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目的:构建含汉坦病毒核蛋白(NP)氨基端编码基因S0.7,CAG启动子及WPRE转录后调节元件的新型重组腺病毒。方法:依据GenBank序列,分别合成CAG序列及WPRE序列,插入S0.7-pShuttle中,构建含S0.7基因,CAG启动子及WPRE转录后调节元件的转移载体pShuttle-S0.7-CAG-WPRE。采用PI-SceI和I-CeuI限制性内切酶将该片段克隆入腺病毒DNA,得到重组腺病毒DNAAdeno-S0.7-CAG-WPRE,使用PacI线性化后转染HEK293细胞,包装重组腺病毒。感染HEK293细胞后用免疫荧光法检测表达产物。结果:转移载体pShuttle-S0.7-CAG-WPRE通过测序证明构建正确,纯化重组腺病毒滴度达1013pfu/L。免疫荧光结果表明,重组腺病毒感染的HEK293细胞被抗汉坦病毒NP的特异性mAb1A8所识别。结论:成功地构建了含汉坦病毒S0.7基因,CAG启动子及WPRE转录后调节元件的新型重组腺病毒,本实验为进一步研制汉坦病毒基因疫苗奠定了良好的基础。
OBJECTIVE: To construct a new recombinant adenovirus containing the S0.7 amino-terminal encoding gene of hantavirus nucleoprotein (NP), CAG promoter and post-transcriptional regulatory elements of WPRE. Methods: According to the GenBank sequence, the CAG sequence and WPRE sequence were synthesized and inserted into S0.7-pShuttle respectively to construct the transfer vector pShuttle-S0.7-CAG-WPRE containing S0.7 gene, CAG promoter and post-transcriptional regulation elements of WPRE . The fragment was cloned into adenoviral DNA using PI-Scel and I-CeuI restriction endonuclease to obtain the recombinant adenovirus DNAAdeno-S0.7-CAG-WPRE. After linearization with PacI, HEK293 cells were transfected and packaged with recombinant adenovirus . After HEK293 cells were infected, the expression products were detected by immunofluorescence. Results: The transfer vector pShuttle-S0.7-CAG-WPRE was proved to be correct by sequencing. The titer of purified recombinant adenovirus was 1013 pfu / L. Immunofluorescence results showed that recombinant adenovirus infected HEK293 cells were recognized by mAblA8 specific for anti-Hantaan virus NP. CONCLUSION: A novel recombinant adenovirus containing S0.7 gene of Hantavirus, CAG promoter and post-transcriptional regulatory elements of WPRE has been successfully constructed. This study laid a good foundation for further development of Hantavirus gene vaccine.