HPV16E7E6重组腺病毒的构建及免疫效果评价

Immunogenicity and antitumor efficacy of the recombinant adenovHPV type 16 in mice

目的构建用于宫颈癌治疗的HPV16E7E6重组腺病毒并评价其免疫效果。方法根据哺乳细胞使用密码子的偏嗜性设计HPV16E7E6融合蛋白表达优势密码子基因序列并合成。将合成的E7E6基因插入腺病毒重组穿棱质粒pCD316后，与Ad5腺病毒骨架质粒共转染293细胞，重组病毒经单斑纯化并进行目的基因插入及表达的鉴定。扩增纯化重组病毒后免疫小鼠，评价其免疫活性。结果PCR试验证明E7E6密码子优化基因成功插入Ad5病毒；Western blot检测结果表明重组腺病毒中E7E6优化基因能高效表达，该重组腺病毒免疫C57小鼠后，可诱发强特异性T细胞免疫应答，在小鼠体内能完全抑制5×10^4TC-1移植瘤细胞的生长。结论重组HPV16E7E6腺病毒可作为治疗HPV慢性感染或宫颈癌的候选疫苗。

Objective To construct one recombinant adenovirus AdE7E6 expressing HPV16 E6 and E7 fusion protein as candidate for HPV16 therapeutic vaccine. Methods The codon-optimized E6 and E7 gene, were fused to create one open reading frame, then inserted into adenovirus vector pCD316. A strain of recombinant adenovirus was constructed through homologous recombinant in 293 cells, and identified by PCR and Western blot. Finally, it was employed to study it＇s immunogenicity and the activity of the tumor growth regression. Rsults The PCR result showed that E6E7 fusion gene had been integrated in recombinant AdS DNA. Western blot test confirmed that the E6E7 fusion protein was highly expressed in 293 cells infected with AdSE7E6 recombinant adenovirus. The recombinant adenovirus elicited significant E7 specific CD8＋ T lymphocyte response in vaccinated mice. These responses could completely prevent the TC-1 tumor cell bearing mice treated with AdE7E6 from developing into tumor. Conclusion These results suggested that rAdSE7E6 could be a potent vaccine candidate for the treatment of HPV16-associated tumors and their precancerous transformations.