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本文以人VEGF cDNA为目的基因,构建成逆转录病毒质粒载体,并进一步包装成重组缺陷型逆转录病毒,以此感染原代兔皮肤成纤维细胞,经G418筛选,获得了具有分泌VEGF能力的抗性细胞克隆,经Southern、PCR、Northern、RT-PCR等检测,证实了外源基因已整合至原代兔皮肤成纤维细胞基因组中,无重排和产生野生型病毒之患。整合的VEGF可在修饰细胞中转录,经内皮细胞增殖实验和血管通透性实验证实其表达产物具有强大的生物活性。为进一步研究VEGF的生物学功能和生理效应提供有用的工具。
In this study, human VEGF cDNA was constructed as a retroviral plasmid vector and further packaged into recombinant defective retroviruses to infect primary rabbit dermal fibroblasts. After screening by G418, VEGF-secreting Resistant cell clones, Southern, PCR, Northern, RT-PCR and other tests confirmed that the foreign gene has been integrated into the primary rabbit skin fibroblast genome, without rearrangement and the production of wild-type virus. The integrated VEGF can be transcribed in the modified cells, and its expression product has strong biological activity confirmed by endothelial cell proliferation test and vascular permeability test. It provides a useful tool to further study the biological functions and physiological effects of VEGF.