垂体腺苷酸环化酶激活肽基因合成表达和产物纯化与鉴定

Gene Synthesis, Expression of Pituitary Adenylate Cyclase Activating Polypeptide and Its Purification and Identification

为利用基因工程技术获得垂体腺苷酸环化酶激活肽 (pituitaryadenylatecyclaseactivatingpolypeptide ,PACAP) ,根据大肠杆菌的密码偏好性 ,设计并人工合成编码 38个氨基酸的PACAP基因 .克隆到表达载体pET 35b(+) ,构建重组质粒pET PACAP ,转化大肠杆菌BL2 1 (DE3)pLysS+ .实现纤维素结合域 (cellulosebindingdomain ,CBD)与PACAP融合蛋白的表达 ,并在两者之间引入 (凝血 )因子Ⅹa识别位点 (Ile Glu Gly Arg↓ ) .融合蛋白CBD PACAP经纤维素亲和层析纯化后 ,因子Ⅹa酶切释放PACAP .在因子Ⅹa识别位点前引入 7个氨基酸的柔性短肽 (Gly Thr Gly Gly Gly Ser Gly)明显提高了融合蛋白对因子Ⅹa的敏感性 .HPLC进一步纯化得到纯度大于 95 %PACAP多肽 .所得的PACAP多肽的Western印迹鉴定为阳性 ;激光飞行质谱测定分子量结果与理论值相符 .生物活性分析表明 ,所制备的PACAP具有促进胰腺癌细胞株SW 1

To produce pituitary adenylate cyclase activating polypeptide (PACAP) using gene engineering technology, a gene coding PACAP was designed and synthesized according to the preference of E.coli, and cloned into the expression vector pET 35b(+). The recombinant plasmid pET PACAP were constructed and transformed into E.coli BL21(DE3)pLysS +. A fusion protein with a recognized site of factor Ⅹa between CBD (cellulose binding domain) and PACAP was expressed and purified by cellulose affinity chromatography. PACAP was released by the cleavage of factor Ⅹa. A short flexible peptide(Gly Thr Gly Gly Gly Ser Gly),was added before the recognized site by factor Ⅹa to improve the sensitivity of fusion protein to factor Ⅹa. PACAP with over 95% purity was purified by HPLC and identified by Western blotting. Laser time of flying mass spectrum showed that the molecular weight was 4536.8 as expected. The preliminary bioactivity assay indicated that the product had the activity promoting cAMP synthesis in the cell line SW1990 of human pancreas carcinoma.