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目的构建携带人甲状旁腺相关激素(PTHLH)基因的慢病毒表达载体pGC-FU/PTHLH。方法酶切载体pGC-FU,根据人PTHLH基因合成特定引物,扩增目的基因片段,将其克隆到pGC-FU质粒(含EGFP基因)上,菌落PCR鉴定及测序分析重组载体,使用Lipofectamine 2000诱导转染pGC-FU、pHelper1.0和pHelper2.0载体三质粒进入293T细胞包装慢病毒,并用带PTHLH的慢病毒感染293T细胞和鼻咽癌CEN1细胞确认慢病毒包装是否成功。结果菌液PCR产物琼脂糖凝胶电泳鉴定显示,与理论预计值阳性转化子735bp,阴性转化子198bp基本相吻合;PCR鉴定阳性的克隆进行测序和比对分析,结果完全匹配,进一步鉴定载体构建成功。分别将质粒包装系统共转染293T细胞,包装产生空白对照慢病毒(pGC-FU)和过表达PTHLH的慢病毒(pGC-FU/PTHLH);或用携带PTHLH和EGFP基因的病毒上清感染CNE1细胞,48h后,倒置荧光显微镜下观察293T和CNE1细胞,均可见绿色荧光,转染成功。结论成功构建携人PTHLH基因慢病毒表达载体,为进一步研究PTHLH基因在鼻咽癌转移中的作用及鼻咽癌发病分子机制奠定了基础。
Objective To construct lentiviral vector pGC-FU / PTHLH carrying human parathyroid hormone (PTHLH) gene. Methods The vector pGC-FU was digested with restriction endonucleases. The target gene fragment was amplified by PCR from human PTHLH gene and cloned into pGC-FU plasmid containing EGFP gene. The recombinant plasmid was identified by PCR and sequenced. The recombinant plasmid was induced by Lipofectamine 2000 The plasmids pGC-FU, pHelper1.0 and pHelper2.0 were transfected into 293T cells and the lentivirus packaging was confirmed by infecting 293T cells and nasopharyngeal carcinoma CEN1 cells with lentivirus with PTHLH. Results The bacterial liquid PCR product agarose gel electrophoresis showed that it was basically consistent with the theoretical expected positive transformant 735bp and the negative transformant 198bp. The positive clones were sequenced and compared with the positive ones by PCR, and the results were completely matched to further identify the vector construction success. 293T cells were cotransfected with plasmid packaging system respectively and packaged to generate blank control lentivirus (pGC-FU) and lentivirus overexpressing PTHLH (pGC-FU / PTHLH); or CNE1 infected with virus supernatant carrying PTHLH and EGFP genes After 48h, 293T and CNE1 cells were observed under an inverted fluorescence microscope. All cells showed green fluorescence and the transfection was successful. Conclusion The recombinant lentiviral vector carrying human PTHLH gene was successfully constructed, which laid the foundation for further study on the role of PTHLH gene in the metastasis of nasopharyngeal carcinoma and the molecular mechanism of the disease.