中华绒螯蟹卵黄磷蛋白单克隆抗体制备及免疫组化应用

Production of Monoclonal Antibody against Vitellin of <i>Eriocheir sinensis </i> and Its Application in Immunohistochemistry

本研究通过对中华绒螯蟹成熟卵巢中的卵黄磷蛋白进行分离,并使用全自动蛋白纯化仪和胶回收的方对其进行纯化,以此为抗原免疫小鼠,使之产生针对性抗体后进行融合筛选,获得稳定额单克隆细胞株,将细胞株培养后打入小鼠腹腔,制备腹水。随后从腹水中纯化收集抗体,并进行Elisa效价监测,结果表明,获得三株单克隆抗体2N17、3G2、2J11效价均高于64 K,抗体浓度为2N17:0.8 mg/ml,3G2:0.8 mg/ml,2J11:0.7 mg/ml。随后通过免疫组化技术对中华绒螯蟹卵黄磷蛋白在其胚胎和幼体中进行定位,结果表明,胚胎发育在囊胚期和原肠期时,卵黄磷蛋白强阳性位点均分布于卵黄物质中,聚集与卵黄岛周边,当胚胎发育至心跳期时,卵黄囊中已无卵黄磷蛋白阳性物质变弱,仅仅分布于卵黄囊外周,围绕肝胰腺前体细胞有弱阳性位点,随后胚胎孵化,卵黄磷蛋白阳性位点消失,大眼幼体的肝胰腺区域无发现,暗示中华绒螯蟹在胚胎发育中期是卵黄磷蛋白动用高峰,而末期卵黄磷蛋白已消耗殆尽,初孵幼体已不带有卵黄磷蛋白。

The vitellogenin protein in the mature ovary of the Chinese mitten crab (Eriocheir sinensis) was separated, and purified by automatic protein purification instrument and native polyacrylamide gel electrophoresis (Native-PAGE). Mice were immunized with vitellogenin as antigen to produce targeted antibodies. After fusion and selection, a stable monoclonal cell line was obtained. The cell line was cultured and injected into the abdominal of mice to prepare ascites. The antibodies were purified and collected from ascites and titer monitored by Elisa. The titers of the three obtained monoclonal antibodies 2N17, 3G2 and 2J11 were all higher than 64 K, and antibody concentrations were 0.8 mg/ml, 0.8 mg/ml, 0.7 mg/ml, respectively. Then, immunohistochemical technology was performed to locate the vitellin in the embryonic and larvae of Eriocheir sinensis. The results showed that the strong positive sites of the vitellin were distributed in the vitellus material and gathered around the vitellus island during the blastula stage and the gastrula stage. When the embryo developed to the heart beating stage, there was no vitellin in the yolk sac and positive material became weaker. It was only distributed on the periphery of the yolk sac. Weak positive sites were around the hepatopancreas precursor cells, and then the positive sites disappeared after embryo hatching. No positive sites were found in the hepatopancreas of Megalopa, suggesting that the peak of vitellin mobilization was in the middle of embryo development. Vitellin was exhausted in the late stage of embryo development and no exist in the newly hatched larvae.