目的: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BDNF mRNA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48 h时,COS-7细胞以分泌proBDNF-GFP融合蛋白为主,在96 h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.%Objective: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-Nl-BDNF cloning was verified by restriction endonuclease digestion and sequencing. The immunoblot analysis indicated that proBDNF-GFP fusion 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-Nl-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.
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