人脑源性神经营养因子和神经营养素3真核双表达载体的构建与鉴定

背景：脑源性神经营养因子（brain-derived ；neurotrophic factor，BDNF）和神经营养素3（Neurotrophines-3，NT-3）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-NT-3并对其进行鉴定。方法： BDNF和NT-3基因核心序列是通过直接PCR的方法从外周血单个核细胞的基因组DNA中获取。然后将BDNF的cDNA片段插入到pIRES2-EGFP多克隆位点构建pIRES2-BDNF-EGFP载体，随后将NT-3 cDNA片段以替换EGFP的方式插入pIRES2-BDNF-EGFP中，最后构建成为含有内部核糖体进入位点（IRES）的pIRES2-BDNF-NT-3双基因共表达载体。实验通过双酶切和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BDNF和NT-3均与基因库报道序列一致。构建的pIRES 2-BDNF-NT-3双基因共表达载体经Eco RⅠ/Bam HⅠ切出BDNF条带，经Bam HⅠ/NotⅠ双酶切后切出IRES-NT-3片段，经Eco RⅠ/NotⅠ双酶切后切出BDNF-IRES-NT-3片段。RT-PCR与Western-blot方法检测显示，此载体转染后的HEK293细胞均能表达BDNF和NT-3 mRNA和蛋白。结果证实，实验成功采用IRES序列构建了能分别表达的 BDNF 和 NT-3双基因真核表达载体。

Construction and identification of bicistronic eukaryotic expression vector of human brain-derived neurotrophic factor and neurotrophine-3

BACKGROUND：Human brain-derived neurotrophic factor （BDNF） and neurotrophine-3 （NT-3） are essential genes for celldifferentiation. Viral vector has been used numerously in clinical practice, but the security is the most important problem. Eukaryotic expressing vector is a way to solve this question.
OBJECTIVE：To construct and identify pIRES2-BDNF-NT-3 bicistronic eukaryotic expression vector. METHODS：BDNF and NT-3 genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. The BDNF cDNA fragment was inserted into the multiple cloning sites of pIRES2-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES2-BDNF-EGFP. Then NT-3 cDNA fragment was cloned into the pIRES2-BDNF-EGFP, instead of EGFP, to create plasmid pIRES2-BDNF-NT-3. Subsequently pIRES2-BDNF-NT-3 was used to transfect HEK293 cells, and RT-PCR and western blot analysis were applied to test the co-expression of double genes.
RESULTS AND CONCLUSION：The DNA sequencing analysis demonstrated that the BDNF and NT-3 were exactly consistent with the sequence recorded in the GenBank. Enzyme digestion analysis indicated that, in the constructed bicistronic eukaryotic expression vector pIRES2-BDNF-NT-3, BDNF band was obtained by Eco RI/Bam HI digestion, IRES-NT-3 fragment was obtained by Bam HI/Not I digestion, and BDNF-IRES-NT-3 was obtained by Eco RI/Not I digestion. RT-PCR and western blot analysis showed that, after the HEK293 cells were transfected with pIRES2-BDNF-NT-3, double gene was expressed at the mRNA and protein level. Experimental findings suggest that, bicistronic eukaryotic expression vector of BDNF and NT-3 genes can be successful y constructed using IRES sequence.