斜带石斑鱼PACAP的原核表达及活性分析

自1989年从绵羊下丘脑提取物发现垂体腺苷酸环化酶激活多肽（Pituitary adenylate cyclase activating polypeptide，PACAP）以来（Miyata et al．，1989），已证明它能促进垂体激素释放，同时还具有神经递质、神经调质和神经营养等作用，使对PACAP的研究成为十分活跃的领域。PACAP属于血管活性肠肽（VIP）-胰高血糖素-生长激素释放因子-分泌素家族（Campbell and Scanes，1992）成员，已鉴别出包含27和38个氨基酸两种类型。对原索动物（McRory et al．，1997）、两栖类（蛙）（Alexandre et al．，2000）、爬行类（蜥蜴）（Pohland Wank，1998）、鸟类（鸡）（McRory et al．，1997），啮齿类（鼠）（Ghatei et al．，1993）等脊椎动物PACAP的研究多集中在结构与进化方面，对功能了解甚少。

The prokaryotic expression and biological activity of the pituitary adenylate cyclase activating polypeptide in groupers Epinephelus coioides

The objective of this study is to produce biologically active grouper recombinant pituitary adenylate cyclase activating polypeptide (PACAP). The cDNA encoding the mature grouper PACAP was isolated by RT-PCR using total RNA extracted from grouper brain. The expression plasmid pTRX-PACAP was constructed by inserting PACAP cDNA into plasmid pTRX and transformed into E.coli BL21(DE3). An expression strain BL21(DE3)-pTRX-PACAP was selected. SDS-PAGE analysis revealed that the grouper PACAP protein was highly expressed and accumulated up to above 25% of the total bacterial proteins, which is present both in inclusion bodies and in soluble form. The soluble PACAP accounts for above 40%. After Ni 2+ -chelation affinity chromatography, the purified protein has a purity of over 95%. Purified PACAP protein could significantly promote the secretion of growth hormone (GH) from hyphathelamas and pituitary mixture incubation.