WANG Zhi-Guang1, GUI Ming2, JIANG Li1, CHEN Xiu-Ying1, HUANG Jun2, CHEN Qi1, and FAN Le-Ming1
1. Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029, China; 2.Cardiovascular Division, Jiangsu Province Hospital, Nanjing 210029, China 在知网中查找 在百度中查找 在本站中查找
Aim To construct recombinant adeno-associated viruses (rAAV) vectors carrying human apolipoprotein AⅠ (apoAⅠ) and apo AⅠ Milano cDNA, and explore a new strategy to prevent and treat the atherosclerotic diseases. Methods Human apo AⅠ cDNA with a his-tag in upward of the cDNA sequence was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR), human apo AⅠ Milano cDNA was then prepared by Site-Directed Mutagenesis. The particle numbers of rAAV vectors after extractd with a most economic and convenient method was assayed by Dot-blot, and the purity was assayed by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The expression efficiency of the apo AⅠ and apo AⅠ Milano in C2C12 after infected by rAAV vectors were detected by ELISA method. Results The titre of the rAAV vectors of apo AⅠ and apo AⅠ Milano was about 2×10 14/L, and the result of SDS-PAGE showed the purity of the rAAV vectors was good. The expressed apo AⅠ level is 0.39±0.04 mg/L and the apo AⅠ Milano is 0.31±0.03 mg/L in the DMEM culture medium. Conclusions The success of the rAAV vectors construction and purification and the expression of apo AⅠ and apo AⅠ Milano in C2C 12 cells mediated by these vectors, makes the injection of rAAV encoding human apo AⅠ (rAAVA) and rAAV encoding human apo AⅠ milano (rAAVAM) vectors in mouse muscular cells possible, and contributes to the hope of finding a safe and effective way to prevent and treat atherosclerotic diseases.