长距离PCR法扩增RHDV全基因组及其序列分析  

Cloning and Sequencing of the Complete Nucleotide Sequence of Rabbit Viral Hemorrhagic Disease Virus Isolated from China

作　　者：张玉颖[1] 刘光清[2] 倪征[2] 云涛[2] 吴润[1] 张淼涛[3] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]浙江省农业科学院农业部病毒学与生物技术研究所重点实验室,浙江杭州310021 [3]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《西北农业学报》2006年第3期1-5,共5页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：浙江省农业科学院博士科学基金资助(S20043300002)

摘　　要：从人工感染RHDV致死的兔肝组织中提取病毒总RNA,应用特异性引物将病毒RNA反转录成cD-NA,以此为模板用长距离PCR法扩增RHDV全基因组,将成功扩增出的RHDV全基因组克隆到pMD18-T载体中,经PCR鉴定及酶切分析后进行序列测定。测序结果表明,JX97全基因组由7 437个核苷酸组成,包括由9个核苷酸组成5′NCR、59个核苷酸组成3′NCR和一个至少含有27个核苷酸的poly(A)尾。序列比较结果显示,RHDVJX97株的衣壳蛋白与已报道的参考毒株具有较高的序列同源性,均在90%以上,提示所有的RHDV分离株具有较近的亲缘关系。Total RNA of RHDV were extracted from liver samples of rabbits killed by RHDV. A special primer was used to reverse transcription and produced the first of cDNA. The full length genome of RHDV were amplified by the Longaccurate polymerase chain reaction （LA-PCR） in a single step amplification. The PCR product was cloned into the pMD18-T vector and the recombinant DNA was identified by restriction enzyme digestion and PCR. The results showed that the full-length genome of RHDV was 7437bp long, containing a 9bp of 5＇NCR ,a 59bp of 3rNCR and a poly （A） tail with 27bp at least. The capsid protein Vp60 gene of JX97 shared high homology compared with those previously published （ranged from 90% to 99.1% ）.

关 键 词：免病毒性出血症病 长距离扩增 全基因组 

分 类 号：Q78[生物学—分子生物学] S852.65[农业科学—基础兽医学]