人白血病抑制因子和血管内皮生长因子165双基因真核表达载体的构建与鉴定

背景：人向血病抑制因子（LIF）和血管内皮生长因子165（VEGF165）对脊髓损伤后神经元存活有重要作用.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.目标：构建双基因共表达载体plRES2-LIF-VEGF165并对其进行鉴定.方法：是采用直接PCR的方法从人外周血单个核细胞的基因组DNA中获取人白血病抑制因子基因,然后将人白血病抑制因子的cDNA片段插入到plRES2-EGFP多克隆位点构建成为plRES2-LIF-EGFP.人血管内皮生长因子165 cDNA片段是通过双PCR的方法从plRES2-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plRES2-LIF-EGFP中,最后构建成为含有IRES（即内部核糖体进入位点）的plRES2-LIF-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,提取的人白血病抑制因子和血管内皮生长因子165均与基因库报道序列一致,片段大小分别为609 bp和576 bp.构建的plRES2-LIF-VEGF165双基因共表达载体经EcoRI/BamHI切出LIF条带,经BamHI/Notl双酶切后切出IRES-VEGF165片段,经EcoRI/ Notl双酶切后切出LIF-IRES-VEGF165片段.RT-PCR与Western-blot方法检测显示,此载体转染后,HEK293细胞均能表达人白血病抑制因子和血管内皮生长因子165 mRNA和蛋白.结果证实,实验成功构建了人白血病抑制因子和血管内皮生长因子165双基因真核表达载体.

Construction and identification of plRES2-LIF-VEGF165 bicistronic eukaryotic expression vector

BACKGROUND： Human leukemia inhibitory factor （LIF） and vascular endothelial growth factor 165 （VEGF165） play an important role in neuronal survival after spinal cord injuries. The clinical application of viral vector has a hidden danger, and eukaryotic expressing vector is a way to solve this question. OBJECTIVE： To construct and identify plRES2-LIF-VEGF165 bicistronic eukatyotic expression vector. METHODS： LIF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by direct PCR. Then LIF cDNA fragment was inserted into the multiple cloning sites of plRES2-EGFP, to generatethe bicistronic eukaryotic expression plasmid plRES2-LIF-EGFP. The VEGF165 gene was obtained from plRES2-VEGF165-EGFP plasmid by twin PCR, then VEGF165 cDNA fragment was cloned into the pIRES2-LIF-EGFP instead of EGFP to create a double gene co-expressing vector plasmid plRES2-LIF-VEGF165. Then plRES2-LIF-VEGF165 was applied to transfect HEK293 cells, and the co-expression of double genes was detected using RT-PCR and western blot analysis. RESULTS AND CONCLUSION： The DNA sequencing analysis demonstrated that the LIF and VEGF165 were exactly consistent with the sequence recorded in the GenBank. The size of LIF gene and VEGF165 gene was respectively was 609 bp and 576 bp. Enzyme digestion analysis indicated that LIF band was found in the double gene co-expressing vector plRES2-LIF-VEGF165 by EcoRI/BamHI, IRES-VEGF165 fragmet was found by BamHI/Notl, and LIF-IRES-VEGF165 fragment was found by EcoRI/Notl. RT-PCR and western blot analysis showed that, after transfection, both LIF and VEGF165 mRNA and protein were expression in HEK293 cells. LIF and VEGF165 bicistronic eukaryotic expression vector was successfully constructed in this study.