【作者】 车军；

【导师】 刘洁生；

【作者基本信息】 暨南大学 ， 动物学， 2002， 硕士

【摘要】 目的：以C-fos启动子为启动子、以绿色荧光蛋白为报告基因，构建重组质粒HF443-GFP；利用重组质粒转染人膀胱移行细胞癌细胞系BIU-87细胞。培养体系中依次加入赤潮毒素和乌头碱，观察乌头碱诱导产生的绿色荧光蛋白表达的变化情况，通过荧光量的变化定量赤潮毒素，建立一种以细胞为基础的赤潮毒素检测方法。 方法：PCR扩增GFP基因并用HindⅢ和XbaⅠ双酶切，与同样经过HindⅢ和XbaⅠ双酶切并纯化的HF443质粒连接，构建重组质粒HF443-GFP。酶切鉴定重组HF443-GFP质粒并测序，对比序列和文献报道是否一致。利用脂质体LipofectAMINE2000将重组质粒HF443-GFP转入体外培养的BIU-87细胞，加入Na~+通道激活剂乌头碱诱导绿色荧光蛋白大量表达，荧光显微镜下观察绿色荧光蛋白的表达情况，检测乌头碱能否诱导BIU-87细胞中绿色荧光蛋白表达。在赤潮毒素检测时，细胞培养体系中先后加入不同浓度的赤潮毒素GTX(0.0033μmol／L、0.033μmol／L、0.33μmol／L)和乌头碱(0.033μmol／L)，观察不同毒素水平下绿色荧光蛋白的表达情况，确定毒素和细胞中绿色荧光蛋白表达的相关性。 结论：成功构建了一个重组质粒HF443-GFP；重组质粒转染BIU-87细胞后加入乌头碱，荧光显微镜下观察到细胞发出绿色荧光并能稳定存在3-4天，提示乌头碱可以诱导BIU-87细胞中绿色荧光蛋白的表达。在赤潮毒素检测实验中，细胞发出的荧光强度随毒素水平的增加而减弱，呈现一定的剂量依赖关系，提示用这种方法定量检测赤潮毒素是可行的。更多还原

【Abstract】 Objection: A novel rapid cell-based assay was established for detection theharmful algal bloom (HAB) toxin. The first, A recombinant plasmid HF443-GFP which contained a c-fos promoter and GFP gene was constructed, and then transfected into Human bladder transitional cell carcinoma BIU-87 cell; The second, GTX, a HAB toxin and aconitine, a sodium channel activator were added into the cell culture system. The expression of GFP induced by aconitine was detected in the system. Based on the changes in the expression of GFP blocked by GTX, the level of GTX was finally deduced.Methods: The green fluorescent protein(GFP) gene amplified by PCR wasdigested by Hind III and Xba I, and then ligated with the plasmid HF443 which digested by the same restriction endonuclease. The resulting plasmid HF443-GFP was identified with sequencing. The BIU-87 cell was transfected by the recombinant plasmid HF443-GFP through LipofectAMINE2000. The behavior of expression of GFP in BIU-87 cell was observed under the fluorescent microscope.Conclusions: A new recombinant plasmid HF443-GFP was constructedsuccessfully. The sequence of the GFP was identified as same as the reported sequence. When aconitine were added into BIU-87 cell culture system transfected with plasmid HF443-GFP, green fluorescence was observed, which suggested that the aconitine can induced the expression of GFP in the BIU-87 cell. In the toxin detection experiment, with the increase of the level of GTX, the intensity of green fluorescence decreased. A certain dose-effect relationship was observed. These results indicated that GTX may inhibit the expression of GFP. This system can be used in detection of HAB toxin.更多还原