脑源性神经营养因子融合蛋白真核表达质粒的构建及表达

Construction and expression of the eukaryotic expression plasmid of BDNF-EGFP fusion protein

目的构建以增强型绿色荧光蛋白(EGFP)为报告基因的脑源性神经营养因子(BDNF)融合蛋白真核表达质粒,并对其表达和定位进行研究。方法以人血白细胞DNA为模板,利用一对删除终止密码子的引物进行PCR获得BDNF基因片段,并将其与pEGFP-N1质粒载体连接,构建BDNF-EGFP融合蛋白真核表达质粒。以脂质体Lipo-fectamine2000转染Hela细胞,提取总RNA,通过RT-PCR方法检测BDNF-EGFP在转录水平的表达,Western blot及荧光显微镜进一步观察融合蛋白的表达定位。结果酶切和PCR鉴定证实BDNF基因片段正确克隆入载体质粒中,转染Hela细胞后,RT-PCR证实BDNF-EGFP融合蛋白在转录水平有表达,荧光显微镜观察显示BDNF-EGFP融合蛋白主要分布于细胞质中,Western blot结果显示Hela细胞培养液中有BDNF-EGFP融合蛋白存在。结论成功构建了BDNF-EGFP融合蛋白真核细胞表达质粒,BDNF-EGFP融合蛋白能够在Hela细胞中表达,主要位于细胞质中并可以分泌到细胞外。

Objective To construct the eukaryotic expression vector of brain-derived neutrophic factor （BDNF-EGFP） fusion protein and to investigate its expression and location. Methods BDNF DNA was amplified from the genomic DNA isolated from the human leucocytes by PCR with a pair of primers without stop codon, and then subcloned into plasmid pEGFP-N1. The recombinant plasmid was transfected into Hela cells with lipofectamine 2000. Total RNA was isolated, and then expression of BDNF-EGFP gene was detected by RT-PCR. The expression and cellular location of BDNF-EGFP protein were examined by Western blot and fluorescence microscopy. Results By analyzing restriction enzyme digestion and PCR, we found that BDNF gene was correctly inserted into the plasmid vector. RT-PCR results confirmed the expression of BDNF-EGFP gene. The fluorescence microscopy indicated that the BDNF-EGFP fusion protein was mainly distributed in cytoplasm. Western blot results showed that BDNF-EGFP fusion protein existed in the medium of Hela cells. Conclusion We have successfully constructed the eukaryotic expression vector of BDNF-EGFP fusion protein, which could be expressed in Hela cells. BDNF-EGFP fusion protein is mainly located in cytoplasm and could be secreted into the outside.