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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
PCR primers were designed and synthesized with appropriate restriction sites. The full-length human THcDNA was used as a template to amplify the full length of tyrosine hydroxylase (TH) and its catalytic core sequence Inserted glutathione S-transferase (GST) fusion expression vector pGEX 4T 1, transformed into E. coli BL21, induced expression by IPTG, SDS PAGE, western blotting analysis of expression products. Results The cDNA fragment coding for tyrosine hydroxylase catalytic domain with N-terminal deletion of 154 amino acids was obtained and the full-length fragment with the recognition site of certain endonuclease was obtained. The expression vector was constructed correctly. The sequence of the insert was exactly the same as that of human THmRNA. The recombinant plasmids transformed into E. coli were induced to express 83,000 and 66,000 exogenous fusion proteins and obtained relatively pure enzyme protein