携人PTHLH基因慢病毒表达载体的构建

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。

Construction of a lentiviral expression vector harboring human PTHLH gene

Objective To generate lentiviral expression vector harboring human PTHLH gene. Methods PTHLH DNA fragment was designed and cloned into the pGC-FU vector (including EGFP). The recombinant vector was analyzed by PCR and DNA sequencing. pGC-FU,pHelper 1.0 and pHelper 2.0 were transfected into 293T cells to package lentivirus. Lentivirus supernatant harboring PTHLH genes were used to infected 293T cells. Results The PCR product from bacteria liquid was identified by agarose electrophoresis. The result coincided with the theoretical predicted value which positive transformant was 735 bp and negative transformant was 198 bp. The positive clone was sequenced and blasted, the result showed that coding sequence of PTHLH was cloned successfully. Recombinant and vector plasmids and helpful packaging plasmids were respectively transfected to 293T cells, then we aquired pGC-FU and pGC-FU /PTHLH virus. CNE1 cells were infected by virus supernatant of pGC-FU /PTHLH which contain PTHLH and EGFP. After 48h, green fluorescence of 293T and CNE1 cells can be observed under inverted microscope, confirmed the successful transfection of PTHLH gene. Conclusions The constructed lentiviral expression vector can be used for further research of nasopharyngeal carcinoma (NPC). It can not only be used in the study of metastasis, but also the molecule pathogenesis of NPC.