人神经营养素-3受体基因的扩增及其重组腺病毒载体的构建

Amplification of Human Neurotrophin-3 Recepter Gene and Construction of Recombinant Adenovirus Expression Vector for TrkC

【目的】构建人神经营养素-3(neurotrophin-3,NT-3)受体(即酪氨酸蛋白激酶受体C,tyrosineproteinkinaseC,TrkC)基因重组的腺病毒表达载体。【方法】从人脑组织mRNA中扩增TrkC基因全长cDNA,定向克隆于穿梭质粒pShuttle中,获得一个带有CMV启动子的表达盒。再将表达盒与腺病毒骨架DNA(Adeno-XviralDNA)体外连接,形成重组腺病毒质粒(pAd-TrkC)。用pAd-TrkC转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(Adeno-TrkC)。【结果】TrkC基因RT-PCR扩增产物为2478bp。Adeno-TrkC经PCR鉴定为正确重组子。【结论】应用体外连接法已构建了人TrkC重组腺病毒表达载体,这为进一步应用NT-3进行基因治疗中枢神经损伤奠定了基础。

[ Objective ] To construct a recombinant adenovirus expression vector carrying human neurotrophin-3 （NT-3） receptor TrkC gene. [Methods] The mRNA were extracted from human brain tissue and the full-length cDNA of human TrkC gene was amplified by reverse transcription polymerase chain reaction （RT-PCR）. Firstly, an expression cassette with CMV promoter was made by cloning NT-3 into pShuttle vector. Secondly, the expression cassette was hgated to Adeno-X viral DNA to form a recombinant adenoviral plasmid （pAd-TrkC）, which was packaged into infectious recombinant adenoviral particles （Adeno-TrkC） by transfecting human embryonic kidney 293 cells. [Results] The amplified TrkC gene was 2 478 bp in length; Adeno-TrkC was proved to be correct by PCR. [Conclusion] A recombinant adenovirus carrying TrkC was constructed successfully by in vitro ligation reaction, which may provide the basis for using NT-3 in gene therapy.