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目的探讨DJ-1L166P突变基因在帕金森病发生中的作用。方法构建pcDNA3.1-myc-his-DJ-1L166P真核表达载体,利用显微注射方法将DJ-1L166P基因片段(约3.9kb)导入BDF1小鼠受精卵雄原核并移植到同期受孕的ICR假孕母鼠输卵管中,对产出仔鼠的鼠尾组织DNA进行聚合酶链反应和Southern blot检测,对Southern blot鉴定阳性的转基因小鼠进行逆转录聚合酶链反应和Western blot检测。结果共产生20只子代小鼠,1号和7号为DJ-1L166P转基因阳性小鼠。1号转基因小鼠大脑、小脑、肝、肺、肾、睾丸有不同程度的DJ-1mRNA表达,均高于正常对照小鼠,但只有大脑中DJ-1mRNA的表达水平显著高于正常对照小鼠(P<0.05);7号转基因小鼠只有肝、肺组织中有DJ-1mRNA表达,也高于正常对照小鼠,但差异无统计学意义。只有1号小鼠大脑有His标签蛋白的表达。结论成功获得1只DJ-1L166P基因突变转基因小鼠模型,为课题的下一步实验研究打下良好的基础。
Objective To investigate the role of DJ-1L166P mutation in the development of Parkinson’s disease. Methods The eukaryotic expression vector pcDNA3.1-myc-his-DJ-1L166P was constructed. The DJ-1L166P gene fragment (about 3.9kb) was introduced into the male pronucleus of BDF1 mouse by microinjection and transplanted into ICF In the fallopian tubes of pregnant rats, PCR was performed on rat tail tissue DNA of the offspring rats by PCR and Southern blot. Reverse transcriptase polymerase chain reaction and Western blot were used to detect the Southern blot. Results A total of 20 offspring mice were produced, and DJ-1L166P transgenic positive mice were numbered 1 and 7. The expression of DJ-1mRNA in brain, cerebellum, liver, lung, kidney and testes of No.1 transgenic mice was higher than that of normal control mice, but only the expression of DJ-1mRNA in brain was significantly higher than that of normal control mice (P <0.05). Only DJ-1 mRNA expression in liver and lung tissue of transgenic mice 7 was also higher than that in normal control mice, but the difference was not statistically significant. Only mouse brain 1 has His-tagged protein expression. Conclusion One transgenic DJ-1L166P transgenic mouse model was successfully obtained, which laid a good foundation for the next experimental study.