Lab in Biotechnology & Biosignal Transduction, Department of Orthodontics, Saveetha Dental College & Hospitals, Chennai 600077, Tamil Nadu, India.
Fisheries Science Institute, Chonnam National University, 50 Daehak-ro, Yeosu 59626, Republic of Korea.
Int J Mol Sci. 2023 Jul 15;24(14):11494. doi: 10.3390/ijms241411494.
The seafood industry plays a huge role in the blue economy, exploiting the advantage of the enriched protein content of marine organisms such as shrimps and molluscs, which are cultured in aquafarms. Diseases greatly affect these aquatic organisms in culture and, hence, there is need to study, in detail, their innate immune mechanisms. Hemocyanin is a non-specific innate defense molecule present in the blood cells of several invertebrates, especially molluscs, arthropods, and annelids. It is concerned with oxygen transport, blood clotting, and immune enhancement. In the present study, this macromolecular metalloprotein was isolated from the hemolymph of the marine snail (Born, 1778) using Sephadex G-100 gel filtration column chromatography. It occurred as a single band (MW 80 kDa) on SDS-PAGE. High-performance liquid chromatography (HPLC) of the purified hemocyanin showed a single peak with a retention time of 4.3 min. The secondary structure and stability of the protein were detected using circular dichroism (CD), and the spectra demonstrated negative ellipticity bands close to 208 nm and 225 nm, indicating β-sheets. Further exploration of the purified hemocyanin revealed remarkable antimicrobial and antibiofilm activities against Gram-positive ( and ) and Gram-negative bacteria ( and ) at a concentration of 1-5 μg/mL. Spectrophotometric and in situ microscopic analyses (CLSM) unveiled the potential of the purified hemocyanin to inhibit biofilm formation in these bacteria with a minimal inhibitory concentration of 40 μg/mL. Furthermore, hemocyanin (10 μg/mL concentration) displayed antifungal activity against . The purified hemocyanin was also assessed for cytotoxicity against human cancer cells using cell viability assays. Altogether, the present study shows that molluscan hemocyanin is a potential antimicrobial, antibiofilm, antifungal, anticancer, and immunomodulatory agent, with great scope for application in the enhancement of the immune system of molluscs, thereby facilitating their aquaculture.
贝类血蓝蛋白的分离纯化及其抗菌、抗生物膜活性研究
贝类血蓝蛋白是一种存在于多种无脊椎动物（特别是软体动物、节肢动物和环节动物）血细胞中的非特异性先天防御分子，它与氧气运输、血液凝结和免疫增强有关。在本研究中，采用 Sephadex G-100 凝胶过滤柱层析法从海洋蜗牛（Born，1778）的血淋巴中分离出这种大分子金属蛋白。SDS-PAGE 显示它为单一带（MW 80 kDa）。纯化的血蓝蛋白的高效液相色谱（HPLC）显示单一峰，保留时间为 4.3 min。使用圆二色性（CD）检测蛋白质的二级结构和稳定性，光谱显示接近 208nm 和 225nm 的负椭圆率带，表明存在β-折叠。进一步探索纯化的血蓝蛋白表明，在 1-5μg/mL 的浓度下，对革兰氏阳性菌（ 和 ）和革兰氏阴性菌（ 和 ）具有显著的抗菌和抗生物膜活性。分光光度法和原位显微镜分析（CLSM）揭示了纯化的血蓝蛋白在这些细菌中抑制生物膜形成的潜力，最小抑制浓度为 40μg/mL。此外，血蓝蛋白（浓度为 10μg/mL）对 表现出抗真菌活性。还使用细胞活力测定法评估了纯化的血蓝蛋白对人癌细胞的细胞毒性。总之，本研究表明，软体动物血蓝蛋白是一种有潜力的抗菌、抗生物膜、抗真菌、抗癌和免疫调节剂，在增强贝类免疫系统方面具有广阔的应用前景，从而促进其水产养殖。
