【作者】 吴敏仪；

【导师】 杨维东；

【作者基本信息】 暨南大学 ， 动物学， 2006， 硕士

【摘要】 目的：构建含c-fos启动子的pEGFP-c-fos重组质粒载体。将重组质粒载体转入膀胱移行细胞瘤T24细胞中，筛选获得稳定表达株。加入贝毒后，利用细胞中绿色荧光蛋白表达水平的变化进行贝毒素的检测，建立一种以细胞为基础的受体水平的贝毒检测方法。 方法：用PCR扩增c-fos启动子片断，进行VspI、EcoRI双酶切，获得含粘性末端的片断。将该片段与同样双酶切的pEGFP-N1质粒进行连接，构建pEGFP-c-fos重组质粒载体。用PCR、双酶切鉴定，最后测序鉴定。利用脂质体法将重组质粒pEGFP-c-fos转入人膀胱移行细胞瘤T24细胞中，通过G418筛选得到稳定表达株。加入梯度神经性贝毒(neurotoxic shellfish poisoning，NSP)，或加入梯度麻痹性贝毒(Paralytic shellfish poison，PSP)后在经10ng／ml的NSP处理，利用激光共聚焦荧光显微镜观察细胞中绿色荧光蛋白表达的变化，并拍摄不同贝毒水平下绿色荧光蛋白的表达图像。利用美国Image-pro Plus专业图像分析软件对图像进行荧光强度定量分析，绘出绿色荧光表达强度与不同浓度NSP、PSP毒素的关系曲线。 结论：构建pEGFP-c-fos重组质粒载体并使之在T24表达。筛选得到的稳定表达株在经过NSP诱导下，发出较强的荧光，表明重组质粒pEGFP-c-fos在T24细胞中成功表达。建立了绿色荧光表达强度与不同浓度NSP毒素或PSP毒素的关系曲线，为构建以细胞为基础的受体水平的NSP和PSP贝毒检测方法奠定了基础。更多还原

【Abstract】 Objective: A recombinant plasmid pEGFP-c-fos with c-fos promoter and EGFP was constructed, and then transfected into T24 cell. The positive transfectants were obtained by screening. Based on the changes of the expression of EGFP in the T24 cell which induced by the HABs toxins, a new method to detect HABs toxins was founded in receptor level.Methods: The c-fos with cohesive terminus was obtained after amplified by PCR and then digested by VspI and EcoRI, and then ligated with the plasmid pEGFP digested by the same restriction enzymes. The recombinant plasmid pEGFP-c-fos, which was identified by PCR, digesting, and sequencing, was transfected into T24 cell through LipofectAMINE2000. The positive transfectants were obtained by G418. The changes of the expression of EGFP in T24 cell with addition of increasing NSP or PSP were detected by Laser scanning confocal microscope, and quantitative analysis was carried out by Image-pro Plus software.Conclusions: A recombinant plasmid pEGFP-c-fos was constructed and a new pEGFP-c-fos-T24 cell strain was established successfully. The dose-effect relationship between the intensity of green fluorescence and the level of NSP or PSP was detected. A rapid cell-based assay for detection HABs toxin was establishedprimarily.更多还原