| 黄莹,唐青,扈荣良.HEP-Flury株病毒辅助质粒包装CTN株病毒基因组拯救狂犬病病毒[J].Chinese journal of Epidemiology,2009,30(5):493-496 |
| HEP-Flury株病毒辅助质粒包装CTN株病毒基因组拯救狂犬病病毒 |
| The function of helper plasmids from HEP-Flury strain rabies virus on encapsidating the full-length genome of CTN strain |
| Received:February 10, 2009 |
| DOI： |
| KeyWord: 狂犬病病毒 基因组 结构蛋白 病毒拯救 |
| English Key Word: Rabies virus Genome Structural proteins Virus rescue |
| FundProject:国家"863"高科技计划(2006AA02Z110,2007AA02Z402);国家自然科学基金(30630049) |
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| Abstract: |
| 目的以CTN株狂犬病病毒全长基因组cDNA重组质粒(pCTN-GFP)和HEP-Flury株病毒N、P、L辅助质粒共转染拯救具有活性的CTN株重组狂犬病病毒(CTN-GFP)。方法将CTN株狂犬病病毒全长基因组分4段进行扩增，体外连接扩增片段并克隆入表达载体中，构建CTN株狂犬病病毒全长基因组cDNA重组质粒，基因组ψ基因被标识基因GFP取代，通过与HEP-Flury株病毒N、P、L3个辅助质粒共转染BHK-21细胞，拯救能够稳定表达GFP的重组病毒CTN-GFP。结果成功扩增全长基因组的4个基因片段，并将其克隆入表达载体，构建了CTN株狂犬病病毒全长基因组cDNA重组质粒pCTN-GFP，经酶切鉴定和序列测定证明pCTN-GFP为正确克隆，可直接用于病毒拯救。将pCTN-GFP与HEP-Flury株病毒辅助质粒共转染BHK-21细胞4d后，成功拯救出CTN株重组狂犬病病毒，重组病毒能够表达标识基因GFP，荧光显微镜下可直接观察到标识基因GFP的表达，设计跨GFP和L基因的特性引物对重组病毒进行RT-PCR检测，结果证明此病毒是从克隆的质粒中拯救的重组病毒。结论HEP-Flury株病毒的结构蛋白能够包装并转录CTN株病毒的基因组RNA。 |
| English Abstract: |
| Objective To identify the helper plasmids from HEP-Flury strain rabies virus that could encapsidate the full-length genome of CTN strain.Methods Four overlapped fragments covering the full-length genome of rabies virus CTN strain were cloned into expression vector. A recombinant full-length genome plasmid (pCTN-GFP) contained the full-length genome of the CTN strain expect for ψ gene which was replaced by GFP gene was then constructed using restriction enzyme cleavage and ligation in vitro. In order to obtain the recombinant rabies virus CTN-GFP, the pCTN-GFP was transfected with helper plasmids carrying N, P, L gene of HEP-Flury strain.Results The four gene fragments of the genome were amplified and cloned into the expression vector. The recombinant genome cDNA plasmid pCTN-GFP was constructed and subjected to restriction endonuclease digestions. After sequenced to assure no absence and mutations compared with their parental viruses, it was ready for virus rescue. After the transfection of both pCTN-GFP and the helper plasmids from HEP-Flury strain into BHK-21 cells, the recombinant rabies virus CTN-GFP was rescued and confirmed by fluorescence analysis and RT-PCR, which demonstrated that the CTN-GFP was recovered from cloned cDNA.Conclusion The proteins of HEP-Flury strain rabies virus could encapsidate and transcribe the CTN strain rabies virus RNA genome. |
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