携带人血红素加氧酶-1基因重组腺病毒的构建及其在大脑皮层神经元中的表达

目的构建人血红素加氧合酶-1（hHO-1）基因的重组腺病毒，并研究其感染大脑皮层神经元的效率。方法采用基因克隆技术，将hHO-1基因克隆到腺病毒穿梭质粒pShuttle—IRES—hrGFP中，构建pShuttle-hHO-1-IRES—hrGFP质粒，利用细菌BJ5183将穿梭质粒与pAdEasy-1进行重组，获得重组的腺病毒质粒。线性化的腺病毒质粒经脂质体转染AD293细胞，进行重组腺病毒的包装和扩增。采用CsCl梯度离心进行病毒纯化。获得的腺病毒感染大脑皮层神经元，观察其感染效率和hHO-1表达水平。结果通过酶切鉴定证明腺病毒载体构建成功，包装出携带hHO-1基因的腺病毒，病毒滴度为2．0×10^10pfu／ml，获得的腺病毒对大脑皮层神经元的感染效率高于95％以上。结论成功地利用细菌内同源重组方法构建了携带hHO-1基因的腺病毒，并能够在大脑皮层神经元中高效地表达，为利用HO—1进行基因治疗提供了材料。

Construction of recombinant adenovirus encoding human heme oxygenase-1 and its expression in cerebral cortical neuron

Objective To construct recombinant adenovirus carrying human heme oxygenase-1 （hHO-1）, and observe its ability of infecting cerebral cortical neuron. Methods hHO-1 gene was subcloned into adenovirus shuttle vector pShuttle-IRES-hrGFP to generate the pShuttle-hHO-1- IRES-hrGFP. The linearized shuttle vector was transformed into recombinant bacteria BJ5183 containing pAdEasy- 1 to produce adenovirus vector encoding hHO- 1. Recombinant adenovirus vector pAd - hHO-1 was transfected into AD293 cells by liposome to generate recombinant adenovirus particles which were purified by CsC1 gradient centrifugation. Adenovirus particles were used to infect cerebral cortical neuron, and the infection efficiency and hHO-1 expression were observed. Results The shuttle and adenovirus vectors were constructed successfully and adenovirus encoding hHO-1 gene was generated with titers of about 2.0 × 10^10 pfu/ml. The infection efficiency of adenovirus to cerebral cortical neuron was more than 95 %. hHO- 1 could be expressed in cerebral cortical neuron. Conclusions The recombinant adenovirus carrying hHO-1 gene was constructed successfully, which can be expressed in cerebral cortical neurons with high efficiency, and that may provide an useful tool for gene therapy.