人骨骼肌TnI-fast基因克隆和体外表达

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-fast cDNA，构建TnI-fast基因克隆质粒，测序证实后将TnI-fast cDNA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-fas cDNA序列与NCBIGenebank TnI-fast cDNA序列完全一致。ELISA检测证实，TnI-fast/pSec Tag2 B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。

Cloning and in vitro expression of human fast-twitch skeletalmuscle troponin I gene

Objective To construct a recombinant eukaryotic expression plasmid encoding human fast twitch skeletal muscle troponin I gene (TnI fast) for anti angiogenesis in tumor gene therapy. Methods The TnI fast cDNA was amplified with standard PCR and RT PCR by using the human adult normal skeletal muscle cDNA and total RNA as templates, respectively, and then cloned into vector pUC18. After being sequenced in both directions, the cDNA was cloned into the eukaryotic expression vector pSecTag2 B. The obtained recombinant plasmid was transferred to COS 1 cells with a lipofectamine method. The expression of TnI fast in eukaryotic cells in vitro was examined with ELISA. Results DNA sequencing revealed that the PCR and RT PCR amplified TnI fast cDNA was identical to our objective. The recombinant TnI fast/pSecTag2 B plasmid was constructed successfully, and the secreting expression of TnI fast gene was detected in the transfected COS 1 cells. Conclusion Our results indicate that the recombinant TnI fast/pSecTag2 B plasmid can be used for animal study of anti angiogenesis in tumor gene therapy.