摘要
目的 克隆人野生型parkin基因并构建真核表达载体pCDNA3.1 parkin ,将重组质粒转染PC12细胞获得高表达人野生型parkin基因的PC12细胞克隆。 方法 从胎脑组织中提取总RNA ,用RT PCR方法获得人野生型 parkin基因的全长cDNA ,插入 pCR2 .1 TA克隆载体中进行序列测定 ,测序正确后将其亚克隆至表达载体pCDNA3.1,利用脂质体将重组质粒转染PC12细胞 ,经G4 18筛选获得抗性细胞克隆 ,采用RT PCR和WesternBlot方法鉴定人野生型 parkin基因在PC12细胞中的过表达。 结果 经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒 ,RT PCR和WesternBlot证明经G4 18筛选得到的转基因PC12细胞克隆中存在人野生型 parkin基因的表达。结论 成功构建了人野生型 parkin基因的真核表达载体 ,获得了稳定表达人野生型parkin基因的PC12细胞克隆 ,为进一步研究parkin的生物学功能以及parkin在帕金森病发病机制中的作用奠定了良好的基础。
Objective To clone human wild-type parkin cDNA, construct its eukaryotic expression vector and obtain positive PC12 cell clones expressing human wild-type parkin gene stably. Methods The total RNA was extracted from aborted fetal brain. The full-length cDNA encoding human wild-type parkin gene was obtained by RT-PCR method and inserted into pCR2.1-TA cloning vector. After the sequencing was confirmed, the gene was subcloned to pCDNA3.1 to construct recombinant eukaryotic expression vector pCDNA3.1-parkin. The recombinant plasmid was transfected into PC12 cells by lipofectamin method and positive cell clones were screened with G418. Overexpression of human wild-type parkin gene in the transfected cells was confirmed with RT-PCR and Western Blot. Results Enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant vector. Overexpression of human parkin gene in the transfected PC12 cells was identified with RT-PCR and Western Blot. Conclusion The eukaryotic expression plasmid containing human parkin gene was successfully constructed. The positive PC12 cell clones expressing human wild-type parkin gene stably were obtained, which may be a promising cell model for studying the biological function of the parkin gene and the role of parkin in the pathogenesis of Parkinson disease.
基金
脑功能和脑重大疾病的基础研究4/4/帕金森病的发病机制和防治基础资助项目 (G19990 5 40 0 8)
国家自然科学基金资助项目 (3 9970 2 63
3 0 1710 2 5 )
上海市卫生系统百名跨世纪优秀学科带头人培养计划资助项目 (97BR0 0 1)
