重组腺辅助病毒载体的制备、纯化及其介导的基因转移

Production and Purification of rAAV and Gene Transfer Introduced by rAAV

目的 :探讨一种制备、纯化重组腺辅助病毒 (r AAV)载体的有效方法。方法 :以质粒 PDG和含有人 apo AIc DNA的AAV载体共转染 2 93T细胞 ,继以 Iodixanol密度梯度离心结合肝素亲和层析法分离、纯化 ,斑点杂交鉴定 r AAV病毒颗粒数 ;并以含人 apo AIc DNA的 r AAV感染 C2 C12细胞。结果 :r AAV颗粒数为 7× 10 1 0 / ml;r AAV成功介导了人 apo AIc DNA在 C2 C12细胞中的表达并持续了 30天 ,在分化为肌管细胞后仍有此表达能力。结论 :本实验采取的制备、纯化 r AAV的方法简便、高效 ,r AAV成功介导 apo AI基因在肌源性细胞 C2 C12中的表达。

Objective:To investigate an efficient way for production and purification of recombinant adeno associated virus vectors. Methods:The 293T cells was cotransfected with PDG and an AAV plasmid containing human apoAIcDNA to produce infectious rAAV, and it was purified by non ionic iodixanol gradients followed by heparin affinity chromatography. The numbers of rAAV particle were assayed by Dot blot, then C2C12 cells was transduced by these vectors. Results:The particle number of rAAV was 7×10 10 /ml and the rAAV successfully mediated the expression of human apoAIcDNA even after differentiation into myotubes. The expression of human apoAIcDNA in C2C12 cells lasted for 30 days. Conclusion:The methods which was used for production and purification of rAAV is an efficient way and the rAAV vector can successfully mediate apoAI gene transfer to C2C12 myoblasts.