摘要
【目的】建立Ⅰ型鸭病毒性肝炎(Duck hepatitis virus,DHV)的反向遗传系统。【方法】根据Ⅰ型鸭肝炎病毒(DHV-Ⅰ)ZJ-V/2006株全基因组序列设计并合成5对特异引物,进而应用RT-PCR技术分5段扩增了DHV全基因组cDNA。将扩增的cDNA重叠片段A、B、C和DE片段分别克隆到载体pBluescriptⅡKS(+)中,获得了DHV-ⅠZJ-V/2006株全长基因组cDNA克隆pBLDHV。在扩增5′末端时,引入ApaⅠ酶切位点和SP6启动子序列;在基因组3′末段PolyA尾引入NruⅠ酶切位点,以供cDNA模板的线性化之用。【结果】核酸序列分析表明,DHV-ⅠZJ-V/2006株基因组全长为7 711个核苷酸,与DHV-ⅠZJ-V BHK-21细胞分离株的同源性为99.9%;全长基因组中有6个核苷酸发生突变,均未导致对应的氨基酸发生改变,为沉默突变。【结论】成功构建了DHV-ⅠZJ-V/2006株基因组全长cDNA。
【Objective】 The study was to develop a reverse genetics system of Duck Virus Hepatitis Type Ⅰ（DHV-Ⅰ）.【Method】 Five pairs of oligonucleotides were designed based on the full length genomic sequence of DHV ZJ-V strain.Using RT-PCR technique,five overlapping cDNA fragments,designated as A,B,C,D and E were amplified respectively.And D and E fragments were fused by PCR designated as ED.Using pBluescript Ⅱ KS（＋） as a plasmid vector,the full-length cDNA clone pBLDHV of DHV ZJ-V strain was obtained by connecting the four cDNA fragments utilizing single restriction endonuclase site.A ApaⅠsite and a SP6 promoter were introduced immediately upstream of 5′ end,while a NruⅠ Site was engineered down stream of 3′ end of DHV poly（A） tail（containing 20 As）.【Result】 The results of sequencing and analysis showed that there were 99.9% identical between the construction of the full-length cDNA sequences and cDNA sequences of DHV-Ⅰ ZJ-V strain.The only differences in sequence were at 6 positions,none of which affected the amino acid sequence.【Conclusion】 Successful construction of full-length cDNA clone of DHV-Ⅰ ZJ-V strain lays a foundation for rescuing DHV effectively and enables further research of DHV at molecular level.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2010年第5期27-31,共5页
Journal of Northwest A&F University(Natural Science Edition)
基金
浙江省自然科学基金项目(Y305031
Y307124)
浙江省重大科技专项(2007C12010)
嘉兴科技局项目(No.2008AY1001)
浙江省农业科学院与中国科学研究院微生物所合作项目(2007R21Y03E01)
