文昌鱼胰岛素样肽的表达、纯化、鉴定及其多克隆抗体制备

为了深入研究胰岛素和胰岛素样生长因子1（IGF－1）的起源和进化以及结构与功能的关系。表达了胰岛素和IGF－1的祖先分子－－文昌鱼胰岛素样肽（ILP）。重组单链ILP的基因用化学方法合成（从cDNA推测的ILPB结构域的C端和A结构域的N端用Ala-Ala-Lys三肽连接起来，并钭B28Arg突变为Lys），克隆到表达载体pVT102-U中，ILP在酿酒酵母中得到有效表达。发酵液经4步分离纯化，得到均一的单链ILP，经质谱测定分子量和氨基酸组成分析证明表达产物正确。通过Lys-C蛋白内切酶处理将重组单链ILP转化成双链形式。虽然双链ILP与人胰岛素受体没有结合活力。但圆二色性光谱显示它与胰岛素的结构非常相似，用表达的单链ILP免疫新西兰大白兔，获得了高滴度的多克隆抗体。

Expression, Purification, Characterization of Amphioxus Insulin-like Peptide and Preparation of Polyclonal Antibody to It

To elucidate the origination and evolution as well as structure function relationship of insulin and IGF 1, the gene of amphioxus insulin like peptide (ILP), a common ancestor of insulin and IGF 1, was chemically synthesized and cloned into the expression vector pVT102 U. In the recombinant ILP, the C terminus of B domain and the N terminus of A domain of amphioxus ILP deduced from cDNA were linked together by a tripeptide, Ala Ala Lys, and its B28Arg residue was also replaced by Lys to facilitate its conversion to a double chain form by Lys C cleavage. The expression vector was transformed into yeast cells and the recombinant ILP was expressed efficiently. The purified single chain and double chain ILP was obtained by fermentation, purification and enzymatic cleavage. Molecular weight measurement and amino acid composition analysis showed that the primary structure of ILP was correct. Circular dichroism analysis showed that the secondary and tertiary structure of double chain ILP was similar to that of insulin, while double chain ILP had no measurable insulin activity in insulin receptor binding assay. Additionally, New Zealand Rabbits were immunized with single chain ILP and high titre polyclonal antibody was obtained. This work was important for further investigating the molecular evolution of insulin and IGF 1 and the distribution and existence of ILP in amphioxus.