携带人S100A1基因的重组腺相关病毒载体的构建及鉴定

Construction of recombinant adeno-associated virus vector expressing hs100a1 gene

目的:构建携带有人S100A1基因的重组腺相关病毒(Adeno-associated viru,AAV)载体,并对其进行鉴定。方法:用BamHⅠ和EcoRⅠ将目的基因pUC57-Simple-S100A1和腺相关病毒骨架质粒pAAV-IRES-ZsGreen1行双酶切,回收酶切质粒的目的片段进行连接,产物转化感受态DH5a,获得重组腺相关病毒骨架质粒pAAV-IRES-ZsGreen1-S100A1。酶切及测序鉴定后,将pAAV-IRES-ZsGreen1-S100A1、包装质粒pAAV-RC和辅助质粒pHelper三质粒共转染AAV-293,包装重组腺相关病毒rAAV-IRES-Zs-Green1-S100A1。收获重组病毒后感染HEK-293细胞,荧光计数法测定病毒滴度,病毒基因组外源基因扩增鉴定重组病毒的包装是否成功。结果:重组腺相关病毒骨架质粒pAAV-IRES-Zs-Green1-S100A1经双酶切和测序鉴定正确;荧光显微镜下观察转染AAV-293细胞72h后,病毒包装效率达95%～100%,荧光计数法测定病毒感染滴度达(2～3)×107 TU/mL;提取重组病毒基因组成功扩增出外源目的基因S100A1片段。结论:成功构建携带有人S100A1的重组腺相关病毒载体rAAV-IRES-ZsGreen1-S100A1,收获的病毒具有较高滴度,为今后利用腺相关病毒载体进行S100A1基因转染治疗慢性心力衰竭的体外及体内研究提供了实验基础。

AIM： To construct the recombi- nant adeno-associated virus （rAAV） vector ex- pressing hS100A1 gene and to verify the correct recombination. METHODS. The hS100A1 plas- mid （pUC57-Simple-S100A1） and AAV frame- work plasmid （ pAAV-IRES-ZsGreenl ） were cleaved by restriction endonuclease BamH I and EcoR I. The target gene fragments were con- nected together to generate a recombinant plas- mid pAAV-IRES-ZsGreenl-S100A1 that was transfected into E. coli DHSa. The plasmid was confirmed to be constructed as expectation by enzyme digestion and sequencing. The recombi- nant plasmid pAAV-IRES-ZsGreenl-S100A1 was co-transfected into AAV-293 cells with pHelper and pAAV-RC for packaging of recom- binant AAV. The efficiency of rAAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured through infecting HEK-293 cells, and the recombinant rAAV-IRES- ZsGreenl-S100A1was verified by PCR of the exogenous interest genes of S100A1. RESULTS： The recombinant plasmid pAAV-IRES-ZsGreenl-S100A1 was ver- ified by double digestion and sequencing. Using the AAV helper-free system, ZsGreenl expres- sion could be observed under fluorescent micro- scope 72 hours after triple plasmid co-transfec- tion and the system provided a high packing ratio of 95%--100%. The rAAV has a high purity and high titer of （2--3）× 10^7 TU/mL. The re- combinant virus was confirmed by PCR of exog- enous S100A1 genes. CONCLUSION： Recombi- nant rAAV-IRES-ZsGreenl-S100A1 was suc- cessfully constructed with a high virus titer, which may offer the basement of in vitro and in vivo experiments of hS100A1 for gene therapy of chronic heart failure.