鼻咽癌相关新基因NPCEDRG真核诱导表达载体的构建及鉴定

目的构建受Tet系统调控人鼻咽癌相关新抑瘤基因NPCEDRG表达的真核诱导表达载体。方法以pcDNA3．1-NPCEDRG重组质粒为模板，采用聚合酶链反应（PCR）技术扩增NPCEDRG基因编码区，亚克隆到pRevTRE载体，PCR及双酶切鉴定，测序验证。结果以pRevTRE—NPCEDRG重组载体为模板PCR扩增以及双酶切重组载体，分别产生530bp和520bp长度的片断，测序结果证实插入片段方向正确，序列与Genebank已知序列一致，NPCEDRG基因编码区成功插入pRevTRE载体。结论成功构建了受Tet系统调控NPCEDRG基因表达的pRevTRE—NPCEDRG真核诱导表达载体，为深入研究NPCEDRG基因功能和摁示鼻咽癌发病分子机制提供实验手段。

Construction of Eukaryotic Expression Recombinant of NPCEDRG Controlled by Tetracycline(Tet)-Inducible System

Objective To construct an inducible eukaryotic expression vector pRevTRE- NPCEDRG containing the open reading frame （ORF） of the NPCEDRG gene for regulating the gene expression by Tet system. Methods The ORF of NPCEDRG gene was amplified from the recombinant plasmid of pcDNA3.1 - NPCEDRG by polymerase chain reaction （ PCR}, and subcloned into the pRevTRE vector after digestion with restriction enzymes BamH I and Hind Ⅲ Then the products were transferred into E. coli DH5α, and the positive clones were screened after being identifid with PCR, restrictive enzymes and sequence analysis. Results The target segment of NPCEDRG was obtained successfully 530 bp in PCR products and 520 bp in digestion ones. The DNA sequence after being sequenced mas consistent with the cDNA sequeuce of NPCEDRG from GeueBank. Conclusion The recombinant plasmid of pRevTRE - NPCEDRG was constructed successfully.