小体鲟卵黄蛋白生化特性及合成途径的研究

STUDY ON THE BIOCHEMICAL CHARACTER AND SYNTHESIS PATHWAY YOLK PROTEIN IN ACIPENSER RUTHENUS

使用葡聚糖凝胶(Sephadex G-200)从小体鲟鱼卵粗提液中,提纯卵黄脂磷蛋白(Lipovitellin,Lv)和卵黄高磷蛋白(Phosvitin,Pv)。卵黄脂磷蛋白(含糖、磷和脂,等电点7.50)具有雌性特异性,分子量为144kD,由97.4kD和30kD的大小2个亚基组成。卵黄高磷蛋白(含糖、磷,等电点8.30)其分子量为66kD,其具有两个亚基,分子量分别为47.6kD、16.8kD。制备卵黄脂磷蛋白兔抗血清,采用免疫组化方法对不同年龄小体鲟的肝脏、肠、卵(Ⅱ—Ⅴ期卵巢)及血涂片,进行免疫组织化学定位研究。小体鲟卵巢发育到Ⅳ期前,卵黄蛋白主要靠卵母细胞自身合成,这个时期内源性合成卵黄蛋白;当卵巢发育到Ⅳ期,卵母细胞自身不合成卵黄蛋白,主要是通过肝脏合成卵黄蛋白原,通过血液循环运送到卵巢,被卵母细胞吸收后,裂解为卵黄蛋白,这个时期外源性合成卵黄蛋白。

Yolk is the primary nutrition material during the development of embryo,of which vitellin is primary constitute element. In order to know the biochemical characters and synthesis pathway of yolk protein from Acipenser ruthenus, the yolk protein was abstracted. The biochemical character and synthesis pathway of yolk protein from Acipenser ruthenus were studied by chromatography with Sephadex G-200, polyacrylamide gel electrophoresis, westeru-blotting and immunohisto- chemistry. The results showed that yolk protein was composed of lipovitelin, yolk immunoglobulin and Phos- vitin. Lipovitellin was a kind of glycosyl, phosphorus and lipid protein and it was same as female serum specific protein （FSSP）. It had female specificity and its antibody had genus and tissue specificity. The molecular weight of Lipovitellin was 144kD,which was composed of two subunits with molecular weight of 97.4kD and 30kD, respectively, its isoelectric point was 7.50. Phosvitin was made up of two subunits with molecular weight of 47.6kD and 16.8kD,respeetively,its mo- lecular weigh was 66kD and isoelectric point was 8.65. The rabbit antiserums of Lipovitellin were prepared, and immunohistochemistry location was investigated in liver,in- testine,egg （ Ⅱ-Ⅴ period ovary） and blood smears at different age. The results showed that liver cells of female sturgeon at Ⅳ stage ovary and above IV stage ovary were immunostained with DAB,which indicated the liver eeUs were positive to special antibody and positive reaction occurred mainly in the cytoplasm of liver cells. No special immunostaining was ob- served either in liver cells of male sturgeon and female sturgeon which ovary was during Ⅰ --Ⅲ period or in control group. The surface of blood cells of female sturgeon which gonad was at Ⅳ- Ⅴ stage was positive to special antibody. In contrast,the surface of blood cells of mature male sturgeon,female sturgeon during Ⅰ--Ⅳ period ovary and negative con- trol were negative to special antibody. The intestine cells of anterior intestine, midintestine and posterior intestine before maturing in both male and female sturgeon were also negative to special antibody. There were a lot of positive reaction sub- stances in nucleus during Ⅱ period ovary. Grand growth phase 3 primary oocyte occupied the greater part of Ⅲ stage ovary which could be divided into nonage, metaphase and anaphase stage. A lot of positive reaction substances existed in nucleus at nonage stage of m stage ovary. When the ovary developed to metaphase,positive reaction substances of nucleus began to decrease, whereas, it appeared in cytoplasm. The cells were oval and the positive reaction substances increased at anaphase stage. There were no positive reaction substances at IV stage ovary but yolk granule appeared positive reaction. A lot of yolk granule which were positive in oocyte appeared in Ⅴ stage ovary. In conclusion,the synthesis style of yolk protein from Acipenser ruthenus Linnaeus was belonged to facuhative. Before Ⅳ stage of ovary,the yolk protein was mainly synthesized by oocyte oneself,which was autosynthesis. When ovary developed to Ⅳ stage,oocytes stoped to synthesize yolk protein and liver cells began to synthesize vitellogenin which was trans- ported to ovary by blood circulation. After endocytosis by developing oocytes,vitellogenin was enzymatieally converted into egg yolk protein,which was endogenesis.