整合siRNA技术的结核核酸疫苗的AAV载体构建与浓缩纯化  

Construction and Purification of Recombinant Adeno-Associated Virus Vector for Tuberculosis DNA Vaccine integrated with siRNA Interference Technology

作　　者：朱伟[1] 曹以诚[1] 杨磊[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《现代食品科技》2011年第7期802-806,755,共6页Modern Food Science and Technology

摘　　要：引进腺病毒相关病毒载体系统(AAV载体系统),并通过PCR、酶切、连接等分子克隆手段将之前对于结核研究的成果转移至该系统中,完成同时具备小干扰RNA(si-Mcl-1)和结核融合抗原(AG85B-ESAT6)表达单位的二重表达载体的构建。转染AAV-293细胞,包装重组AAV病毒颗粒(rAAV)并收集病毒。分别设计另外两组rAAV表达载体:rAAV-EGFP与rAAV-siEGFP-EGFP。两组表达载体转染AAV-293包装病毒并感染HT1080细胞。荧光显微镜检测,感染后HT1080细胞48 h后表达荧光蛋白,证明病毒包装成功。通过流式细胞仪检测,前者荧光表达占总细胞2.78%,后者荧光表达占总细胞0.75%。证明rAAV表达载体中siRNA表达部分正常发挥其抑制作用。为进一步提高感染效率,利用超滤浓缩方法浓缩纯化rAAV载体。感染HT1080,48 h至72 h通过流式细胞仪检测,前者荧光表达细胞占细胞总数10.18%,后者荧光表达细胞占细胞总数1.55%。A new double-expression tuberculosis DNA vaccine possessing both siRNA and fusion antigen expression units was constructed,which was finally turned into virus-vector form by applying AAV-helper free system.Meanwhile rAAV-EGFP and rAAV-siEGFP-EGFP were designed and constructed to infect HT1080 cell line.After infection for 48 h,the expression of green fluorescent protein was confirmed by fluorescence microscope and flow cytometry.The difference of EGFP expression between HT1080 infected by rAAV-EGFP and HT1080 infected by rAAV-siEGFP-EGFP were significant.The former reached 2.78% and the later reached 0.75%.Besides ultrafiltration was applied to concentrate and purify rAAV to improve infection results.After infection for 48 h to 72 h,the results were obtained through flow cytometry.The former reached 10.18% and the later reached 1.55%.

关 键 词：腺病毒相关病毒载体系统 结核疫苗 RNA干扰技术 

分 类 号：R392.1[医药卫生—免疫学]