体内靶向树突状细胞的腺病毒通用肿瘤疫苗的构建

A Novel Strategy for Development of Universal Tumor Vaccine:A DC-targeted Adenovirus Encoding hTRT

目的采用细胞菌内同源重组法构建人端粒酶逆转录酶(hTRT)重组腺病毒,并将其以甘露聚糖进行表面修饰,以探索构建一种新型的体内靶向树突状细胞(DC)的通用型肿瘤抗原。方法将编码hTRT的cDNA序列自pBABE-PURO-hTERT质粒亚克隆至腺病毒质粒pAdTrack-CMV中,将后者与骨架质粒pAdEasy-1经电冲击共转化宿主菌BJ5183进行同源重组。经过克隆筛选获得正确的重组腺病毒质粒后,将其线性化转染293细胞包装获得重组腺病毒。将重组腺病毒扩增纯化,以甘露聚糖进行表面修饰。最后将甘露聚糖修饰后的腺病毒免疫小鼠。结果重组腺病毒能在293细胞产生细胞病变效应(CPE),腺病毒转染后的细胞通过PCR及免疫印迹能测到hTRT的表达。重组腺病毒经过甘露聚糖修饰后,经高碘酸席碱染色呈阳性。重组腺病毒能在小鼠体内靶向脾脏DC细胞。结论成功构建能体内靶向DC细胞的hTRT重组腺病毒,为通用型肿瘤疫苗的研发作出了新的探索。

Objective To explore the feasibility of constructing a dendritic cell targeted adenovirus as a possible universal tumor vaccine. Methods The cDNA of human telomerase reverse transcriptase （hTRT） from pBABE-PURO-hTERT was subcloned into pAdTrack-CMV. Recombinant adenovirus for hTRT was constructed in AdEasy system. The confirmed recombinant adenovirus plasmid was transfected into 293 cells to generate the recombinant adenovirus. Later the recombinant adenovirus was multiplied, purified and modified with mannan as a vaccine to immunize mice. Results An obvious cytopathic effect was observed with the rescue of recombinant adenovirus. The expression of hTRT was confirmed by PCR and Western blot analysis. Mannan modified adenovirus stained positively by periodic acid Schiff stain, and expression of the adenovirus was found mainly restricted in dendritic cells （DC） in spleen of mice. Conclusion A DC-targeted adenovirus was constructed. This might be helpful for the development of universal tumor vaccine in future.