hβ-NGF基因真核表达载体构建及亚细胞定位

Construction and subcellular localization of hβ-NGF eukaryotic expression vector

目的构建含EGFP标签的人β-神经生长因子(hβ-NGF)的真核表达载体,观察和分析其在HEK293细胞中的定位,为后续基因转移实验奠定基础。方法PCR扩增hβ-NGF基因编码框,连接到pEGFP-N1载体,将HEK293细胞分为对照组(不转染)、空载组(转染pEGFP-N1质粒)和实验组(转染pEGFP-N1-hβ-NGF质粒),后两组采用脂质体法转染,24h后荧光显微镜下观察EGFP瞬时表达及细胞定位,通过蛋白序列数据库和生物信息学软件进一步分析亚细胞定位。结果经双酶切和测序鉴定,pEGFP-N1-hβ-NGF真核表达载体构建成功。与空载组比较,实验组融合EGFP只少量分布于胞质中(P<0.05),不存在于细胞核内。生物信息学分析表明,hβ-NGF基因编码长241个氨基酸的肽链,由信号肽、前导肽和功能肽3段构成,含有2个N-糖基化位点,6个高度保守半胱氨酸残基,可以形成3个二硫键,是可溶、不稳定的分泌型蛋白;亚细胞定位该蛋白主要位于细胞外,部分分布于胞吞内体和高尔基体,以模序二聚体形式发挥生物学功能。结论成功构建了有活性的hβ-NGF真核表达载体,观察到融合蛋白在HEK293真核细胞系分泌表达,其亚细胞分布与生物信息学分析一致。

Objective To construct the eukaryotic expression vector of hβ-NGF gene labeled with EGFP tag and to study its subcellular localization in HEK293 cells.Methods hβ-NGF gene was obtained from the human peripheral blood by PCR and cloned into pEGFP-N1 vector.HEK293 cells were divided into control,vector and experimental group.The plasmid was transfected into last two group cells by liposome,transiently expression and localization was detected by fluorescence microscope.The subcellular localization was further analyzed by bioinformatic tools.ResultsThe result of double digestion and sequencing showed that pEGFP-N1-hβ-NGF eukaryotic expression vector was constructed.The compared with the vector group,results of fluorescence microscope revealed that pEGFP-N1-hβ-NGF fusion protein was partial expressed at cytoplasm of HEK293 cells in experimental group（P〈0.05）,but not nucleus.The most of hβ-NGF protein was subcellular located in extracellular space,a small number of its was located endosome and golgi apparatus.Conclusion hβ-NGF eukaryotic expression vector is constructed successfully,which is expression in eukaryotic cells in line with bioinformatics analysis.