O型口蹄疫病毒O/LZ株基因组全长cDNA的构建

Construction of foot-and-mouth disease virus O/LZ genome cDNA

目的构建O型口蹄疫病毒(foot-and-mouth disease virus,FMDV)O/LZ株基因组全长cDNA。方法根据FMDV基因组和克隆载体的限制性内切酶酶切位点,将FMDV基因组设计为4段,进行反转录和扩增(RT-PCR),得到的片段分别克隆到质粒pMD18T-vetcor,筛选阳性重组质粒,测序正确的重组质粒(pMD18-S、pMD18-I、pMD18-P1和pMD18-P2)保存备用。然后对S区、I区和P2片段重新设计引物,其中S区的上游引物引入T7启动子序列;I区上游引物引入6个C,下游引物带有FMDV基因组中唯一酶切位点NruⅠ;用于3′端扩增的下游引物引入16个T,并以pMD18-S和pMD18-I为模板,对S区和I区进行融合PCR获得IS,以pMD18-P2为模板对P2片段进行2次PCR,获的带16个T的P3片段。将扩增片段克隆到pMD18T-vetcor,在pBuescriptⅡSK(-)中进行长片段的连接,获得基因组全长cDNA克隆。结果获得O型FMDV O/LZ株全基因组,并在此基础上构建了O/LZ株FMDV全长cDNA。结论成功构建了O型FMDV O/LZ基因组全长cDNA克隆,为进一步获得具有感染性的FMDV分子克隆,并研究该病毒基因组结构和功能奠定了基础。

Objective To construct the cDNA of foot-and-mouth disease virus（FMDV） O/LZ genome. Methods Four eDNA fragments covering the FMDV O/LZ genome were amplified and cloned into pMD18T-vector, constructing four recombinant plasmid pMD18-S, pMD18-I, pMD18-P1 and pMD18-P2. The sequences of eDNA were analyzed by TaKaRa Biotechnology（Dalian） Co. , Ltd. When the above sequences were right by analysis, the IS was amplificated by fusing PCR with the pMD18-S and pMD18-I as template, and the IS specific primers was designed, which T7 promoter was introduced upstream of S, 6 C were introduced upstream of I. As same time, the antisense primer of I include the on- ly Nru I of the eDNA of FMDV genome. The P3 was amplificated with the pMD18-P2 as template, and the 15 A was added to at the 3- utmost sequence of P3 by renew designing primers of P3. The amplified fragments were cloned into the pMD18T-vector at first and then subcloned into pBluescript Ⅱ SK（-）. The recombinant plasmid CKS-F containing a full-length genomic eDNA of FMDV. Results The genome of FMDV O/LZ isolate was cloned by RT-PCR, and se- quenced. The full-length genomic eDNA of FMDV O/LZ was constructed on the basis of genome of FMDV O/LZ. Con- elusion The construction of a full-length genomic cDNA clone of FMDV is a crucial step to obtain the infectious clone. Further study will be going to test infectivity of the cDNA clone and look insight the structure and function of FMDV ge- home.