抗肿瘤血管生成VEGFR2 DNA疫苗的构建

Construction of a VEGFR2 DNA Vaccine Against Tumor Angiogenesis

目的构建一种新的抗肿瘤血管生成DNA疫苗pcDNA3.1+/flk-1(n1-4).方法采用RT-PCR技术,从BALB/c胎鼠组织中扩增出鼠血管内皮生长因子受体flk-1胞外区1-4个袢的cDNA片段,并将其插入真核表达载体质粒pcDNA3.1+中,构建成重组质粒pcDNA3.1+/flk-1(n1-4),经DNA序列分析证实后,将重组质粒经脂质体法转染真核表达系统COS-7细胞,通过W estern b lot实验检测重组质粒在真核表达系统COS-7细胞中蛋白的表达.结果RT-PCR扩增产物为1 248 bp大小的基因片段,经过DNA序列分析证明与Gene Bank所公布的flk-1胞外区1-4结构域碱基一致,成功构建了重组质粒pcDNA3.1+/flk-1(n1-4),并且该重组质粒在真核表达系统COS-7细胞中成功得到表达.结论成功地制备了抗肿瘤血管生成DNA疫苗pcDNA3.1+/flk-1(n1-4),为进一步开展有关抗肿瘤血管生成的动物实验和临床应用奠定了基础.

Objective To construet a novel DNA vaccine pcDNA3.1 ＋/flk - 1 （nl-4） against tumor angiogenesis. Methods flk - 1 （nl-4） cDNA amplified by RT PCR was inserted into eucaryotic expression vector pcDNA3.1 ＋ and the recombinant plasmids pcDNA3.1 ＋/ilk- 1（nl-4） was constructed. Confirmed by DNA se- quence analysis, the recombinant plasmids was transfected into eucaryotic expression system COS -7 cells by lipofectamine. The protein expression of flk - 1 （nl-4） was detected by Western blot. Results Extracellular loop 1 -4 of flk - 1 was cloned and the recombinant plasmid was successfully constructed, which has been proved by DNA sequencing and compared with the data in Gene Bank. The protein expression of flk - 1 （nl-4） was detected in eucaryotic expression system COS -7 ceils. Conclusions The DNA vaccine against tumor angiogenesis was successfully constructed, which found a basis for the further study on animal experiment and clinical application.