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目的 构建人骨骼肌TnI fast基因分泌型真核表达载体 ,为利用TnI fast基因进行抗肿瘤血管生成基因治疗奠定基础。方法 采用PCR和RT PCR制备TnI fastcDNA ,构建TnI fast基因克隆质粒 ,测序证实后将TnI fastcDNA插入到pSecTag2B构建分泌型重组真核表达质粒。然后用TnI fast基因重组表达质粒转染COS 1细胞 ,检测TnI fast基因体外表达。结果 DNA测序证实 ,我们从人骨骼肌总RNA和cDNA文库中扩增的TnI fastcDNA序列与NCBIGenebankTnI fastcDNA序列完全一致。ELISA检测证实 ,TnI fast pSecTag2B转染后目的基因可在真核细胞中分泌表达。结论 本研究成功地构建了TnI fast基因重组表达质粒 ,可进一步用于抗肿瘤血管生成基因治疗动物实验
Objective To construct the secretory eukaryotic expression vector of TnI fast gene of human skeletal muscle and lay a foundation for gene therapy of anti-angiogenesis using TnI fast gene. Methods TnI fast cDNA was prepared by PCR and RT PCR. TnI fast cloned plasmid was constructed. After sequencing, TnI fast cDNA was inserted into pSecTag2B to construct secreting recombinant eukaryotic expression plasmid. Then, the TnI fast gene was transfected into COS 1 cells with TnI fast recombinant plasmid to detect the expression of TnI fast gene in vitro. Results DNA sequencing confirmed that the TnI fastcDNA sequence we amplified from human skeletal muscle total RNA and cDNA libraries was identical to the NCBIGenebankTnI fastcDNA sequence. ELISA test confirmed that TnI fast pSecTag2B transfected target gene can be secreted in eukaryotic cells. Conclusion This study successfully constructed TnI fast gene recombinant expression plasmid can be further used for anti-angiogenic gene therapy in animal experiments