GDNF基因克隆及表达载体的构建

Cloning of GDNF Gene and Construction of Expression Vector

目的 克隆大鼠胶质细胞源性神经营养因子 (GDNF)全长基因 ,构建真核细胞表达质粒。方法 从孕17d大鼠胚胎脑组织中提取RNA ,参照分子克隆指南 ,合成cDNA第 1、2链 ,加入引物PCR扩增目的基因 ,与pcDNA3质粒连接 ,构建表达质粒。结果 提取的总RNA经纯化后电泳出现 18s和 2 8s两条带 ,PCR扩增的目的基因为 6 30bp大小的条带 ,测序结果为 6 33bp。 结论 正确地克隆了大鼠GDNF基因全序列 ,为进一步获得重组活性的GDNF蛋白和神经系统疾病的基础和临床研究奠定了基础。

Objective To clone the glial cell line derived neurotrophic factor total gene of rat and construct expression of prokaryotic cell. Methods Total RNA was extracted from fetal rat brain of timed pregnant mom at E17, according to the guide of molecular clone, the first and second cDNA chains were synthesized and GDNF gene was amplified by PCR using primers. The DNA product was then linked with pcDNA3 plasmid and the expression vector was built. Results Purified RNA showed two bands in electrochromatography (18 s and 28 s), GDNF gene showed a 630 bp band by RT PCR amplification,and the DNA fragment size was 633 bp. Conclusion The total sequence of GDNF gene is cloned correctly has established the fundation for obtaining the protein of GDNF with recombination activity and the basic and clinical research in nervous system diseases.