CAJ | 학술논문

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 factor 165，VEGF 165)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pIRES 2-BDNF-VEGF 165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pIRES 2-EGFP多克隆位点构建为pIRES 2-BDNF-EGFP。人血管内皮生长因子165 cDNA片段是通过双PCR的方法从pIRES 2-VEGF 165-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pIRES 2-BDNF-EGFP中，最后构建成为含有即内部核糖体进入位点的pIRES 2-BDNF-VEGF 165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pIRES 2-BDNF-VEGF 165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 IRES-VEGF 165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。
배경：인뇌원성신경영양인자(Brain-derived neurotrophic factor ， BDNF)화혈관내피생장인자165(vascular endothelial growth factor 165，VEGF 165)재세포분화과정중유중요작용。병독재체림상응용존재안전은환，이용진핵표체재체표체단백위해결안전성문제제공료일충방법。 　　목적：구건쌍기인공표체재체pIRES 2-BDNF-VEGF 165병대기진행감정。 　　방법：채용PCR적방법종인외주혈단개핵세포적기인조DNA중획취인뇌원성신경영양인자기인，연후장인뇌원성신경영양인자적cDNA편단삽입도pIRES 2-EGFP다극륭위점구건위pIRES 2-BDNF-EGFP。인혈관내피생장인자165 cDNA편단시통과쌍PCR적방법종pIRES 2-VEGF 165-EGFP질립중획취，접착장혈관내피생장인자165 cDNA편단이체환 EGFP 적방식삽입pIRES 2-BDNF-EGFP중，최후구건성위함유즉내부핵당체진입위점적pIRES 2-BDNF-VEGF 165쌍기인공표체재체。통과쌍매절화 DNA측서방법대기감정，장중조적쌍기인공표체재체감염HEK293세포，이용 RT-PCR 여Western-blot 방법검측쌍기인적표체。 　　결과여결론：DNA측서현시，제취적인뇌원성신경영양인자화혈관내피생장인자165균여기인고보도서렬일치，편단장도분별위744 bp화576 bp。구건적pIRES 2-BDNF-VEGF 165쌍기인공표체재체경Eco RⅠ/Bam HⅠ절출BDNF조대，경BDNF/NotⅠ쌍매절후가견 IRES-VEGF 165기인편단，경 Eco RⅠ/NotⅠ쌍매절후가견BDNF-IRES-VEGF165기인편단。RT-PCR 여Western-blot 방법검측현시，차재체전염후，HEK293세포균능표체인뇌원성신경영양인자화혈관내피생장인자165 mRNA화단백。결과증실，실험성공구건료인뇌원성신경영양인자화혈관내피생장인자165쌍기인진핵표체재체。
BACKGROUND:Brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor 165 (VEGF 165 ) are essential genes for celldifferentiation. Virus mediated method has been used numerously in researches, but the security is the most important problem. Eukaryotic expressing vector is a way to solve this question. OBJECTIVE:To construct and identify pIRES 2-BDNF-VEGF 165 bicistronic eukaryotic expression vector. METHODS:BDNF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. Then, the BDNF cDNA fragment was inserted into the multiple cloning sites of pIRES 2-EGFP to generate the bicistronic eukaryotic expression plasmid pIRES 2-BDNF-EGFP. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by double PCR. Next step was that VEGF 165 cDNA fragment was cloned into the pIRES2BDNF-EGFP instead of EGFP to create a double gene co-expressing vector plasmid pIRES 2-BDNF-VEGF 165 . Then, pIRES 2-BDNF-VEGF 165 was used to transfect HEK293 cells, and RT-PCR and western-blot assay were employed to test the co-expression of double genes. RESULTS AND CONCLUSION:BDNF and VEGF 165 genes were cloned in this study. The DNA sequencing analysis demonstrated that the BDNF and VEGF 165 were exactly consistent with the sequence recorded in the GenBank. The size of BDNF gene was 744 bp. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by PCR, and the size of VEGF 165 gene was 576 bp. Enzyme digestion analysis indicated that BDNF and VEGF 165 genes were inserted into the expression vector pIRES 2-EGFP correctly and the BDNF and VEGF165 co-expression plasmid was successful y constructed. Then, by transfecting pIRES 2-BDNF-VEGF 165 into HEK293 cells, double genes were expressed at the mRNA and protein level. It provides a novel expression system, which enables further study on the functions of BDNF and VEGF 165 genes.