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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[1] URSULA HEFFELS-REDMANN, MULLER H, KALETA E F. Structural and biological characteristics of reoviruses isolated from muscovy ducks (Cairina moschata)[J]. Avian Pathology, 1992, 21:481-491. doi: 10.1080/03079459208418866 [2] KUNTZ-SIMON G, LE GALL-RECULE G, DE BOISSESON C, et al. Muscovy duck reovirus sigma C protein is atypically encoded by the smallest genome segment[J]. J Gen Virol, 2002, 83(5):1189-1200. doi: 10.1099/0022-1317-83-5-1189 [3] LE GALL-RECULEG, CHERBONNEL M, ARNAULD C, et al. Molecular characterization and expression of the S3 gene of muscovy duck reovirus strain 89026[J]. J Gen Virol, 1999, 80(1), 195-203. doi: 10.1099/0022-1317-80-1-195 [4] 胡奇林, 陈少莺, 林锋强, 等.番鸭呼肠孤病毒的鉴定[J].病毒学报, 2004, 20(3):241-248. http://www.cnki.com.cn/Article/CJFDTOTAL-BDXB200403009.htm [5] YUN T, YU B, NI Z, et al. Isolation and genomic characterization of a classical Muscovy duck reovirus isolated in Zhejiang China[J].Infect Genet Evol, 2013, 20:444-453. doi: 10.1016/j.meegid.2013.10.004 [6] 张云, 欧阳岁东, 刘明, 等, 番鸭呼肠孤病毒非结构基因的克隆和序列分析[J].中国预防兽医学报, 2005, (2):139-143. http://www.cnki.com.cn/Article/CJFDTOTAL-ZGXQ200502013.htm [7] 林锋强, 陈少莺, 王劭, 等.番鸭呼肠孤病毒M (M1~M3)基因克隆及特性分析[J].福建农业学报, 2012, 27(9):951-956. http://d.wanfangdata.com.cn/Periodical/fjnyxb201209008 [8] ZHANG Y, GUO D. Characterization of M-class genome segments of muscovy duck reovirus S14[J]. Virus Res, 2007, 125(1):42-53. doi: 10.1016/j.virusres.2006.12.004 [9] KUNTZ-SIMON G, BASDLKLANCHARD P, CHERBONNEL M, et al. Bachulovirus-expressed muscovy duck reovirus σC protein induces serum neutralizing antibodies and protection against challenge[J]. Vaccine, 2002, 20:3113-3122. doi: 10.1016/S0264-410X(02)00264-5 [10] 莫武宁, 唐安洲, 周玲. Ad5F35重组腺病毒载体研究进展[J].病毒学报, 2006, 22(6):480-483. http://www.cnki.com.cn/Article/CJFDTOTAL-BDXB200606013.htm [11] 张云, 欧阳岁东, 胡奇林, 等.番鸭呼肠孤病毒MW9710δC蛋白基因克隆及其在E.coli中表达[J].畜牧兽医学报, 2005, 36(4), 376-380. http://www.cnki.com.cn/Article/CJFDTOTAL-XMSY200504016.htm [12] 余旭平, 刘慧芳, 何世成, 等.番鸭呼肠孤病毒ZJ99株σC基因在甲醇酵母中的表达[J].黑龙江畜牧兽医, 2006, (12):15-18. http://www.cnki.com.cn/Article/CJFDTOTAL-HLJX200612006.htm [13] 张云, 郭东春, 耿宏伟, 等.番鸭呼肠孤病毒σC编码基因在昆虫细胞中的表达[J].水禽世界, 2007, (6):32-35. http://www.cnki.com.cn/Article/CJFDTOTAL-SQSJ200706021.htm -
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