哇巴因结合肽氨基酸序列的测定

Investigation of amino acid sequence of specific ouabain conjugated peptides

目的确定能与哇巴因特异性结合的多肽氨基酸及基因序列,为实现阻断或拮抗哇巴因与钠泵的作用奠定实验基础。方法采用噬菌体随机肽库表面呈现技术筛选出哇巴因特异性结合肽,通过测序,获得氨基酸序列,进行同源性分析、合成筛选出的12肽,人工合成哇巴因结合肽,并检测其与哇巴因的结合能力。结果从噬菌体12肽库中筛选出3种多肽。肽A(12肽)的筛选一致率达到667%(8/12);肽B(8肽)筛选一致率为167%(2/12);肽C(12肽)为83%(1/12);仅1例未见插入的多肽片段。GenBank中蛋白质同源性分析结果肽A、B、C均未见同源蛋白。获得特异性哇巴因结合肽的氨基酸序列为Leu-Leu-Ala-Asp-Thr-Thr-His-His-Arg-Pro-Trp-Thr。结论哇巴因特异性结合肽氨基酸及DNA序列的获得,为哇巴因的研究提供了实验基础;同时,本研究所采用的方法也为使用噬菌体随机肽库进行甾体类激素特异性受体(配体)的筛选提供了可靠的实验依据。

AIM: The purpose of the study was to investigate the gene and amino acid sequence of specific ouabain conjugated peptides (OCP) in order to get an experimental bases to block or antagonist the actions between endogenous ouabain(EO) and sodium pump in hypertension. METHODS: Screening the phage displayed 12-peptide library by biopaning for OCP. The sequence of each selected peptide was determined and the sequence was analyzed through internet. The bioactivity was determined by erythrocyte [ 86 Rb] uptaking. RESULTS: Three kinds of peptides were screened out. Peptide A (12 peptide) was occupied in 66 7%(8/12), peptide B (8 peptide) 16 7% (2/12) and peptide C (12 peptide) 8 3% (1/12). There was only one case without insertron. The analysis of protein showed that there were no homogenous between peptide A, B, C and sodium pump. The amino acid sequence of specific OCP was Leu-Leu-Ala-Asp-Thr-Thr-His-His-Arg -Pro-Trp-Thr. CONCLUSIONS: Determination of the sequence of OCP supplies an important experimental foundation for ouabain research. The results also show that phage display peptide library is an effective, simple and efficient method to select specific steroid receptors. [