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背景:人脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)和血管内皮生长因子165(vascular endothelial growth factor165,VEGF165)在细胞分化过程中有重要作用。病毒载体临床应用存在安全隐患,利用真核表达载体表达蛋白为解决安全性问题提供了一种方法。目的:构建双基因共表达载体pIRES2-BDNF-VEGF165并对其进行鉴定。方法:采用PCR的方法从人外周血单个核细胞的基因组DNA中获取人脑源性神经营养因子基因,然后将人脑源性神经营养因子的cDNA片段插入到pIRES2-EGFP多克隆位点构建为pIRES2-BDNF-EGFP。人血管内皮生长因子165 cDNA片段是通过双PCR的方法从pIRES2-VEGF165-EGFP质粒中获取,接着将血管内皮生长因子165 cDNA片段以替换EGFP的方式插入pIRES2-BDNF-EGFP中,最后构建成为含有即内部核糖体进入位点的pIRES2-BDNF-VEGF165双基因共表达载体。通过双酶切和DNA测序方法对其鉴定,将重组的双基因共表达载体感染HEK293细胞,利用RT-PCR与Western-blot方法检测双基因的表达。结果与结论:DNA测序显示,提取的人脑源性神经营养因子和血管内皮生长因子165均与基因库报道序列一致,片段长度分别为744 bp和576 bp。构建的pIRES2-BDNF-VEGF165双基因共表达载体经Eco RⅠ/Bam HⅠ切出BDNF条带,经BDNF/NotⅠ双酶切后可见IRESVEGF165基因片段,经Eco RⅠ/NotⅠ双酶切后可见BDNF-IRES-VEGF165基因片段。RT-PCR与Western-blot方法检测显示,此载体转染后,HEK293细胞均能表达人脑源性神经营养因子和血管内皮生长因子165 mRNA和蛋白。结果证实,实验成功构建了人脑源性神经营养因子和血管内皮生长因子165双基因真核表达载体。
BACKGROUND: Brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor 165 (VEGF165) play an important role in cell differentiation. There are potential safety problems in the clinical application of virus vectors, and the use of eukaryotic expression vectors to express proteins provides a solution to the problem of safety. Objective: To construct a double gene co-expression vector pIRES2-BDNF-VEGF165 and identify its. Methods: Human brain-derived neurotrophic factor gene was obtained from genomic DNA of human peripheral blood mononuclear cells by PCR. The cDNA fragment of human brain-derived neurotrophic factor (EGFR) was inserted into the multiple cloning site of pIRES2-EGFP pIRES2-BDNF-EGFP. Human Vascular Endothelial Growth Factor 165 cDNA fragment was obtained from the pIRES2-VEGF165-EGFP plasmid by double PCR, and then inserted into pIRES2-BDNF-EGFP in a manner to replace the EGFP cDNA fragment. Finally, Namely the internal ribosome entry site pIRES2-BDNF-VEGF165 double gene co-expression vector. Double enzyme digestion and DNA sequencing were used to identify the recombinant plasmids. HEK293 cells were infected with the recombinant double gene co-expression vector and the double gene expression was detected by RT-PCR and Western-blot. RESULTS AND CONCLUSION: DNA sequencing showed that the extracted human brain-derived neurotrophic factor and vascular endothelial growth factor 165 were consistent with the reported gene sequences. The length of the fragments was 744 bp and 576 bp, respectively. The co-expression vector pIRES2-BDNF-VEGF165 was digested by Eco RⅠ / Bam HⅠand the IRESVEGF165 gene fragment was digested by BDNF / NotⅠ. After double digestion with Eco RⅠ / NotⅠ, BDNF-IRES -VEGF165 gene fragment. RT-PCR and Western-blot showed that HEK293 cells could express human brain-derived neurotrophic factor and vascular endothelial growth factor 165 mRNA and protein after transfection. The results confirmed that the successful construction of human brain-derived neurotrophic factor and vascular endothelial growth factor 165 double gene eukaryotic expression vector.