【摘要】 采用人的全长酪氨酸羟化酶 c DNA为模板 ,设计特定的引物进行 PCR扩增反应 ,获得编码 N-端缺失 14 1个氨基酸的催化结构域 c DNA片段 ,将其插入谷胱甘肽巯基转移酶融合表达载体 p GEX-4 T-1中 ,构建表达质粒 p GEXTHc,同时构建其全长片段的表达质粒 p GEXTHa。两者分别转化大肠杆菌 BL2 1,以 IPTG诱导可高效表达 ,产物以可溶形式存在 ,经亲和层析纯化后以蛋白酶裂解 ,再经亲和层析分离得到较纯的酶蛋白 ,采用高效液相色谱法 ( HPLC-ECD)检测酶活性 ,结果证明催化结构域活性高于全酶 ,是全酶活性的 2 .96倍更多还原

【Abstract】 In order to obtain catalytic domain of human tyrosine hydroxylase(TH) cDNA, the full length of cDNA from human TH was used as template and the specific oligonucleotide primers were designed for PCR. The target fragments were cloned into vector pGEX 4T 1 respectively, called pGEXTHc(including the catalytic domain of TH gene) and pGEXTHa(including the full length of TH gene). They were transformed into the E.coli BL21 and the expression of target protein was induced with IPTG. The fusion protein of TH gene expression was detected by Western blot. The products(TH fusion protein) were cleaved by thrombin and purified by affinity system. In the mean time, the specific activities of the two forms of TH were determined by HPLC ECD. The results showed that the specific activity of TH catalytic domain was 2.96 fold higher than that of the wild type enzyme. The results of the present study may provide a new form of TH for gene therapy of Parkinson’s desease.更多还原