狂犬病病毒修饰基因组的构建  

Construction of Genomes-Rabies Virus Modified

作　　者：魏玉荣[1] 易忠[1] 符子华[1] 马素贞[2] 简子健[2] 胡尔玛西[1] 王海烽[1,2] 魏婕[1,2] 朱晶晶[3] 

机构地区：[1]新疆畜牧科学院兽医研究所,乌鲁木齐830000 [2]新疆农业大学动物医学学院,乌鲁木齐830052 [3]塔里木大学,新疆阿拉尔843300

出　　处：《新疆农业科学》2010年第6期1236-1241,共6页Xinjiang Agricultural Sciences

基　　金：新疆维吾尔自治区高技术研究发展计划项目(200611107)

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。[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 glyeoprotein 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 eytochrome C gene was obtained as expected. [ Conclusion]Sequencing of full - length genomes showed that genomes - rabies virus modified was constructed successfully.

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 

分 类 号：S118[农业科学—农业基础科学] S851.655