草魚泌乳激素cDNA的選殖與核酸定序

Abstract

本研究主要目的在純化鼠類子宮分泌的Lactotransferrin，並製備專一性高的Anti-Lactotransferrin抗體，作為酵素免疫分析(ELISA)及西方墨點法(Western blotting)方法使用。

酵素免疫分析法的結果知 (l) 血清中，雌雄老鼠都有但雌的略高於雄的，但其量每ug血清中約為1ng的Lactotransferrin。 (2) 生殖器官中，雄老鼠只有貯精囊有且量極高，睾丸、副睾、前攝護腺都沒有，雌老鼠卵巢、陰道、乳線存在有，子宮則未測試到。(3)其他組織腦、肝、心、肺、胰、腎、脾、胸線、十二指腸中，雌雄老鼠二者都存在的有肝、脾、腎，雌的含量略高於雄的含量。另外，雌的胸腺有而雄的沒有。雌的腦中有時可以測到。西方墨點法(Western blotting)的結果，除腦外其餘組織均可測試到有一 65K的蛋白質會和Anti-Lactotransferrin抗體反應。有人報告此65K可能是Lactotransferrin的Low Molecular weight型式，而有待進一步證實。

Among the hormones of fish pituitary gland, prolactin (PRL) plays an important role in osmoregulation and freshwater adaption. For the purpose of undersdanding the amino acid sequence of grass carp (Ctenopharyngodon idellus) PRL, a cDNA library was constructed from the pituitary gland using λ gt10 as cloning vector. The carp PRL cDNA, which was obtained by screening the carp pituitary cDNA library with 32-mer synthetic probe, was labeled by nick translation and used as probes to screen the cDNA library for grass carp PRL. Four positive clones was obtained and the longest cDNA was subjected to nucleotide sequence analysis. It is shown that the cDNA is 633 bp in length and contains a coding region of 273 bp and a non-coding region of 357 bp. While the deduced amino acid sequence of this 91 amino acid residue polypeptide shows high extent of homology (94.5%) with the carboxyl terminal portion of carp PRL( i.e. amino acid residue 97 to 187), it is 64% similar to salmon PRL and only 30% homolgous to human PRL.