Construction of TH-GDNF vector and its expression in a dopaminergic cell line MES23.5

刘天蔚; 谢俊霞

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Construction of TH-GDNF vector and its expression in a dopaminergic cell line MES23.5

更新时间：2026-01-09

- Construction of TH-GDNF vector and its expression in a dopaminergic cell line MES23.5
- Journal of Army Medical University Vol. 30, Issue 1, Pages: 53-56(2008)
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  CLC： R394-33;R394.3;R742.5
- Published：2008
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刘天蔚,谢俊霞.TH和GDNF双基因表达载体的构建及其在MES23.5细胞中的表达[J].陆军军医大学学报(原第三军医大学学报),2008,30(01):53-56.

　LIU Tian-wei,XIE Jun-xia.Construction of TH-GDNF vector and its expression in a dopaminergic cell line MES23.5[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(01):53-56.
刘天蔚,谢俊霞.TH和GDNF双基因表达载体的构建及其在MES23.5细胞中的表达[J].陆军军医大学学报(原第三军医大学学报),2008,30(01):53-56. DOI：

　LIU Tian-wei,XIE Jun-xia.Construction of TH-GDNF vector and its expression in a dopaminergic cell line MES23.5[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(01):53-56. DOI：

摘要

目的构建酪氨酸羟化酶（tyrosine hydroxylase

TH）和胶质细胞源性神经营养因子（glial cell line-derived neurotrophic factor

GDNF）双基因表达载体并检测其在体外的表达，为帕金森病提供新的治疗措施。方法采用分子克隆技术，从pWAV2-TH质粒酶切获得人TH基因片段，将其克隆入真核表达质粒载体pIRES的上游，构建出pIRES-TH。PCR法扩增小鼠GDNF基因片段，克隆入pIRES-TH的下游，构建出携带TH和GDNF双基因的重组质粒pIRES-TH-GDNF。用酶切电泳验证重组结果的正确性，测序检测质粒重组后的序列。随后将重组质粒用Lipofectamine2000转染至MES23.5细胞，经G418筛选后，以RT-PCR及免疫荧光法检测TH和GDNF基因在mRNA及蛋白水平的表达。结果重组质粒酶切后其片段大小与预期结果相同，测序发现基因序列完全正确。转染后MES23.5细胞中TH和GDNF基因的水平明显升高（P0.05）。结论重组pIRES-TH-GDNF质粒构建成功并能在体外正确表达TH和GDNF基因。

Abstract

Objective To construct a vector carrying tyrosine hydroxylase (TH) and glial cell line-derived neurotrophic factor (GDNF) in order to establish a new gene therapy method in Parkinson’s disease. Methods Human TH gene fragment from the plasmid pWAV2-TH was cloned into pIRES to construct pIRES-TH. The mouse GDNF gene

amplified by PCR was inserted into pIRES-TH to construct pIRES-TH-GDNF. Restriction analysis and nucleotide sequencing were used to confirm the structure of pIRES-TH-GDNF. Then MES23.5 cells were transfected with this eukaryon vector using Lip

ofectamine

2000. The expression of TH and GDNF in mRNA and protein levels were detected by RT-PCR and immunofluorescence after G418 selection. Results The 2 objective fragments were inserted into pIRES correctly. RT-PCR and immunofluorescence results showed that TH and GDNF were highly expressed in MES23.5 cells. Conclusion The plasmid pIRES-TH-GDNF is constructed successfully and can express TH and GDNF in vitro.

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