摘要
目的利用CMV启动子构建CVB3感染性克隆载体。方法将质粒pcDNA3.1(+)的CMV启动子下游区域通过PCR技术替换成设计好的多克隆位点区,分别插入PCR合成的HdvRz核心序列和polyA尾片段,形成框架载体pc-H-A,通过瞬时转染HeLa细胞初步鉴定pc-H-A的可用性;用本室保存的CVB3(nancy株)感染HeLa细胞,取感染液上清提取病毒RNA,利用长链RT-PCR和长链PCR技术反复优化条件扩增病毒基因组全长片段,将此全长片段克隆至设计好的框架载体pc-H-A中,筛选阳性克隆,并对其进行测序鉴定。结果与结论构建了带有CMV启动子的框架载体pc-H-A并初步鉴定了该载体可用,利用此框架载体构建了与基因库中序列相似性为99%的CVB3全长cDNA克隆pc-CVB3-H-A。该研究为进一步鉴定所构建的CVB3全长克隆的感染性提供了基础,并为后续在病毒学研究和基因治疗方面的研究提供了手段。
Objective To construct a CMV promoter-based infectious clone of Coxsackie virus B3.Methods First,the plasmid pcDNA3.1（＋）was used and the downstream sequence of its CMV promoter was replaced by a multiple clone site sequence we designed.Two elements（the core sequence of HdvRz and polyA tail）were inserted into its multiple clone site to produce plasmid pc-H-A.We transfected pc-H-A into HeLa cells to evaluate its availability.Second,HeLa cells were infected with wild type CVB3（nancy）,and the viral RNA was extracted from the supernatant.Then,the full length CVB3 cDNA was amplified by long RT-PCR and long PCR through condition optimization.Finally,this full length cDNA was inserted into plasmid pc-H-A and the recombinant particles were verified by sequencing.Results and Conclusion The framework vector pc-H-A is constructed based on CMV promoter and its availability is initially evaluated.A full length pc-CVB3-H-A cDNA clone is obtained with a sequence similarity of 99% compared with CVB3 sequence in GenBank.This work will be of great value for the further research on the infectivity of the clone we constructed and will provide new methods for the study of virus and gene therapy.
出处
《军事医学科学院院刊》
CSCD
北大核心
2010年第4期356-360,共5页
Bulletin of the Academy of Military Medical Sciences
