人乳头状瘤病毒16亚型E6E7基因重组腺病毒的构建及其在小鼠骨髓源树突状细胞中的表达

Construction of Recombinant Adenovirus with Human Papillomavirus 16 E6E7 Gene and Its Expression in Mouse Bone Marrow-derived Dendritic Cells

目的 构建人乳头状瘤病毒16亚型(Human papillomavirus 16,HPV16)E6E7基因重组腺病毒,并在小鼠骨髓源树突状细胞(Dendritic cell,DC)中表达。方法双酶切质粒pET-32a(+)-E6E7获得E6E7,基因片段,插入腺病毒穿梭质粒pAd-Track-CMV中,转染HEK293细胞,扩增病毒,经同源重组、包装后获得重组腺病毒pAd-E6E7,转染体外培养的小鼠骨髓源DC,激光共聚焦显微镜观察转染的小鼠DC的形态,流式细胞术检测转染前后小鼠DC表面标志物(CD40、CD86、MHCⅡ和CD11C),Western blot检测E6蛋白的表达。结果双酶切及测序证实,重组腺病毒质粒pAd-E6E7构建正确,插入的E6E7基因片段序列正确;转染HEK293细胞48 h,倒置荧光显微镜下可见绿色荧光蛋白的表达,重组腺病毒的滴度为3×107 CCID50/ml;重组腺病毒pAd-E6E7感染后DC的状态与成熟DC表面标志物(CD40、CD86、MHCⅡ和CD11C)相符合,感染后的DC表面有许多树枝状、刺状突起,符合成熟DC的表面形态;感染后的DC中存在E6蛋白的表达。结论已成功构建了HPV16 E6E7基因重组腺病毒表达质粒,其能在小鼠骨髓源DC中表达。

Objective To construct the recombinant adenovirus carrying human papillomavirus 16 （HPV16） E6E7 gene and express in mouse bone marrow-derived dendritic cells （DCs）. Methods E6E7 gene was obtained by digestion of plasmid pET-32a （＋）-E6E7 with restriction endonuclease and inserted into adenovirus shuttle plasmid pAdTrack-CMV. The constructed recombinant plasmid was transfected to HEK293 cells for amplification, based on which recombinant adenovirus pAd-E6E7 was obtained by ho- mologous recombination and packaging and transfected to mouse bone marrow-derived DCs cultured in vitro. The morphology of trans- fected DCs was observed by laser confocal microscopy. The surface markers on DCs before and after transfection, such as CD40, CDs6, MHC lI and CDtlc, were determined by flow cytometry. The expression of E6 protein was determined by Western blot. Results Both restriction analysis and sequencing proved that recombinant adenovirus pAd-E6E7 was constructed correctly, in which an E6E7 gene fragment with correct sequence was inserted. The expression of green fluorescent protein （GFP） was observed under invert micro- scope in the HEK293 cells 48 h after transfection. The titer of recombinant adenovirus pAd-E6E7 reached 3 x 107 CCIDso/ml. The ex- pression levels of surface markers of DCs after transfection were consistent with those of mature DCs. Many dendritic or spiniform processes were observed on the surface of transfected DCs, which were consistent with the surface morphology of mature DCs. E6 protein was expressed in the transfected DCs. Conclusion The expression vector for recombinant adenovirus carrying HPV16 E6E7 gene was successfully constructed and expressed in mouse bone marrow-derived DCs.