School of Marine Biomedical Science (BK21-PLUS), Jeju National University, 102 Jejudaehakno, Jeju, Republic of Korea.
Department of Chemistry, Faculty of Engineering and Technology, NSHM Knowledge Campus, Durgapur, West Bengal, India.
Protein J. 2018 Feb;37(1):82-92. doi: 10.1007/s10930-017-9753-7.
A polyclonal antibody specific to an egg protein of Suminoe oyster Crassostrea ariakensis was previously developed in our laboratory to assess the reproductive life cycle of the oyster. The present study was undertaken to investigate vitellin of C. ariakensis (CAVt). Vitellin is an essential component of egg proteins in marine invertebrates as it provides energy and nutrients to the embryo and larvae. CAVt was purified from eggs of the oyster using ammonium sulfate precipitation followed by affinity chromatography with Concanavalin A-agarose. Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE showed that CAVt is a high molecular weight [532 kiloDaltons (kDa)] protein, with multiple subunits. Similar to other vitellin proteins, it is a phospholipoglycoprotein composed of phospholipids (12.06%), carbohydrates (mannose, 10.08% or glucose, 9.84%), and alkali-labile phosphates (4.16%). Affinity chromatography, enzyme-linked immunosorbent aasay (ELISA) and western blot analysis revealed that CAVt is only present in the ovary, and two subunits of CAVt (72 and 35 kDa) are believed to be incorporated from the hemolymph into the oocyte. The antibody specific to CAVt (anti-CAVt), raised in rabbit, strongly cross reacted with the egg proteins of oyster species and scallops, suggesting that the antigenic epitopes are highly conserved among species. Our results suggest that the anti-CAVt antibody can be used to develop a tool similar to ELISA or western blotting for investigation of the effect of microorganisms on reproduction as well as the effect of chemicals on the endocrine system in C. ariakensis.
先前，我们实验室开发了一种针对日本囊对虾卵蛋白的多克隆抗体，用于评估牡蛎的生殖生命周期。本研究旨在研究日本囊对虾卵黄蛋白（CAVt）。卵黄蛋白是海洋无脊椎动物卵蛋白的重要组成部分，因为它为胚胎和幼虫提供能量和营养。CAVt 是从牡蛎卵中通过硫酸铵沉淀和 Concanavalin A-琼脂糖亲和层析纯化得到的。天然聚丙烯酰胺凝胶电泳（PAGE）和十二烷基硫酸钠 PAGE 显示，CAVt 是一种高分子量（532 千道尔顿（kDa））的蛋白质，具有多个亚基。与其他卵黄蛋白蛋白一样，它是一种由磷脂（12.06%）、碳水化合物（甘露糖，10.08%或葡萄糖，9.84%）和碱不稳定的磷酸盐（4.16%）组成的磷糖蛋白。亲和层析、酶联免疫吸附测定（ELISA）和 Western blot 分析表明，CAVt 仅存在于卵巢中，CAVt 的两个亚基（72 和 35 kDa）被认为是从血液中进入卵母细胞的。针对 CAVt 的抗体（抗 CAVt）在兔中产生，与牡蛎和扇贝的卵蛋白强烈交叉反应，表明抗原表位在物种间高度保守。我们的结果表明，抗 CAVt 抗体可用于开发类似于 ELISA 或 Western blot 的工具，以研究微生物对生殖的影响以及化学物质对日本囊对虾内分泌系统的影响。
