Construction and Certification of Recombinant Adenovirus Vector for σC Gene in Muscovy Duck Reovirus
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摘要: 应用RT-PCR方法扩增出番鸭呼肠孤病毒（Muscovy duck reovirus，MDRV）的σC基因，通过XhoⅠ+HindⅢ双酶切后把该基因插入到经过同样双酶切的穿梭质粒pShuttle-CMV载体中。重组穿梭载体经过双酶切和PCR鉴定后进行测序，序列测定正确，同源性为99.8%。将获得的重组穿梭质粒与腺病毒骨架质粒共转染293细胞后获得重组腺病毒。PCR和RT-PCR鉴定的结果证明σC蛋白基因已成功插入到重组腺病毒载体中。Abstract: The σC gene of muscovy duck reovirus (MDRV) was amplified by RT-PCR and cloned into pShuttle-CMV plasmid after the amplicon was digested by XhoI+Hind III enzyme. Sequencing analysis confirmed the gene to be σC. Subsequently, the recombinant plasmid pShuttle-CMV-σC and the adenovirus backbone vector, pAdEasy-1, were co-transfected into 293 cells to construct a recombinant adenovirus. The obtained recombinant adenovirus pAd-σC was positively identified by PCR and RT-PCR. The information obtained could be used for genetic engineering a vaccine against MDRV.
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Key words:
- muscovy duck reovirus /
- σC gene /
- recombinant adenovirus vector
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图 1 pMD18-T-σC PCR结果
注：1为PCR产物；2为DL2000Marker。
Figure 1. PCR result on pMD18-T-σC plasmid
图 2 pMD18-T-σC酶切结果
注：1为DL2000Marker；2为XhoⅠ和Hind Ⅲ双酶切pMD18-T-σC质粒。
Figure 2. Enzymatically digested pMD18-T-σC plasmid
图 3 pShuttle-CMV-σC的Kpn Ⅰ、Not Ⅰ双酶切结果
注：1为DL15000Marker；2为pShuttle-CMV-σC byKpnⅠ & NotⅠ；3为DL2000Marker。
Figure 3. Enzymatically digested pShuttle-CMV-σC plasmid by KpnⅠ and NotⅠ
图 4 pShuttle-CMV-σC质粒PCR结果
注：1为DL2000Marker；2为引物1和2PCR产物；3为引物3和4PCR产物；4为对照；5为DL2000Marker。
Figure 4. PCR result of pShuttle-CMV-σC
图 5 pAdCMV-σC Pac Ⅰ酶切结果
注：1为DL15000Marker；2为pAdCMV-σC digested by Pac；3为DL6000Marker。
Figure 5. Enzymatically digested pAd CMV-σC plasmid by Pac Ⅰ
图 7 转染后8 d AD293T细胞病变(200×)
Figure 7. Transfected AD293T cells cultured for 8 days, 200×
图 8 引物P1、P2 PCR结果
注：M为DL2000 Marker；1~6为pAd-σC A (5~10) DNA。
Figure 8. PCR result on vector amplified using primers, P1 and P2
图 9 引物P1、P2 RT-PCR结果
注：M为DL2000 Marker；1~6为pAd-σC A (5~10) RNA。
Figure 9. RT-PCR result on vector amplified using primers, P1 and P2
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