腺病毒载体介导密码子优化型HPV 16 L1基因在哺乳动物细胞中的高效表达及病毒样颗粒的装配

为研究重组腺病毒载体作为HPV16预防性疫苗的可行性,构建了含密码子优化型HPV 16 L1基因的重组腺病毒,并对优化基因在哺乳动物细胞中的表达进行研究。首先按照哺乳动物密码子偏好对野生型HPV16 L1基因进行改造并合成优化基因,命名为mod.HPV16L1。将mod.HPV16L1基因克隆到穿梭质粒PDC316上,与骨架质粒共转染293细胞,在细胞内包装重组腺病毒rAd-mod.HPV16L1。用免疫印迹法检测病毒感染的293T细胞中HPV16L1蛋白的表达。通过Optiprep密度梯度超速离心法纯化HPV16 L1病毒样颗粒(VLPs)。用磷钨酸负染,在电子显微镜下观察HPV16 L1蛋白自我装配形成的VLPs。结果显示,重组腺病毒载体可介导mod.HPV16 L1基因在哺乳动物细胞内的高效表达,L1蛋白可自我装配形成VLPs。

Highly Efficient Expression of Codon-modified HPV16 L1 Gene in Mammalian Cell Using Adenovirus Vector and Assembly of VLPs

To investigate the feasibility of using recombinant adenovirus vector as prophylactic vaccine against HPV infection,a recombinant adenovirus containing codon modified HPV16 L1 gene was constructed,and the expression of the optimized gene in mammalian cells mediated by adenovirus was evaluated.We optimized the codon usage of wild type HPV 16 L1 gene according to the codon bias of mammalian,and synthesized the full-length optimized L1 gene,named mod.HPV16L1,which was later cloned to shuttle plasmid PDC316,and co-transfected with the backbone plasmid into 293 cell for rAd production.We detected the expression of HPV16 L1 protein in rAd infected 293T cell by Western blot,and purified the HPV16 virus-like particles that self-assembled in 293T cell using Optiprep density gradient ultracentrifugation.The purified HPV16 L1 virus-like particles were seen under the electron microscope.The results showed the recombinant adenovirus,rAd-mod.HPV16L1,mediated highly efficient expression of codon-modified HPV16L1 gene in mammalian cell.The L1 protein could self-assemble into VLPs in mammalian cell.