重组腺相关病毒介导的Dystrophin小基因载体构建及在mdx鼠中表达

Recombinant Adeno-associated Virus Vector Carrying Human Minidystrophin Gene SMCKA3999 Vector Construction and Expression in mdx Mice

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 study the method of recombinant adeno-associated virus vector carrying human minidystrophin gene SMCKA3999 vector construct and expression in mdx mice (an animal model of DMD).Methods:We constructed recombinant adeno-associated virus vector carrying human minidystrophin gene SMCKA3999 vector (rAAVSMCKA3999) by three plasmids co-infection into 293 cells method and measured the titers of rAAVSMCKA3999 by dot blot hybridization. When injected into the skeletal muscle of mdx mice, gene expression was proved in mdx mice by Western BLot. Results:We can construct recombinant adeno-associated virus vector carrying human minidystrophin gene SMCKA3999 vector (rAAVSMCKA3999) by three plasmids co-infection into 293 cells method.The titers of rAAVSMCKA3999 will reach 5.0×10 10 .rAAVSMCK3999 resulted in efficient and stable expression persisting for 7 months. Conclusion:rAAVSMCKA 3999 we constructed has important significance for further gene therapy researches of DMD.