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人41型腺病毒(Human adenovirus type 41,HAdV-41)为难养腺病毒,E1区缺失的HAdV-41无法在293细胞中拯救并扩增,本研究试图通过反向遗传学操作获得能够在293细胞中拯救并扩增的重组HAdV-41。首先,对原有的骨架质粒pAdbone41进行改造:通过重叠延伸PCR方法将HAdV-41的E3区基因替换为HAdV-5E4orf6编码区;将HAdV-5E1A增强子克隆至HAdV-41E4区启动子上游,获得新的骨架质粒pAdbone41E4EE。该骨架质粒与线性化的穿梭质粒pSh41-GFP在E.coli BJ5183菌株同源重组得到腺病毒质粒pAd41E4EE-GFP。pAd41E4EE-GFP经Pme I酶切线性化后转染293细胞,拯救出重组病毒HAdV-41-E4EE-GFP。经过7轮扩增,超速离心纯化后获得1.0ml浓度为8.0×10~(10) vp/mL的重组腺病毒,其感染滴度为1.7×10~9 IU/mL,电子显微镜下观察可见形态完整的病毒颗粒,限制性内切酶酶切分析及PCR鉴定结果均表明携带HAdV-5E4orf6基因与E1A增强子基因的重组腺病毒载体构建正确。本研究利用反向遗传学技术对病毒载体进行改造,实现了在293细胞中培养E1区缺失的HAdV-41的目的。
Human adenovirus type 41 (HAdV-41) is a hard-to-support adenovirus, and HAdV-41 lacking in the E1 region can not be rescued and expanded in 293 cells. In this study, Rescued and expanded recombinant HAdV-41 in cells. First, the original backbone plasmid pAdbone41 was modified: the E3 region gene of HAdV-41 was replaced by the HAdV-5E4orf6 coding region by overlap extension PCR; the HAdV-5E1A enhancer was cloned upstream of the promoter of HAdV-41E4 region to obtain The new backbone plasmid pAdbone41E4EE. This backbone plasmid was homologously recombined with the linearized shuttle plasmid pSh41-GFP in the E. coli BJ5183 strain to obtain the adenovirus plasmid pAd41E4EE-GFP. The pAd41E4EE-GFP was linearized with Pme I and transfected into 293 cells to rescue the recombinant virus HAdV-41-E4EE-GFP. After 7 rounds of amplification, 1.0 ml of recombinant adenovirus with the concentration of 8.0 × 10 ~ (10) vp / mL was obtained after ultracentrifugation and the infection titer was 1.7 × 10 ~ 9 IU / mL. The morphology was observed under an electron microscope The complete virus particles, restriction endonuclease analysis and PCR identification showed that the recombinant adenovirus vector carrying HAdV-5E4orf6 gene and E1A enhancer gene was correctly constructed. In this study, the use of reverse genetics technology to transform the virus vector to achieve the purpose of cultivating HAdV-41 deleted in the E1 region in 293 cells.