辣椒脉斑驳病毒侵染性克隆构建的简易方法

Simple methods for constructing infectiouscDNA clone of ChiVMV

本文介绍了一种构建辣椒脉斑驳病毒(Chilli veinal mottle virus,ChiVMV)侵染性克隆的简易方法.基于辣椒脉斑驳病毒的全长确定序列,根据基因内部的单一限制性酶切位点,设计引物,将ChiVMV分成KL1,KL2,KL3三个片段通过RT-PCR和TA克隆的方法将3个片段构建到PMD19-T载体上.通过双酶切将ChiVMV前面7000bp的片段构建到载体上,从而将全部的ChiVMV cDNA分段构建到两个重组质粒中,成功地避免了全长病毒序列在大肠杆菌中不稳定的现象.进行体外转录时,先通过PCR扩增出两个彼此有一定重叠的平末端片段,再利用两个重叠片段为模板用重叠PCR的方法扩增出全长带T7启动子的ChiVMV cDNA,并通过T7RNA聚合酶成功转录出ChiVMV的病毒RNA.

In this study, a simple method for constructing Chilli veinal mottle virus （Chilli veinal mot-tle virus, ChiVMV） infectious clone was described. Primers were designed based on single restrictionsites inside the gene of full-length ChiVMV determined sequences, and divided ChiVMV into KL1,KL2, KL3 three fragments. These three fragments were built into the PMD19-T vector by RT-PCR andTA cloning. Then the front 7000bp fragments of ChiVMV were construct to the vector by restrictionenzyme digestion. So that the entire ChiVMV eDNA were segmented constructed to two recombinantplasmids and successfully avoided the phenomenon of full-length viral sequence instability in E. coli.When in vitro transcription proceed, firstly two blunt end fragment overlap each other amplified by PCR Then with two overlapping fragments used as template, full-length ChiVMV eDNA with the T7 pro-moter was amplified by overlapping PCR. Finally the viral RNA of ChiVMV was successfully tran-scribed in vitro by T7 RNA polymerase.