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目的构建含有hCD40L基因的重组腺病毒载体,为其在动物模型体内表达和肿瘤基因治疗提供基础。方法用XhoⅠ、SwaⅠ双酶切质粒pORF_hCD40L,回收1 900 bp基因片段并定向克隆插入穿梭质粒pShuttle中XhoⅠ、EcoRⅤ双酶切位点,得到重组质粒pShuttle_hCD40L。经PmeI酶切与腺病毒骨架质粒pAdEasy_1共转化BJ5183细菌,同源重组后用选择培养基筛选阳性克隆,提取质粒PacI酶切线性化后用脂质体介导转染293细胞。酶切分析和PCR鉴定重组的腺病毒。结果酶切分析、PCR验证表明,hCD40L基因成功克隆到腺病毒pAdEasy_1载体中。结论成功构建表达hCD40L基因的重组腺病毒载体,为进一步研究其在哺乳动物内表达及基因治疗提供了基础。
Objective To construct a recombinant adenovirus vector containing hCD40L gene and provide a basis for its expression in vivo and tumor gene therapy in animal models. Methods Plasmid pORF_hCD40L was digested by XhoⅠand SwaⅠ, and a fragment of 1 900 bp was recovered and cloned into the shuttle plasmid pShuttle. The recombinant plasmid pShuttle_hCD40L was constructed. BJ5183 was co-transformed by PmeI and adenovirus backbone plasmid pAdEasy_1. After homologous recombination, the positive clones were screened by selection medium. The recombinant plasmid was digested with PacI and linearized and transfected into 293 cells by liposome. Restriction analysis and PCR identification of recombinant adenovirus. Results Enzyme digestion analysis and PCR analysis showed that hCD40L gene was successfully cloned into adenovirus pAdEasy_1 vector. Conclusion The successful construction of recombinant adenovirus vector expressing hCD40L gene provides the basis for further study of its expression in mammals and gene therapy.