，源自螃蟹的 Ss-β-GBP 可能被广泛用于抑制病原菌的生长。
