重组腺相关病毒载体介导的人载脂蛋白AⅠ及载脂蛋白AⅠMilano在肌源性细胞C2C12中的表达

Recombinant Adeno-Associated Viruses Mediated Apolipoprotein A1 and Apolipoprotein A1 Milano Expression in Myogenic C2C12 Cells

摘要:

目的 以重组2型腺相关病毒为载体介导人载脂蛋白A_Ⅰ及载脂蛋白A_ⅠMilano在小鼠肌源性细胞C₂C₁₂中的表达,探索一种崭新的预防、治疗动脉粥样硬化性疾病的途径。方法 以正常人肝组织中抽提的RNA为模板,用逆转录聚合酶链反应获得人载脂蛋白A_ⅠcDNA ,点突变制备载脂蛋白A_ⅠMilanocDNA。分别将载脂蛋白A_Ⅰ、载脂蛋白A_ⅠMilano的cDNA插入重组2型腺相关病毒载体质粒,并与辅助质粒共转染包装细胞2 93T获得编码人载脂蛋白A_Ⅰ和载脂蛋白A_ⅠMilano的重组腺相关病毒载体,粗提后以冰乙醇法进行浓缩。斑点杂交检测重组2型腺相关病毒载体滴度,十二烷基磺酸钠—聚丙烯酰胺凝胶电泳检测其纯度。分别用含载脂蛋白A_Ⅰ或载脂蛋白A_ⅠMilanocDNA的编码人载脂蛋白A_Ⅰ和载脂蛋白A_ⅠMilano的重组腺相关病毒对小鼠肌源性细胞C₂C₁₂进行感染,酶联免疫吸附法检测蛋白表达情况。结果 聚合酶链反应产物序列正确,点突变成功。编码人载脂蛋白A_Ⅰ和载脂蛋白A_ⅠMilano的重组腺相关病毒的滴度均在2×10 14 个/L左右。重组2型腺相关病毒纯度良好。载脂蛋白A_Ⅰ及载脂蛋白A_ⅠMilano分别获得0 .39±0 .0 4mg/L和0 .31±0 .0 3mg/L的表达水平。结论 成功制备表达载脂蛋白A_Ⅰ或载脂蛋白A_ⅠMilano的重组腺相关病毒载体,并在C₂C₁₂细胞中获得有效表达,为进一步探索方便、安全的预防和治疗动脉粥样硬化性疾病的方法打下了基础。

Abstract:

Aim To construct recombinant adeno-associated viruses (rAAV) vectors carrying human apolipoprotein A_Ⅰ (apoA_Ⅰ) and apo A_Ⅰ Milano cDNA, and explore a new strategy to prevent and treat the atherosclerotic diseases. Methods Human apo A_Ⅰ 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 A_Ⅰ 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 A_Ⅰ and apo A_Ⅰ Milano in C₂C₁₂ after infected by rAAV vectors were detected by ELISA method. Results The titre of the rAAV vectors of apo A_Ⅰ and apo A_Ⅰ 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 A_Ⅰ level is 0.39±0.04 mg/L and the apo A_Ⅰ 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 A_Ⅰ and apo A_Ⅰ Milano in C2C 12 cells mediated by these vectors, makes the injection of rAAV encoding human apo A_Ⅰ (rAAVA) and rAAV encoding human apo A_Ⅰ 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.