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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 full-length cDNA of foot-and-mouth disease virus (FMDV) O / LZ strain. Methods The FMDV genome was designed into four segments according to the restriction enzyme sites of FMDV genome and cloning vector. The FMDV genome was cloned into plasmid pMD18T-vetcor by reverse transcription and amplification (RT-PCR) Positive recombinant plasmids were screened and sequenced. The recombinant plasmids (pMD18-S, pMD18-I, pMD18-P1 and pMD18-P2) were reserved for future use. The primers of S region, I region and P2 fragment were redesigned. The upstream primer of S region introduced the T7 promoter sequence. The upstream primer of I region introduced 6 C and the downstream primer contained Nru Ⅰ of the FMDV genome. The downstream primer amplified at the 3 ’end introduced 16 T’s, and fused the PCR products of S and I regions using pMD18-S and pMD18-I as templates, and the P2 fragment was subjected to 2 times using pMD18-P2 as a template PCR, obtained P3 fragment with 16 T’s. The amplified fragment was cloned into pMD18T-vetcor, and the long fragment was ligated in pBuescriptⅡSK (-) to obtain a full-length cDNA clone. Results The whole genome of FMDV O / LZ strain O was obtained, and the full-length FMDV cDNA of O / LZ strain was constructed. Conclusion The full-length cDNA clone of O-type FMDV O / LZ genome was successfully constructed, which laid the foundation for obtaining infectious FMDV molecular clone and studying its structure and function.