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【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 an adenovirus expression vector containing the gene of human neurotrophin-3 (NT-3) receptor (tyrosine protein kinase C, TrkC). 【Method】 The full-length cDNA of TrkC gene was amplified from human brain mRNA and cloned into shuttle plasmid pShuttle to obtain an expression cassette with CMV promoter. The expression cassette was then ligated with Adeno-Xviral DNA in vitro to form a recombinant adenovirus plasmid (pAd-TrkC). 293 cells were transfected with pAd-TrkC to construct recombinant adenovirus (Adeno-TrkC). 【Result】 The TrkC gene RT-PCR product was 2478bp. Adeno-TrkC was PCR-identified as the correct recombinant. 【Conclusion】 Human TrkC recombinant adenovirus expression vector has been constructed by in vitro ligation method, which lays the foundation for the further application of NT-3 for gene therapy of central nervous system injury.