狂犬病病毒CVS-11株全序列测定及其感染性克隆的构建

Sequencing the complete genome of rabies virus CVS-11 strain and constructing its full-length infectious cDNA clone

摘要:

摘要:【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。

Abstract:

Abstract:［Objective］To sequence the complete genome of CVS-11 strain and establish a reverse genetic system of CVS-11 to further study its pathogenic mechanism，virulence genes and antigenic sites．［Methods］We amplified12 fragments covering the complete genome of the CVS-11 strain by RT-PCR，and then cloned to pEASY-Blunt vector for sequencing the complete genome of CVS-11．We analyzed single restriction enzyme sites of the full length cDNA of the CVS-11 strain by DNAMAN and designed 4 pairs of specific primers．We amplified the full-length cDNA of CVS-11 by RT-PCR．We obtained four fragments and cloned into pcDNA3.1．We named the full-length cDNA plasmid pcDNA3. 1-CVS-11．We also cloned helper plasmids pcDNA3. 1-N，P，L and G expressing N，P，L and G protein of CVS-11 strain，respectively．We co-transfected NA cells with the full-length plasmid and four helper plasmids．We identified the supernatant of the transfected and then passaged NA cells by immunofluorescence staining and RT-PCR and found the recombinant virus rCVS-11 rescued successfully．［Results］Sequencing results showed that the complete genome of CVS-11 was composed of 11 927 nucleotides． The complete genome of CVS-11 encoded 5 structure proteins and gene array was the same as other reported rabies viruses． We successfully constructed a reverse genetic system of CVS-11，namely the full length plasmid pcDNA3. 1-CVS-11 and 4 help plasmids pcDNA3. 1-N，P，L，G and rescued the rCVS-11 from a full-length infectious cDNA clone．［Conclusion］ The reverse genetic system of the CVS-11 strain laid the foundation for future studies on rabies virus．