The packaging and purification of recombinant adeno-associated virus loaded with anti-amyloid ? peptide single-chain antibody gene / 基础医学与临床

ABSTRACT

Objective To produce and purify recombinant adeno-associated virus(AAV) loaded with anti-amyloid ? 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 ? 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?1012vg/mL.The latency of treated mice in water maze test were reduced significantly.Conclusion The adeno-associated virus carrying anti-amyloid? peptide single-chain antibody gene was produced by HSV1system and purified.The animal behavior test demonstrated the recombinant AAV waseffective in the gene therapy of Alzheimer's disease.