| 狂犬病病毒修饰基因组的构建 |
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| 引用本文： | 魏玉荣,易忠,符子华,马素贞,简子健,胡尔玛西,王海烽,魏婕,朱晶晶.狂犬病病毒修饰基因组的构建[J].新疆农业科学,2010,47(6):1236-1241. |
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| 作者姓名： | 魏玉荣 易忠 符子华 马素贞 简子健 胡尔玛西 王海烽 魏婕 朱晶晶 |
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| 作者单位： | 新疆畜牧科学院兽医研究所,乌鲁木齐,830000;新疆农业大学动物医学学院,乌鲁木齐,830052;新疆畜牧科学院兽医研究所,乌鲁木齐,830000;新疆农业大学动物医学学院,乌鲁木齐,830052;塔里木大学,新疆阿拉尔,843300 |
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| 基金项目： | 新疆维吾尔自治区高技术研究发展计划项目 |
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| 摘 要： | 目的]利用反向遗传学技术在DNA水平上构建了狂犬病病毒修饰基因组.方法]提取狂犬病病毒(Rabies Virus,RV)口服疫苗株SRV9基因组总RNA作为RT-PCR的扩增模板,并根据GenBank已发表的SRV9基因组序列设计引物, 缺失狂犬病基因组Ψ区并在缺失位置插入人细胞色素C基因,再设计引物删除糖蛋白(G)胞质区(CD区).缺失Ψ区及删除CD区时采用基因同源重组的方法进行.在缺失的Ψ区插入人细胞色素C基因时,利用引物引入酶切位点NheI和EcoRI, 用PCR扩增出人细胞色素C基因并插入NheI和EcoRI位点.结果]获得含预期的人细胞色素C基因,同时缺失了CD区与Ψ区的质粒pBSK- LA.结论]全基因组测序结果表明已成功构建了狂犬病病毒修饰基因组.
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| 关 键 词： | 狂犬病病毒 胞质区 Ψ区 同源重组 人细胞色素C基因 反向遗传学 |
| 收稿时间： | 2010-06-25 |
Construction of Genomes-Rabies Virus Modified |
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| WEI Yu-rong,YI Zhong,FU Zi-hua,MA Su-zhen,JIAN Zi-jian,Huermaxi,WANG Hai-feng,WEI Jie,ZHU Jing-jing.Construction of Genomes-Rabies Virus Modified[J].Xinjiang Agricultural Sciences,2010,47(6):1236-1241. |
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| Authors: | WEI Yu-rong YI Zhong FU Zi-hua MA Su-zhen JIAN Zi-jian Huermaxi WANG Hai-feng WEI Jie ZHU Jing-jing |
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| Abstract: | 【Objective】Genomes-rabies virus modified was constructed at the DNA level by the reverse genetics technology.【Method】Genomic total RNA as an amplification template for RT-PCR was extracted from oral vaccine strain SRV9 of rabies virus and several pairs of primers were designed according to SRV9 genomic sequence published on GenBank.Ψ region of rabies virus genome were lacked by designed primers and then the human cytochrome C gene was introduced into the lacked Ψ region and cytoplasmic region of glycoprotein gene was deleted by designed primers.When Ψ region was lacked and cytoplasmic region was deleted,the method of gene homologous recombination was used.When the human cytochrome C gene was introduced into the lacked Ψ region,NheI and EcoRI enzyme sites were introduced using the primers.Human cytochrome c DNA was introduced into the sites of NheI and EcoRI after it was amplified by PCR.【Result】The result showed that the plasmid pBSK-LA was lacked in Ψ region and CD region.Human cytochrome C gene was obtained as expected.【Conclusion】Sequencing of full-length genomes showed that genomes-rabies virus modified was constructed successfully. |
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| Keywords: | rabies virus cytoplasmic region Ψ region homologous recombination human cytochrome C gene reverse genetics |
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