论文部分内容阅读
目的 :研究重组腺相关病毒载体 (rAAV)介导的人Dystrophin小基因 (SMCKA3 999)载体构建及在DMD模型鼠 (mdx鼠 )的表达。方法 :将SMCKA3 999质粒 ,包装质粒pXX2、腺病毒成分辅助质粒pXX6共转染 2 93细胞 ,包装重组腺相关病毒载体介导的SMCKA3 999基因 (rAAVSMCKA3 999) ,以斑点杂交法测定病毒滴度 ,将rAAVSMCKA3 999单点注射到mdx鼠腓肠肌 ,于注射后 7个月取肌肉提取蛋白质行Westernblot检测。结果 :经三质粒共转染法构建的rAAVSMCKA3 999病毒滴度为 5 0× 10 10 ,在mdx鼠骨骼肌表达持续 7个月以上。结论 :构建的rAAVSMCKA3 999载体为进一步DMD基因治疗研究奠定了基础。
AIM: To investigate the construction of recombinant adeno-associated virus vector (rAAV) -mediated human Dystrophin minigene (SMCKA3 999) and its expression in DMD model mice (mdx mice). Methods: SMCKA3 999 plasmid, packaging plasmid pXX2 and adenovirus component plasmid pXX6 were co-transfected into 293 cells and the recombinant adeno-associated virus vector-mediated SMCKA3 999 gene (rAAVSMCKA3 999) was packaged. The virus titer, RAAVSMCKA3 999 single injection into the mdx rat gastrocnemius muscles, 7 months after injection of muscle extracted protein Western blot detection. Results: The titers of rAAVSMCKA3999 virus constructed by the three plasmid co-transfection method were 50 × 10 10, and the expression of skeletal muscle in mdx rats persisted for more than 7 months. CONCLUSION: The constructed rAAVSMCKA3999 vector lays a foundation for the further study of DMD gene therapy.