人酪氨酸羟化酶全长及催化核心序列的克隆与表达

设计合成含适当酶切位点的PCR引物 ,以人的全长THcDNA为模板 ,进行PCR扩增反应 ,得到人酪氨酸羟化酶 (tyrosinehydroxylase,TH)全长及催化核心序列 ,将其分别插入谷胱甘肽巯基转移酶 (GST)融合表达载体pGEX 4T 1 ,转化入大肠杆菌BL2 1 ,以IPTG诱导表达 ,SDS PAGE、westernblotting分析表达产物。结果获得了编码N 端缺失 1 4 5个氨基酸的酪氨酸羟化酶催化结构域的cDNA片段及带一定内切酶识别位点的全长片段 ,表达载体构建正确。插入片段序列与人THmRNA相应序列完全一致。转入重组质粒的菌经诱导后 ,有相对分子质量约为 8.3万及 6.6万的外源融合蛋白表达 ,并得到较纯的酶蛋白

Clonging and Expression for DNA Fragment of Complete Sequence and Catalytic Domain of Human Tyrosine Hydroxylase

In order to obtain complete sequence and catalytic core 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.The recombinant plasmids were transferred into E.coli BL21,and the expression of target protein was induced with IPTG.The expressed protein was analysed by SDS-PAGE and western-blotting.The result showed that the cDNA fragments of complete sequence and catalytic core of human TH were harvested.The sequencing for them showed that the cloned target gene fragments were correct.SDS-PAGE indicated that the proteins with Mr about 83000 and 66000 were expressed with the recombinant plamids pGEXTHa and pGEXTHc respectively.