海鲜产业在蓝色经济中发挥着巨大的作用,利用虾和软体动物等海洋生物蛋白质含量丰富的优势,在水产养殖场培养。疾病极大地影响了培养中的这些水生生物,因此,有必要学习,在细节上,它们的先天免疫机制。血蓝蛋白是一种存在于几种无脊椎动物血细胞中的非特异性先天防御分子,尤其是软体动物,节肢动物,和环节动物。它与氧气运输有关,血液凝固,和免疫增强。在本研究中,这种大分子金属蛋白是从海洋蜗牛半毛猪的血淋巴中分离出来的(Born,1778)采用SephadexG-100凝胶过滤柱层析。它在SDS-PAGE上作为单条带(MW80kDa)出现。纯化的血蓝蛋白的高效液相色谱(HPLC)显示单峰,保留时间为4.3min。利用圆二色性(CD)检测蛋白质的二级结构和稳定性,光谱显示负椭圆率带接近208nm和225nm,表示β-折叠。对纯化的血蓝蛋白的进一步探索显示,浓度为1-5μg/mL时,对革兰氏阳性(粪肠球菌和金黄色葡萄球菌)和革兰氏阴性细菌(铜绿假单胞菌和寻常变形杆菌)具有显着的抗菌和抗生物膜活性。分光光度和原位显微镜分析(CLSM)揭示了纯化的血蓝蛋白以40μg/mL的最小抑制浓度抑制这些细菌中生物膜形成的潜力。此外,H.pugilinus血蓝蛋白(10μg/mL浓度)对黑曲霉具有抗真菌活性。还使用细胞活力测定评估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潜在的抗菌药物,抗生物膜,抗真菌药,抗癌,和免疫调节剂,在增强软体动物的免疫系统方面具有很大的应用范围,从而促进他们的水产养殖。
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 Hemifusus pugilinus (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 (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa and Proteus vulgaris) 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, H. pugilinus hemocyanin (10 μg/mL concentration) displayed antifungal activity against Aspergillus niger. 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.