人酪氨酸羟化酶催化核心DNA片段的克隆与表达

获得编码人酪氨酸羟化酶催化核心ｃＤＮＡ片段，并观察其表达。方法采用人的全长ＴＨｃＤＮＡ为模板，设计特定的引物进行ＰＣＲ扩增反就并构建真核表达载体－质粒ｐＣＭＶＴＨｃ。在对目的片段测序后，将重组质粒转染到大鼠原代培养骨骼肌细胞内，用免疫组织化学方法检测其表达。

CLONGING AND EXPRESSION FOR DNA FRAGMENT OF CATALYTICDOMAIN OF HUMAN TYROSINE HYDROXYLASE

Objective\ In order to obtain catalytic core of human tyrosine hydroxylase(TH) cDNA and observe its expression.Methods\ The full length of cDNA from human TH was used as template and the specific oilgonucleotide primers were designed for Polymerase Chain Reaction(PCR).An eukaryotic vector,plasmid pCMVTH C was then constructed.The newly constructed pCMVTH C was transfected in vitro to the cultured primary muscle cells from the rat after DNA sequence analysis.The expression of TH was tested by using a method of immunocytochemistry.Results\ The cDNA fragment containing a catalytic domain of human TH(TH C) with encoded N terminal removal of 145 amino acid residues was harvested.An eukaryotic vector,plasmid pCMVTH C was then proved correct.DNA sequence analysis indicated that the inserted fragment was consistent with that of human TH mRNA.The expression of TH was shown in muscle cells by immunocytochemistry analysis.Conclusion\ The results of the present study may provide a new way for gene therapy of Parkinsonism.