大鼠脑源性神经营养因子基因真核表达载体的构建及其在COS-7细胞中的表达

Expression of rat brain-derived neurotrophic factor in COS-7 cells following its eukaryotic expression vector construction

构建脑源性神经营养因子（BDNF）基因真核表达载体，检测其转染COS-7细胞后的表达。方法：从大鼠海马组织中提取总RNA，采用RT-PCR方法获得目的基因片段，克隆至pGEM-T载体中。经测序确证后将BDNFcDNA片段与pEGFP-N1载体定向连接。将鉴定正确的重组体pEGFP-N1-BDNF以脂质体法转染至COS-7细胞中，用RT-PCR鉴定BDNFmRNA的表达，免疫印迹法检测BDNF蛋白质表达水平。结果：克隆了BDNF的cDNA，并构建了其真核表达载体pEGFP-N1-BDNF，经限制性内切酶酶切鉴定及测序分析证实了其序列的正确性；免疫印迹法分析显示，转染48h时，COS-7细胞以分泌proBDNF-GFP融合蛋白为主，在96h以分泌成熟BDNF-GFP融合蛋白为主。结论：成功构建pEGFP-N1-BDNF真核表达载体，并且在COS-7细胞中得到高效表达，BDNF前体及成熟体同时分泌到胞外，在不同时间点分泌组合不同。

To construct eukaryotic expression vector of BDNF and verify BDNF expression in transfected COS-7 cells. Methods： BDNF cDNA fragment was cloned by RT-PCR with the total RNA from rat hippocampus as the template, and subsequently cloned into pGEM-T vector. The BDNF fragment with its sequence confirmed was then cloned into pEG- FP-N1 vector directionally. The right recombinant was transfected into COS-7 cells by lipofectamine 2000. The expression of BDNF in COS-7 cells was detected by RT-PCR and immunoblot analysis. Results： The correct pEGFP-N1-BDNF cloning was verified by restriction endonuclease digestion and sequencing. The immunoblot analysis indicated that proBDNF-GFP fu- sion protein was mainly secreted in transfected COS-7 cells at 48 h and mature BDNF-GFP fusion protein was mainly secreted at 96 h. Conclusion： pEGFP-N1-BDNF was constructed successfully and could be abundantly expressed in COS-7 cells. proBDNF-GFP and mature BDNF-GFP fusion protein were both secreted at the same time in transfected COS-7 cells and the secretion combination was different at different time.