Recombinant Adeno-associated Virsues Mediated ApoA-I And ApoA-IMilano Expression In Myogenic C2C12 Cells

Objective: The apoA-IMilano, with a cysteine to arginine substitution at the position 173, is a naturally occurring mutant of apoA-I, and has been the first described as molecular variant of human apolipoprotein. The antiatherogenic and vasculoprotective effect of recombinant apoA-IMilano is widely accepted. We construct rAAV vectors carrying human apoA-I/apoA-IMilano cDNA, and explore a new strategy to prevent and treat the atherosclerotic diseases.Methods: Human apoA-I cDNA with a his-tag in upward of the cDNA sequence was obtained by RT-PCR and PCR, human apoA-IMilano cDNA was then prepared by Site-Directed Mutagenesis. The particle numbers of rAAV vectors after extracted with a most economic and convenient method is assayed by Dot-blot, and the purity is assayed by SDS-Page. The expression efficiency of the apoA-I and apoA-IMilano in C2C12 after infected by rAAV vectors were detected by ELISA method.Results: The titre of the rAAV vectors of apoA-I and apoA-IMilano is about 2×10~14/L, and the result of SDS-Page showed the purity of the rAAV vectors was good. The expressed apoA-I level is 0.39 ± 0.04ug/mL and the apoA-IMilano is 0.31 ± 0.03ug/mL in the DMEM culture...