吳郭魚腦下腺泌乳激素之研究

Abstract

吳郭魚腦下腺取出後置於乾冰中，以研缽磨碎，加水溶解，以Ca(OH)2調pH值至9.5，於4 ℃攪拌3小時後，離心取上層液，此粗萃液經透析、冷凍乾燥後，溶於100 mM NH4HCO3緩衝液中，通過 Sephacry l-200管柱，得到3個主要的蛋白質吸收?。由SDS-PAGE檢測得知第 3 個吸收?的蛋白質分子量約在22 KD 左右，故回收此部份，冷凍乾燥。再以HPLC C18 管柱分離，最後得到 3 個相當純的蛋白質（A 、B 、C ) ，其分子量分別為24.2 KD、25.5 KD、22.6 KD 。此3個蛋白質的 N－端部份胺基酸序列及胺基酸組成與其他數種硬骨魚的泌乳激素非常類似。經由泌乳激素的生物活性分析，證實此 3 種蛋白質確實都具有魚類泌乳激素的主要功能—鈉離子保留的能力。 以三者中含量最多的 B 注射到兔子體內，誘發抗體、製備抗血清。並以此建立吳郭魚泌乳激素的放射免疫分析系統。由放射免疫分析及免疫沈澱擴散分析，發現 A 和 C 都可和 B 有交叉反應，顯示出此 3 種泌乳激素在免疫結構上的類似性。

Tilapia pituitaries were dissected out, frozen on dry ice, then homogenized in a mortar with cold distilled water, adjusted to pH 9.5 with Ca(OH)2 and stirred at 4 ℃ for three hours. After centrifugation the alkaline extract was dialyzed and lyophilized. This extract was dissolved in 100mM NH4HCO3 buffer and chromatographed on Sephacryl S-200 column. Three major protein peaks were obtained. Each peak was examined by SDS-PAGE. The third peak which contained the proteins of about 22kD was recovered and lyophilized. This fraction was further purified by HPLC on a reverse-phase C18 column. The resulting three pure proteins (named A, B and C in order) have molecular weights of 24.2kD, 25.5kD and 22.6kD, respectively. The N-terminal partial amino acid sequences and amino acid compositions of the three proteins are similar to those of other teleost prolactins. Through prolactin bioassay it was proved that the three proteins do have the major function of fish prolactin—sodium retaining activity. Antiserum was prepared by immunizing a rabbit with Tilapia PRL B which had the highest yield in three proteins. This antiserum was used to construct radioimmunoassay system of Tilapia prolactin. Both PRLA and C showed cross reaction in radioimmunoassay and double diffusion test, indicating the similarity among these prolactins in immunological property.