草鱼垂体生长激素分离纯化及其抗体制备的研究

ISOLATION AND PURIFICATION OF GROWTH HORMONE FROM GRASS CARP (CTENOPHARYNGODON IDELLUS) AND IT'S PREPARATION OF ANTIBODY AGAINST THE gcGH.

应用碱性抽提、亲和层析、凝胶过滤和离子交换层析等技术从草鱼垂体中分离提纯出纯度高的 草鱼生长激素（ｇｃＧＨ）。用酶联免疫吸附测定法（ＥＬＩＳＡ）检测表明草鱼ＧＨ与大马哈鱼ＧＨ抗血清 具有明显的免疫交叉反应，而与大马哈鱼催乳素（ＰＲＬ）抗血清没有交叉反应，聚丙烯酰胺凝胶电 泳表明草鱼 ＧＨ为单一蛋白带，其分子量约为２２５００道尔顿；等电聚焦揭示出草鱼 ＧＨ主要由等电 点为４．７和５．０的两条蛋白带组成。用纯化的草鱼ＧＨ制备了其特异的兔抗血清。在ＥＬＩＳＡ测定中， 该抗血清与大马哈鱼ＧｔＨ及牛ＧＨ完全没有交叉反应，而与大马哈鱼ＧＨ只有当其浓度高达２ｕｇ／ ｍｌ时，才有微弱的交叉反应．草鱼 ＧＨ抗血清的效价确定为１： ４００００左右。应用草鱼 ＧＨ及其抗血清 建立了酶标受体测定法（ＥＬＩＳＡ－ＲＡ）并检测了草鱼ＧＨ的生物活性。结果表明，草鱼ＧＨ具有与草 鱼肝细胞ＧＨ受体特异结合的生物活性。免疫细胞化学研究表明草鱼ＧＨ抗血清只与草鱼垂体间 叶（ＰＰＤ）的生长激素分泌细胞特异结合，而与前叶（ＲＰＤ）及后叶（ＰＩ）细胞均无染色反应。

A highly purified growth hormone (GH) was isolated from the pituitary of grass carp by alkaline extraction, affinity chromatography on Con A-Sepharose 4B, gel filtration on Sephadex G-100 and ion-exchange chromatography on DE-52 cellulose. The GH was monitored by enzyme-linked immunosorbent assay (ELISA) using salmon GH and prolactin (PRL) antiserum, ELISA revealed that grass carp GH (gcGH) showed significant cross reaction with salmon GH antiserum when the coating concentration of gcGH was high. No cross reaction of gcGH with salmon PRL antiserum was observed, gcGH migrated as a single protein band with molecular weight of 22500 dalton in reduced SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Isoelectric focusing revealed that gcGH consisted of two bands with isoelectric points of 4. 7 and 5. 0 Specific gcGH antiserum was prepared in rabbits with a titre of about 1: 40000, ELISA indicated that gcGH antiserum did not show cross reaction with salmon GH、 gonadotropin (GtH) and bovine GH. ELISA-receptor assay (ELISARA) was used to monitor gcGH bioactivity. gcGH showed significant binding capacity with grass carp liver membrane receptor preparation. The specificity of the gcGH antiserum was confirmed by immunocytochemstry studies. gcGH antiserum specifically stained cells in the proximal pars distalis (PPD). No cross reaction with the cells in the rostral pars distalis (RPD) and pars intermedia (PI) was observed.