神经营养素3基因克隆及其真核表达载体的构建

Cloning and construction of eukaryotic expression vector for rat neurotrophin 3 gene

目的克隆大鼠神经营养素-3(NT-3)基因的表达序列,构建大鼠NT-3基因真核表达载体。方法应用RT-PCR从大鼠脑组织总RNA中扩增NT-3基因cDNA,将其克隆到真核表达载体pcDNA3中构建重组质粒pcDNA3-N3,用限制酶酶切鉴定和DNA序列分析鉴定重组质粒。结果RT-PCR产物为822bp的特异片段,重组质粒酶切后产生822bp和5.2kb片段,DNA测序证实822bp片段的碱基序列与大鼠NT-3cDNA序列完全一致。结论成功克隆大鼠NT-3基因表达序列并构建了NT-3基因真核表达载体pcDNA3-N3。

Objective To clone and construct the eukaryotic expression vector for rat Neurotrophin 3 （NT-3） gene. Methods The rat NT-3 cDNA was amplified by RT-PCR from rat brain tissue. By gene recombination technique, rat NT-3 cDNA was inserted into eukaryotic expression vector pcDNA3 to construct recombinant plasmid pcDNA3-N3. The recombinant plasmid was identified with restriction enzyme digestion and DNA sequencing. Results The RT-PCR product is 822bp specific segment. By restriction enzyme digestion, the recombinant plasmid was digested into 822bp and 5200bp fragments. The DNA sequence of the 822bp fragment was identical with rat NT-3 cDNA in GenBank. Conclusion The NT-3 gene was cloned, and the eukaryotic expression vector for NT-3 gene was constructed successfully, which will provide the foundation for the further research.