摘要
目的构建能同时表达HPV16,18,58三亚型E6-E7优化融合基因的重组腺病毒,为下一步获得适合国情的宫颈癌HPV三价治疗性候选疫苗打基础。方法常规扩增、构建HPV16,18,58的E6-E7融合基因,PCR定点突变消除其转化活性后以IRES相连并重组入pAdeno-X腺病毒载体系统;体外细胞、软琼脂培养及裸鼠成瘤实验检测突变基因转化活性去除情况;重组腺病毒电击转染293包装细胞,Western blot分析外源基因表达情况。结果三型重组融合基因序列与设计完全一致,其中突变基因的转化活性显著降低,Western blot等显示所构建重组腺病毒在293细胞中高效表达外源基因。结论成功构建表达HPV16,18,58三型E6-E7融合基因的重组腺病毒,并有效消除了其转化活性,为下一步疫苗研发打下了基础。
Objective To construct a recombinant adenovirus which could expressing E6-E7 fusion proteins of human papillomavirus subtypes 16,18, and 58, for developing therapeutic candidate vaccines best suited to Chinese patients in the next step. Methods E6-E7 fusion genes of human papillomavirus subtypes 16, 18, and 58 were amplified and constructed through conventional molecular biology technology. Site-directed mutagenesis was introduced for abolishing the transforming activity of E6-E7 fusion, while the effects of mutation were also confirmed by cell culture test, anchorage independent growth, and tumorigenesis in nude mice. These mutated fusion genes were then ligatod with Internal ribozyme entry site（IRES） and cloned into pAdeno-X vector system. The recombinant adenovirus was transfected into 293 cells and the expression of heterologons protein was analyzed by Western blotting and fluorescence microscopy. Results Sequences in the constructed plasmids were correct. Transforming activity of mutated E6 and E7 genes were significantly degraded in vitro and in vivo. E6-E7 fusion proteins of all three subtypes were successfully detected in eultivation supernatant of recombinant adenovirus-infected 293 cell by Western blotting. Conclusion The constructed recombinant adenovirus can express E6-E7 fusion protein of human papillomavirus subtypes 16,18, and 58 with transforming activity abolished, which might pave a way for developing a series of HPV multivalent therapeutic vaccines in the future.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2008年第4期459-463,共5页
Immunological Journal
基金
深圳市重点实验室提升计划资助项目
关键词
人乳头瘤病毒
腺病毒
转化活性
Human papillomavirus
Adenovirns
Transforming activity
