Kato Y, Imai K, Hanaoka Y
Endocrinol Jpn. 1986 Dec;33(6):883-9. doi: 10.1507/endocrj1954.33.883.
This study was performed to improve the purification of Xenopus vitellogenin and establish the radioimmunoassay. The procedure of purification consisted of ammonium precipitation, DEAE-Sephadex chromatography and Sephadex G-200 gel chromatography. Using this procedure, 934 mg vitellogenin was purified from 49 ml of estradiol treated female Xenopus plasma (about 19 mg/ml). Vitellogenins purified from male and female plasma after a single injection of estradiol showed good correspondence in electrophoretic patterns and amino acid compositions, indicating that vitellogenin synthesis in the male occurs in four different genes as in the female. The radioimmunoassay for vitellogenin was established using an antibody in the plasma obtained from rabbits injected with purified Xenopus female vitellogenin. The titer was 20,000 times dilution of the plasma, and the minimum detectable amount of vitellogenin was 0.1 microgram. The cross-reactivity of this antibody with newt vitellogenin was about 65% and that of chick 6%. The cross-reaction was also observed in female bullfrog plasma. Vitellogenin content was increased gradually during the first 6 days after injection of estradiol in female and the elevated level of vitellogenin dropped afterward.
本研究旨在改进非洲爪蟾卵黄蛋白原的纯化方法并建立放射免疫测定法。纯化过程包括硫酸铵沉淀、DEAE-葡聚糖凝胶色谱和葡聚糖G-200凝胶色谱。采用该方法，从49毫升经雌二醇处理的雌性非洲爪蟾血浆（约19毫克/毫升）中纯化得到了934毫克卵黄蛋白原。单次注射雌二醇后，从雄性和雌性血浆中纯化得到的卵黄蛋白原在电泳图谱和氨基酸组成上显示出良好的对应性，这表明雄性中卵黄蛋白原的合成与雌性一样，是由四个不同的基因进行的。利用从注射了纯化的非洲爪蟾雌性卵黄蛋白原的兔子血浆中获得的抗体，建立了卵黄蛋白原的放射免疫测定法。效价为血浆稀释20000倍，卵黄蛋白原的最低可检测量为0.1微克。该抗体与蝾螈卵黄蛋白原的交叉反应性约为65%，与鸡的交叉反应性为6%。在雌性牛蛙血浆中也观察到了交叉反应。在雌性中，注射雌二醇后的前6天卵黄蛋白原含量逐渐增加，之后升高的卵黄蛋白原水平下降。
