整合siRNA技术的结核核酸疫苗的AAV载体构建与浓缩纯化

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...

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

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%.