| 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。 |
| 关键词: Ⅰ型鸭病毒性肝炎 全长cDNA克隆 感染性克隆 |
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| 基金项目: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) |
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| Construction and analysis of full length cDNA of Duck Virus Hepatitis Type Ⅰ |
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| Abstract: |
| 【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-ⅠZJ-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-ⅠZJ-V strain lays a foundation for rescuing DHV effectively and enables further research of DHV at molecular level. |
| Key words: Duck virus hepatitis ( DHV) full length cDNA clone infectious cDNA clone |
