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【目的】测定狂犬病病毒标准攻击毒CVS-11株全基因组序列,构建CVS-11株全长cDNA感染性克隆。【方法】RT-PCR扩增CVS-11株全基因组得到有重叠的12个片段,分别克隆至平端载体pEASY-Blunt,测定CVS-11株全基因组核苷酸序列。用软件DNAMAN分析CVS-11全序列单一性酶切位点,设计引物,分4段扩增CVS-11全基因组,扩增产物经多步酶切、连接逐步插入至真核表达载体pcDNA3.1,获得全长质粒pcDNA3.1-CVS-11。pcDNA3.1-CVS-11与其辅助质粒pcDNA3.1-N、P、L、G共转染NA细胞,经免疫荧光染色、RT-PCR鉴定,拯救得到重组病毒rCVS-11。【结果】CVS-11全基因组序列由11 927个核苷酸组成,编码5个结构蛋白,结构基因排列同已知的其他狂犬病病毒一致。成功构建了CVS-11全长cDNA重组质粒pcDNA3.1-CVS-11和其辅助质粒pcDNA3.1-N、P、L和G。经共转染,成功拯救了重组病毒rCVS-11。【结论】CVS-11株感染性克隆的构建为从分子水平上进一步研究狂犬病病毒奠定了基础。
【Objective】 The complete genome sequence of CVS-11 strain of rabies virus and the full-length cDNA clone of CVS-11 strain were constructed. 【Method】 The whole genome of CVS-11 was amplified by RT-PCR. Twelve fragments were obtained and cloned into the blunt-end vector pEASY-Blunt respectively. The complete nucleotide sequence of CVS-11 was determined. CVS-11 was cloned into the eukaryotic expression vector pcDNA3.1 by DNAMAN, and the entire CVS-11 genome was amplified by 4 steps. The amplified product was digested with multiple steps and inserted into the eukaryotic expression vector pcDNA3.1 To obtain the full-length plasmid pcDNA3.1-CVS-11. The pcDNA3.1-CVS-11 and its helper plasmids pcDNA3.1-N, P, L and G were cotransfected into NA cells, and the recombinant virus rCVS-11 was rescued by immunofluorescence staining and RT-PCR. 【Result】 The complete genome sequence of CVS-11 consists of 11 927 nucleotides and encodes five structural proteins. The structural gene sequence is consistent with other known rabies virus. CVS-11 full-length cDNA recombinant plasmid pcDNA3.1-CVS-11 and its helper plasmids pcDNA3.1-N, P, L and G were successfully constructed. After co-transfection, the recombinant virus rCVS-11 was successfully rescued. 【Conclusion】 The construction of infectious clone of CVS-11 strain lays the foundation for the further study of rabies virus at the molecular level.