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

作　　者：杨兴春[1] 白转丽[1] 王瑞[1] 柏宏亮[1] 

机构地区：[1]西安交通大学医学院第一附属医院烧伤整形科,西安710061

出　　处：《陕西医学杂志》2015年第3期263-267,共5页Shaanxi Medical Journal

基　　金：陕西省科技研究发展(攻关)计划项目(2011K12-12)

摘　　要：目的:构建共表达NEP1-40和PRP-1多顺反子重组双链腺相关病毒(scAAV)。方法:PCR扩增NEP1-40、PRP-1及FMDV2A的cDNA,建立NEP1-40-FMDV 2A-PRP-1嵌合肽cDNA。两端加入Flag肽和6×His肽后合成融合基因cDNA,克隆到PES载体构建出重组质粒PES/NEP1-40-FMDV 2A-PRP-1。双酶切重组质粒PBV220-NT4-NAP和PES/NEP1-40-FMDV 2A-PRP-1,连接得到重组质粒pBV220-NT4/NEP1-40-FMDV 2A-PRP-1。以腺相关病毒pSS-CMV构建双链腺相关病毒穿梭质粒ssAAV pSSHG-NT4/NEP1-40-FMDV 2A-PRP-1。经细胞内同源重组得到共表达NEP1-40和PRP-1的重组双链腺相关病毒scAAV pSSHG-NT4/NEP1-40-FMDV 2A-PRP-1。测定病毒滴度并利用鸡胚背根神经节检测其生物活性。结果:克隆了共表达NEP1-40和PRP-1的多顺反子融合基因cDNA,基因测序及核苷酸序列同源性比较结果与设计序列一致;构建了重组质粒pBV220-NT4/NEP1-40-FMDV 2A-PRP-1,酶切鉴定、核酸序列测定结果与理论值一致;重组的双链腺相关病毒scAAV pSSHG-NT4/NEP1-40-FMDV 2APRP-1应用于鸡胚背根神经节显示明显促进神经突起生长。结论:成功构建了共表达NEP1-40和PRP-1的重组双链腺相关病毒scAAV pSSHG-NT4/NEP1-40-FMDV 2A-PRP-1,并显示出促进鸡胚背根神经节神经突起生长作用。Objective：To construct the multicistronic scAAV containing the FMDV 2Acleavage factor and co-expressing NEP1-40 and PRP-1.Methods：PCR technology was used to amplify the NEP1-40,PRP-1and FMDV2 AcDNA.The chimeric peptide cDNA containing NEP1-40-FMDV 2A-PRP-1was 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-1was 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-1was constructed basing on the AAV pSS-CMV.Finally the recombinant scAAV pSSHG-NT4/NEP1-40-FMDV 2A-PRP-1was 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 ganglion（EDRG）.Results：The fusion gene cDNA for co-expressing NEP1-40 and PRP-1was cloned.The gene sequencing and nucleotide sequence homology comparison show consistent with the design sequence.The recombinant plasmid pBV220-NT4/NEP1-40-FMDV 2A-PRP-1was 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 after using the recombinant scAAV in chicken EDRG.Conclusion：The recombinant scAAV pSSHG-NT4/NEP1-40-FMDV 2A-PRP-1is 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 

分 类 号：R392.3[医药卫生—免疫学]