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

1、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         11-01-07 16:05:00     编辑：studa20                作者：石秦东，张蓬勃，田玉梅，张军峰，陈新林，刘勇 【摘要】  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

2、(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pEGFPN1质粒载体连接，构建BDNFEGFP融合蛋白真核表达质粒。以脂质体Lipofectamine2000 转染Hela细胞，提取总RNA，通过RTPCR方法检测BDNFEGFP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，RTPCR证实BDNFEGFP融合蛋白在转录水平有表达，荧光显微镜观察显示BDNFEG

3、FP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BDNFEGFP融合蛋白存在。 结论 成功构建了BDNFEGFP融合蛋白真核细胞表达质粒，BDNFEGFP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。 【关键词】  脑源性神经营养因子；绿色荧光蛋白；融合蛋白；Hela细胞    ABSTRACT: Objective  To construct the eukaryotic expression vector of brainderived neutrophic factor (BD

4、NFEGFP) 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 pEGFPN1. The recombinant plasmid was transfected into

5、Hela cells with lipofectamine 2000. Total RNA was isolated, and then expression of BDNFEGFP gene was detected by RTPCR. The expression and cellular location of BDNFEGFP protein were examined by Western blot and fluorescence microscopy. Results  By analyzing restriction enzyme digestion and PCR,

6、 we found that BDNF gene was correctly inserted into the plasmid vector. RTPCR results confirmed the expression of BDNFEGFP gene. The fluorescence microscopy indicated that the BDNFEGFP fusion protein was mainly distributed in cytoplasm. Western blot results showed that BDNFEGFP fusion protein exist

7、ed in the medium of Hela cells. Conclusion  We have successfully constructed the eukaryotic expression vector of BDNFEGFP fusion protein, which could be expressed in Hela cells. BDNFEGFP fusion protein is mainly located in cytoplasm and could be secreted into the outside.    KEY

8、WORDS: brainderived neurotrophic factor; green fluorescence protein; fusion protein; Hela cells     脑源性神经营养因子(brainderived neurotrophic factor, BDNF)是神经营养因子(neurotrophin family)家族成员之一，与其受体trkB 酪氨酸激酶广泛分布于脑组织，包括皮层、纹状体、海马、丘脑、小脑等1。BDNF除参与神经系统发育过程中神经元的存活、生长和分化外，越来越多的证据表明也参与了成年神经系统神经元的

9、修复与重塑，诱导成年脑内源性神经前体细胞迁移至纹状体等部位23。因此，BDNF对神经损伤性疾病如脑血管病、脑外伤及神经退行性疾病如Alzheimer病的治疗具有潜在的应用前景。为进一步探索BDNF对内源性神经干细胞增殖分化的影响，我们通过基因重组技术构建了BDNF与增强型绿色荧光蛋白(EGFP)的融合蛋白真核表达质粒，并对其在真核细胞的表达及是否具有分泌功能进行了鉴定。    1  材料与方法    1.1  材料  质粒载体pEGFPN1(BD Biosciences Clontech公司)，PMD18

10、T载体(TaKaRa公司)，限制性内切酶、T4DNA连接酶、胶回收试剂盒(TaKaRa公司)，Taq DNA聚合酶(Promega公司)，1640培养基(Invitrogen 公司)，Lipofectamine 2000转染试剂(Invitrogen 公司)，Top10大肠杆菌、Hela细胞株(本实验室保存)，抗人BDNF抗体(Santa Cruz公司)。常用试剂为本室配制。    1.2  方法CGGGTACCATGACCATCCTTTTCCTT3，P2：5GCGGATCCACTCTTCCCCTTTTAATG3。PCR在50L的总体积中进行，反应条件为94变性4min后开始循环，94变性1min，55退火1min，72延伸1min 30s，共30个循环后，再于72延伸10min，用DNA纯化试剂盒从10g/L琼脂糖凝胶回收、纯化所需PCR产物BDNF，用 T4连接酶将其亚克隆入T载体，酶切、PCR鉴定，由上海华诺公司测序。    2  结    果