论文部分内容阅读
目的构建由具有肿瘤特异性启动作用的端粒酶逆转录酶启动子(mTERT-promotor)驱动m4-1BBL(CD137Ligand)基因的腺病毒载体。方法用PCR和RT-PCR的方法,分别从C57BL/6小鼠组织中克隆mTERT启动子和共刺激分子m4-1BBL基因,通过T载体和转移载体将目的基因亚克隆到腺病毒载体上,293A细胞包装成病毒颗粒rAD-mTERT-m4-1BBL,流式细胞仪检测m4-1BBL基因在Hepa1-6及L929细胞上的表达。结果通过PCR检测、直接测序证实目的基因克隆成功并亚克隆到表达载体上,细胞免疫化法证实病毒包装成功,流式细胞仪检测发现重组腺病毒载体rAD-mTERT-m4-1BBL能特异性地在肿瘤细胞Hepa1-6上高表达m4-1BBL。结论由mTERT-promotor驱动m4-1BBL基因的具有肿瘤特异性的腺病毒载体构建成功,为下一步进行体内外抗肿瘤实验打下了基础。
Objective To construct an adenoviral vector carrying m4-1BBL (CD137Ligand) gene by telomerase reverse transcriptase promoter (mTERT-promotor) with tumor-specific promoter. Methods The mTERT promoter and costimulatory molecule m4-1BBL gene were cloned from C57BL / 6 mice by PCR and RT-PCR respectively. The target gene was subcloned into adenoviral vector by T vector and transfer vector, 293A The cells were packaged into virus particles rAD-mTERT-m4-1BBL, and the expression of m4-1BBL gene on Hepa1-6 and L929 cells was detected by flow cytometry. The results of PCR detection, direct sequencing confirmed the successful cloning of the target gene and subcloned into the expression vector, cell immunization confirmed virus packaging, flow cytometry found that the recombinant adenovirus vector rAD-mTERT-m4-1BBL can specifically High expression of m4-1BBL on tumor cells Hepa1-6. Conclusion The successful construction of the tumor-specific adenoviral vector containing m4-1BBL gene by mTERT-promotor lays the foundation for further antitumor in vitro and in vivo experiments.