陕西医学杂志Issue(3)：263-267,5.DOI:10.3969/j.issn.1000-7377.2015.03.002
共表达NEP1-40和PRP-1多顺反子重组scAAV的构建
Construction of Multicistronic scAAV containing the FMDV 2A cleavage factor and co-expressing NEP1-40 and PRP-1
摘要
Abstract
Objective:To construct the multicistronic scAAV containing the FMDV 2A cleavage factor and co‐expressing NEP1‐40 and PRP‐1 .Methods:PCR technology was used to amplify the NEP1‐40 ,PRP‐1 and FMDV2A cDNA .The chimeric peptide cDNA containing NEP1‐40‐FMDV 2A‐PRP‐1 was set up .Then the fusion gene cDNA was synthetized after adding the Flag peptides and 6 × His peptides at ends .The recombinant plasmid PES/NEP1‐40‐FMDV 2A‐PRP‐1 was constructed by cloning the fusion gene into the PES vector .The recombinant plasmid pBV220‐NT4 / NEP1‐40‐FMDV 2A‐PRP‐1 was obtained after digesting and connecting the plasmid PBV220‐NT4‐NAP and PES/ NEP1‐40‐FMDV 2A‐PRP‐1 . The double‐stranded AAV shuttle plasmid ssAAV pSSHG‐NT4 /NEP1‐40‐FMDV 2A‐PRP‐1 was constructed basing on the AAV pSS‐CMV .Finally the recombinant scAAV pSSHG‐NT4 /NEP1‐40‐FMDV 2A‐PRP‐1 was obtained by homologous recombination in cells .The titer of the recombinant scAAV was measured and its biological activity was detected in the chicken embryo dorsal root gan‐glion (EDRG) .Results :The fusion gene cDNA for co‐expressing NEP1‐40 and PRP‐1 was cloned .The gene se‐quencing and nucleotide sequence homology comparison show consistent with the design sequence .The recombinant plasmid pBV220‐NT4 /NEP1‐40‐FMDV 2A‐PRP‐1 was constructed and the results of enzyme identification and the nucleic acid sequence were consistent with the theoretical value .The growth of neural processes showed obviously af‐ter using the recombinant scAAV in chicken EDRG .Conclusion :The recombinant scAAV pSSHG‐NT4 /NEP1‐40‐FMDV 2A‐PRP‐1 is constructed successfully and displays promoting role in the chicken EDRG .关键词
富含脯氨酸多肽/NEP1-40/口蹄疫病毒/多顺反子载体/自互补型腺相关病毒Key words
Proline-rich peptide PRP-1/NEP1-40/Foot and mouth disease virus/Multicistron-ic vector/Self-complementary AAV分类
医药卫生引用本文复制引用
杨兴春,白转丽,王瑞,柏宏亮..共表达NEP1-40和PRP-1多顺反子重组scAAV的构建[J].陕西医学杂志,2015,(3):263-267,5.基金项目
陕西省科技研究发展（攻关）计划项目 （）
