Adeno-associated virus mediated apoA-I and apoA-IMilano expression in skeletal muscular cells

Ming Gui; Zhiguang Wang; Li Jiang; Leming Fan; Kejiang Cao; Qi Chen; Jun Huang

摘要：Objective: To construct recombinant adeno-associated virus type 2(rAAV2) vectors encoding human apoA-I/apoA-IMilano protein, and explore an effective and safe method to prevent and treat the atherosclerotic diseases. Methods: Human apoA-I cDNA with a His-tag in the upward stream of cDNA sequence were obtained with RT-PCR and PCR, and human apoA-IMilano cDNA was prepared by QuikChange[ ] Site-Directed Mutagenesis Kit. After extracted rAAV vectors with a most economic and convenient method, the particle numbers of rAAV vectors were measured by Dot-blot, and the purity was assayed by SDS-Page. The expression efficiency of the apoA-I or apoA-IMilano in C2C12 infected by rAAV vectors were detected by ELISA method. Results: ApoA-I cDNA was gained by RTPCR and a His-tag was added in the upward stream of apoA-I cDNA successfully. ApoA-I cDNA was mutanted to apoA-IMilano cDNA successfully by QuikChange[ ] Site-Directed Mutagenesis Kit. The both titres of the rAAV vectors of apoA-I and apoA-IMilano were about 2×1014/L, and the result of SDS-Page showed that the purity of the rAAV vectors was satisfied. The expression level of apoA-I was (0.39±0.04) μg/ml and the apoA-IMilano was (0.31 ±0.03) μg/ml in the DMEM culture medium at the first 24 h after transfection.Conclusion: The success of the rAAV vectors construction, purification and the expression of apoA-I and apoA-IMilano in C2C12 cells mediated by these vectors, makes possible to inject rAAVA and rAAVAM vectors into mice muscle, and rises a new hope on finding a new way to prevent and treat atherosclerotic diseases and cardiovascular disease.