CVB3-VP1基因与IL-15双表达质粒的构建

Construction of the coxsackie virus VP1 gene and human interleukin-15 gene expression plasmid pairs

目的构建IL-15/CVB3-VP1的基因双表达重组质粒。方法从人胚肺二倍体细胞中提取IL-15的mR-NA,逆转录后将产物与pGEM-Teasy载体连接,转化DH5a大肠杆菌,筛选重组子并提取质粒进行酶切鉴定和测序。取双酶切的目的基因片断,与经过同样两种酶切的pcDNA3载体连接,经过转化、筛选和酶切鉴定后,构建成真核表达重组质粒PcDNA3-IL-15。用自行设计的引物从PcDNA3-VP1质粒上扩增出带有启动子的VP1基因,插入到PcDNA3-IL-15质粒上,构建成真核重组质粒PcDNA3-IL-15/CVB3-VP1。结果构建的PcDNA3-VP1/IL-15重组质粒目的基因与CVB3-VP1和IL-15的标准序列相同。结论成功构建了柯萨奇病毒VP1基因和人白细胞介素15基因双表达质粒PcDNA3-IL-15/CVB3-VP1。

Objective To construct IL-15/CVB3-VP1 the gene-expression plasmid.Extract the mRNA of IL-15 from human embryonic lung diploid cells,after reverse transcription,ligate the product with pGEM-T easy vector,transform E.coli DH5a,then select the recombinant and extract the plasmid to be identificated and sequenced with the approach of restriction enzyme digestion.Use double-digested target gene fragments to ligate with the pcDNA3 vector through the same restriction enzyme digestion,after transformation,screening and restriction enzyme digestion,construct the eukaryotic expression recombinant plasmid PcDNA3-IL-15.Use self-designed primers to amplify VP1 gene with the prometer from PcDNA3-VP1 plasmid,and construct the eukaryotic recombinant plasmid PcDNA3-IL-15/CVB3-VP1 with the insertion into the PcDNA3-IL-15 plasmid.The constructed recombinant plasmid PcDNA3-VPI/IL-15 gene has the same sequence as the standard sequence of CVB3-VP1 and IL-15.Successfully construct the coxsackie virus VP1 gene and human interleukin-15 gene expression plasmid pairs PcDNA3-IL-15/CVB3-VP1.