hIL-2与ZsGreen双基因共表达腺相关病毒的包装及鉴定

摘要

Objective To pack and identify the recombinant adeno-associated virus type 5(rAAV5) vector co-expressing hIL2 and ZsGreen for offering the basement of gene therapy in pancreatic cancer. [Methods] The IRES-ZsGreen and hIL2 genes were amplified by PCR and inserted into pAAV-MCS by enzyme digestion sites respectively. The recombinant expression plasmid pAAV-hIL2-IRES-Zs Green was co-transfected into AAV293 cells with pHelper and pAAV-RC for packaging of recombinant AAV5. The efficiency of rAAV packaging was monitored under fluorescent microscope and re-combinant viral particles were harvested from infected host cells, and titer was measured through qPCR after concentration and purification. HEK293 cells were infected with the rAAV5 and the recombinant AAV5 was verified by PCR of the exogenous interest genes of hIL2. [Results] The recombinant plas-mid pAAV-hIL2-IRES-ZsGreen was verified by double digestion. Using the AAV helper-free system, ZsGreen expression could be observed under flu-orescent microscope 72 hours after triple plasmid co-transfection and the system provided a high packing ratio of 90%. The rAAV5 had a high titer of 2.62 ×1012vp·mL-1. Live HEK293 cells could be infected by rAAV5 through measuring the expression of exogenous ZsGreen gene. The recombinant virus was confirmed by PCR of exogenous hIL-2 genes. [Conclusion] Recombinant rAAV-hIL2-IRES-ZsGreen was successful y constructed with 2.62 ×1012 titer.%[目的]包装并鉴定带有人白介素2（hIL-2）及ZsGreen的双基因共表达的重组5型腺相关病毒（recombinant adeno-associated virus 5, rAAV5），为今后利用5型重组腺相关病毒载体进行胰腺癌基因治疗提供研究基础。[方法]以5型腺相关病毒包装系统（helper-free system）为模版，PCR法扩增pLVX-IRES-ZsGreen和PES-IL-2模板上的IRES-ZsGreen和IL-2基因，利用酶切位点插入重组腺相关病毒骨架质粒，获得重组质粒pAAV-hIL-2-IRES-ZsGreen。经三质粒共转染AAV293细胞包装重组腺相关病毒rAAV5-hIL-2-IRES-ZsGreen。荧光显微镜下检测病毒包装效率，超速离心纯化、浓缩重组病毒，qPCR测定病毒的滴度。重组病毒转导细胞经荧光显微镜检测、外源基因经PCR检测证实病毒包装结果。[结果]5型重组腺相关病毒骨架质粒pAAV-IL-2-IRES-ZsGreen经双酶切和测序鉴定，确定其序列正确。荧光显微镜下观察三质粒共转染AAV293细胞72h后，病毒包装效率达90%以上。5型重组病毒感染HEK 293细胞48h有荧光出现，重组病毒基因组成功扩增出外源基因IL-2，证明有感染力的重组病毒包装成功。[结论]成功包装带有hIL-2及ZsGreen标记的双基因共表达重组腺相关病毒，滴度达到2.62×1012。