| 牛病毒性腹泻病毒HB-DCZ株NS2-3区基因克隆及序列分析 |
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| 引用本文： | 赵月兰,郭红斌,张磊,秦建华,张宁,张保宁.牛病毒性腹泻病毒HB-DCZ株NS2-3区基因克隆及序列分析[J].中国农学通报,2007,23(6):1-1. |
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| 作者姓名： | 赵月兰 郭红斌 张磊 秦建华 张宁 张保宁 |
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| 作者单位： | 1. 河北农业大学中兽医学院,定洲,073000
2. 河北农业大学动物科技学院,保定,071000 |
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| 基金项目： | 国家“十一五”科技攻关计划‘华北农区奶牛疾病综合防治技术应用与示范》部分内容,课题编号（2006BAD04A10-4）;河北省百牧局科攻关计划,课题编号2002-4 |
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| 摘 要： | 【目的】对牛病毒性腹泻病毒HB-DCZ株NS2-3区基因进行克隆及序列分析，为阐明本病致病机理，更好的控制BVDV感染、流行提供理论依据。【方法】以牛病毒性腹泻病毒河北分离株（HB-DCZ）基因组RNA为模板，扩增BVDV HB-DCZ株基因组NS2-3片段cDNA。将PCR产物克隆后进行PCR及双酶切鉴定。克隆产物进行序列测定及分析。【结果】HB-DCZ cDNA体外扩增获得特异性条带，大小约为665bp，表明该分离毒株基因组中无插入序列。PCR产物克隆，提取重组质粒，扩增获得特异性的DNA条带，长度约为665bp，用EcoRⅠ和Hind Ⅲ双酶切，获得两条DNA片段，长度分别为2600bp和702bp左右，与理论值相符。序列测定结果表明，克隆得到的NS2-3重要区扩增片段共665核苷酸，NS2-3重要区的蛋白质由208个氨基酸残基组成。HB-DCZ毒株NS2-3重要区核苷酸序列与已公开发表的BVDV其他毒株相比的同源性依次为184株99.1%，ZM195株97.4%，Osloss株92.3%，C24V株77%，NADL株76.4%。HB-DCZ在NS2-3扩增区域中既没有外源序列的插入，也没有基因重组、重排或缺失，但存在某些核苷酸的替换。【结论】HB-DCZ与Osloss株、国内的184株、ZM195株遗传距离较近，与C24V株、NADL株遗传距离较远。HB-DCZ株应为BVDV Ib基因亚型。
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| 关 键 词： | 扩张蛋白 扩张蛋白 |
| 修稿时间： | 2007-03-25 |
Cloning and Sequence Analysis of Genetic Variation on NS2-3 of Bovine Viral Diarrhea Virus HB-DCZ Strain |
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| Zhao Yuelan,Guo Hongbin,Zhang Lei,Qin Jianhu,Zhang Ning,Zhang Baoning.Cloning and Sequence Analysis of Genetic Variation on NS2-3 of Bovine Viral Diarrhea Virus HB-DCZ Strain[J].Chinese Agricultural Science Bulletin,2007,23(6):1-1. |
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| Authors: | Zhao Yuelan Guo Hongbin Zhang Lei Qin Jianhu Zhang Ning Zhang Baoning |
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| Institution: | 1College of Chinese Traditional Veterinary Science, Hebei Agriculture University, Dingzhou 073000; 2College of Animal Science and Technology. Hebei Agriculture University,. Baoding 071000 |
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| Abstract: | 【Objective】Bovine Viral Diarrhoea-Mucosal Disease Virus HB strain was a field strain isolated from a dairy cows flock in HeBei province . The study were progressed in order to realizing characters of HB strain, providing theory base for illuminating pathogeny, controlling infection and prevalence of BVDV;【Method】Genetic Variation on NS2-3 of Bovine viral diarrhea Virus HB-DCZ strain were amplified by RT-PCR. The product of PCR was cloned into pMD18-T vector, and then transfected into JM109. The recombinant plasmids were extracted and amplified by PCR and identificated by EcoRⅠand Hind Ⅲ enzyme digestion. The nucleotide sequence of the isolated virus NS2-3 was sequenced and amino acid sequence was inferred. Sequence analysis was performed with the aid of DNAstar softerware；【Results】The result showed that the obtained fragment was approximate 665bp by RT-PCR. There was no insertion in the isolated virus genome. EcoRⅠand Hind Ⅲ digestion of the recombinant plasmids resulted in two bands with sizes 702bp and 2635bp, indicating the specificity of the PCR. The cloned NS2-3 segment of HB-DCZ strain contains 665bp nucleotides and 208 amino acid. The homologies of nucleotide sequence of NS2-3 gene of HB-DCZ strain with other BVDV strains were 99.1%(184)，97.4% (ZM195), 92.3% (Osloss), 77% (OregonC24V ), 76.4% (NADL), respectively. The HB-DZC virus strain had no exogenous sequence insertion, gene recombination, gene rearrangement or gene deficiency. However, some nucleotide sequences were replaced. 【Conclusion】The HB-DZC was closed related to BVDV strains Osloss, 184, ZM195 nor the strains C24V, NADL; All the above results proved this virus was BVDV and which belongs to subtype Ib. |
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| Keywords: | Bovine Viral Diarrhea Virus HB-DZC Virus Strain Cloning Sequencing NS2-3 gene RT-PCR |
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