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【目的】构建携带 SARS 冠状病毒刺突蛋白 N 端基因片段的重组腺病毒。【方法】从 SARS 病毒 cDNA 扩增刺突蛋白 N 端基因片段(-45～1469nt,截短的 S1区,S_N),插入腺病毒穿梭质粒 pShuttle,通过体外连接法构建重组腺病毒Ad-S_N。用 Ad-S_N 感染 Vero-E6细胞,通过 RT-PCR 和 Western blot 法检测 S_N 的表达。【结果】克隆的 S_N 基因序列与文献报道基本一致;Ad-S_N 基因组结构与预期一致。在 Ad-S_N 感染的 Vero-E6细胞中存在 S_N 基因的转录和分泌型 S_N 蛋白的表达。【结论】体外连接法是一种简便易行的重组腺病毒载体构建方法;重组腺病毒 Ad-S_N 能正确表达分泌型 S_N 蛋白,可用于诱导抗 SARS 免疫的动物实验研究。
【Objective】 To construct a recombinant adenovirus harboring N-terminal gene fragment of SARS coronavirus. 【Method】 The adenovirus shuttle plasmid pShuttle was inserted into the N-terminal gene fragment of spike protein (-45 ~ 1469nt, truncated S1 region, S_N) from SARS virus cDNA and the recombinant adenovirus Ad-S_N was constructed by in vitro ligation. Vero-E6 cells were infected with Ad-S_N and the expression of S_N was detected by RT-PCR and Western blot. 【Result】 The results showed that the sequence of cloned S_N gene was consistent with that reported in the literature. The structure of Ad-S_N genome was consistent with the expectation. The S_N gene transcription and secretion S_N protein expression exists in Ad-S_N infected Vero-E6 cells. 【Conclusion】 In vitro ligation method is a simple and convenient method for constructing recombinant adenovirus vector. Recombinant adenovirus Ad-S_N can express secretory S_N protein correctly and can be used to induce anti-SARS immunity in animal experiments.