Aβ单链抗体基因腺相关病毒的包装和纯化

The packaging and purification of recombinant adeno-associated virus loaded with anti-amyloid β peptide single-chain antibody gene

目的包装和纯化携带Aβ单链抗体基因的腺相关病毒,为阿尔茨海默病的基因治疗创造条件。方法用Li-pofectamine2000将pSNAV2.0-Abeta-scFv质粒转染BHK-21细胞,并用G418筛选稳定细胞系。培养稳定细胞系,用辅助病毒HSV1-rc/ΔUL2感染包装携带Aβ单链抗体基因的腺相关病毒。用氯仿-PEG/NaCl-氯仿法和离子交换层析法纯化重组腺相关病毒,SDS-PAGE和PCR方法检测纯化的病毒。用地高辛标记探针测定重组腺相关病毒的物理滴度。利用水迷宫测试重组病毒对转基因阿尔茨海默病小鼠模型的治疗效果。结果带Aβ单链抗体基因的腺相关病毒的纯度为98%,物理滴度为1×1012vg/mL。经过治疗的小鼠模型潜伏期明显缩短。结论利用HSV1系统成功包装和纯化了携带Aβ单链抗体基因的腺相关病毒,动物实验表明对阿尔茨海默病具有治疗作用。

Objective To produce and purify recombinant adeno-associated virus （AAV） loaded with anti-amyloid β peptide single-chain antibody gene for gene therapy of Alzheimer＇s disease. Methods The plasmid pSNAV2.0-Abeta-scFv was used to transform BHK-21 cell by Lipofectamine 2000 for stable package cell line establishment. The helper virus HSV1-rc/△UL2 was used to transfect package cell for anti-amyloid β peptide single-chain antibody gene loaded recombinant adeno-associated virus production. The chloroform-PEG/NaCl-chloroform extraction and ion-exchange chromatography were employed for recombinant AAV purification. SDS-PAGE and PCR amplification were adopted for purified virus identification. The final virus physical titer was determined by digoxin-labeled DNA probes. The effect of gene therapy was tested with transgenic mice by water maze test. Results The purity of recombinant adeno-associated virus with our target gene reach up to 98% after stable cell package and serial purification. The physical titer of the final virus was 1×10^12vg/mL. The latency of treated mice in water maze test were reduced significantly. Conclusion The adeno-associated virus carrying anti-amyloid β peptide single-chain antibody gene was produced by HSV1 system and purified. The animal behavior test demonstrated the recombinant AAV was effective in the gene therapy of Alzheimer＇s disease.