雄激素受体功能表位表达载体的构建

Construction of recombinant expression vectors for function epi-position of androgen receptor

  • 摘要: 目的:构建雄激素受体(AR)功能表位表达载体,为深入研究AR与LRP16体外相互作用机制奠定基础。方法:用真核表达质粒pCMV-AR为模板进行常规PCR扩增,产物纯化后将连入pGEM-T载体,挑取阳性克隆扩增培养后测序并进行酶切鉴定;回收酶切后AR目的片断最终与BamHⅠ/XhoⅠ双酶切的pcDNA3.1(+)载体相连接。结果:测序结果表明四个AR功能表位表达载体插入片段与GenBank中报道的序列完全一致。结论:在克隆AR基因的基础上,成功构建了一组雄激素受体AR功能表位表达载体,可以用于AR与LRP16在体外转录翻译方面的研究。

     

    Abstract: Objective: To construct recombinant expression vectors for function epi-position of androgen receptor so as to provide foundation for further investigation and analysis interaction mechanism in vitro beween LRP16 and AR.Methods: The plasmid pCMV-AR was amplified by using PCR technique.PCR product was cloned into the pGEM-T expression vector.Clones containing the vectors were selected on LB-plus ampicillin(100 μg·mL-1) plates and sequenced.Plasmid DNA was isolated and purification after enzymatic digestion.AR fragments were initially connected with pCDNA3.1 vector.The recombinant plasmids pcDNA3.1-ARN1,pcDNA3.1-ARN2,pcDNA3.1-ARN3,pcDNA3.1-ARC1 were extracted and confirmed by digestion with BamHⅠand XhoⅠenzymes.Results: Sequence analysis showed the ARN1,ARN2,ARN3,ARC1 gene fragments cloned were 100% sequence homology with that of corresponding gene coding sequence in GenBank.Conclusion: The recombinant plasmid pcDNA3.1-ARN(1-3),pcDNA3.1-ARC1 was successfully constructed,and they could be used in vitro transcription and interpretation studies beween LRP16 and AR.

     

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