不同型别登革病毒融合外膜基因重组腺病毒的构建及表达

Construction and expression of recombinant adenovirus encoding EDⅢs fusion gene of dengue virus type 1 and 2

目的构建登革病毒1型和2型外膜蛋白(E蛋白)DⅢ区融合基因的重组腺病毒,在BHK-21细胞中表达。方法用PCR法扩增登革病毒1型和2型EDⅢ基因,依次克隆入pCDNA3.1(+),构建重组质粒pCDNA-D1/D2EDⅢ。以此为模板,扩增包含CMV启动子-D1/D2EDⅢ-BGHpA的表达片段,与pENTR4载体连接形成质粒pENTR4-D1/D2EDⅢ,与pAd/PL-DEST进行体外同源重组,形成重组腺病毒载体pAd-D1/D2EDⅢ,在293A细胞系中包装成具有感染性的重组腺病毒,进一步感染BHK-21细胞,用间接免疫荧光法和Westernblot法检测重组蛋白在细胞中的表达情况。结果重组腺病毒构建成功,转染293A细胞后获得2×109pfu/mL滴度的重组腺病毒,感染哺乳动物细胞后能够表达1型和2型EDⅢ区目的蛋白。结论不同型别融合基因的重组腺病毒表达载体能有效地表达相应型别的目的蛋白,为进一步构建单一的四价重组腺病毒应用于登革疫苗研究奠定基础。

To construct recombinant adenovirus encoding EDⅢs fusion gene of dengue virus type 1 and 2 and to induce its expression in BHK-21 cells, envelope domain Ⅲ s of dengue serotypes 1 and 2 were amplified by PCR and cloned into pCDNA3. 1（＋） to generate recombinant plasmid pCDNA-D1/D2EDⅢ. A fragment encoding CMV promoter-D1/D2EDⅢ-BGH pA was amplified from pCDNA-D1/D2EDⅢ and cloned into vector pENTR4 to generate plasmid pENTR4-D1/D2EDⅢ, then was transfer into pAd/PL-DEST by LR recombination reaction. The recombinant adenovirus pAd-D1/D2EDⅢ was transfeeted into 293A cells to package recombinant adenovirus to detect the expression of the recombinant proteins by indirect immunofluorescent assay and western blotting. The results showed that the recombinant adenovirus pAd-D1/D2EDⅢ was obtained with a titer of 2 × 10^9 pfu/mL in transfected 293A cells and the recombinant EDⅢ proteins either of type 1 or type 2 could be expressed efficiently in mammalian cells. In conclusion, the recombinant adenovirus was constructed successfully, which could be valuable for development of a tetravalent adenovirus.