巨噬细胞炎性反应蛋白3β复制缺陷型重组腺病毒的构建与鉴定

Construction and characterization of replication deficient adenovirus containing macrophage inflammatory protein 3β gene

目的构建小鼠巨噬细胞炎性反应因子-3β(mMIP3β)的复制缺陷型重组腺病毒(Ad-mMIP3β),并在体外表达MIP3β。方法通过在细菌BJ5183内同源重组,利用AdEasyTM系统构建Ad-mMIP3β,经过噬斑筛选、PCR鉴定,然后用CsCl不连续密度梯度离心纯化。利用RT-PCR和Western blot方法研究Ad-mMIP3β的体外表达情况。结果成功构建了MIP3β的复制缺陷型重组腺病毒,并检测到分泌到胞外的MIP3β蛋白。结论MIP3β的复制缺陷型重组腺病毒构建成功,为下一步的肿瘤基因治疗研究奠定了基础。

[Objective] To construct the replication deficient adenovirus containing murine macrophage inflammarion protein-3β （Ad-mMIP3β） and express MIP3β in vitro. [Methods] The mMIP3β gene was constructed into the adenoviral backbone plasmid of AdEasyTM system by homologous recombination in bacteria BJ5183. After transfection of such plasmid into mammalian packaging cell lines （HEK293）, the viral production （Ad-mMIP3β） was subsequently screened by plaque assay and PCR, eventually purified by double cesium chloride gradient ultracentrifugation. The Ad-mMIP3β expression in vitro was assayed by RT-PCR and Western Blot methods. [Results] The replication deficient adenovirus containing mMIP3β gene was constructed correctly and the protein expressiion was identified. [Conelusion] These results suggested that Ad-mMIP3β constructed correctly would be applied for the following study of cancer gene therapy.