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亚洲Ⅰ型口蹄疫病毒VP1基因重组犬2型腺病毒的构建
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张秀芹1，2，夏咸柱1，卫广森2，3，常惠芸3，高玉伟1
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（1．解放军军事医学科学院 军事兽医研究所，吉林 长春130062； 2．沈阳农业大学 畜牧兽医学院，
辽宁 沈阳110161； 3．中国农业科学院 兰州兽医研究所，甘肃 兰州730046）
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摘要：
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应用PCR方法扩增出亚洲Ⅰ型口蹄疫病毒的VP1基因，继而构建出真核表达载体pVAXVP1。再将含有巨细胞启动子CMV的目的基因表达盒连接到穿梭载体pVAX△E3上，筛选出VP1表达盒方向与E3区转录方向一致的重组子，获得转移载体。再经酶切，连接等方法获得重组腺病毒质粒。利用脂质体介导将重组质粒转染DK细胞，转染3次后，细胞出现明显的腺病毒病变。经PCR扩增、测序检测及基因组酶切鉴定，证明该病毒即为重组犬2型腺病毒。命名为CAV2/FMDV。经验证，该重组毒可稳定遗传，其毒价为103.5TCID50/mL。 |
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关键词：
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亚洲Ⅰ型口蹄疫病毒； 犬2型腺病毒； 重组； VP1基因 |
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中图分类号：S 852.659.6: Q 786 文献标识码：
A 文章编号：1673-4696(2006)02-0118-04 |
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Construction of recombinant canine adenovirus2 vector containing
VP1 gene of footandmouth disease virus AsiaⅠ |
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ZHANG Xiuqin1,2, XIA
Xianzhu1, WEI Guangsen2,3, CHANG
Huiyun3, GAO Yuwei1 |
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（1. Military Veterinary Research Institute ,PLA Academy of Military Medicine ,Changchun130062,China；
2.College of Animal Science and Veterinary Medicine, Shenyang Agricultural University,Shenyang110161,China；
3. Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences,Lanzhou730046,China） |
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Abstract: n eukaryotic expression vector pVAXVP1 was constructed with VP1 gene of footandmouth disease virus AsiaⅠ,which was amplified by PCR,then the expression vector and a CMV promoter was ligated into a pVAX△E3,and the transvector was obtained by selecting the recombinants which had the same transcription directions of E3 region to the VP1 expression vector .The recombinant adenovirus plasmid was obtained by a serial molecular biology methods.The recombinant was confirmed and then transfected DK cells for 3 times, and the obvious adenovirus CPE was found on DK cells. PCR amplification, sequencing and digestion confirmed that the recombinant virus was canine adenovirus2, and was named CAV 2/FMDV. Results revealed that CAV 2/FMDV could inherit steadily and the viral titer was 103.5 TCID50/mL. |
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Key words: footandmouth disease virus AsiaⅠ; CAV 2; recombination； VP1 gene |
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