小鼠CD40L重组腺病毒载体的构建

目的构建含有小鼠CD40L基因的重组腺病毒载体,为研究mCD40L的生物学特性及肿瘤基因治疗提供基础。方法用XhoI、SwaI双酶切质粒pORF-mCD40L,回收1955bp基因片断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pSh-mCD40L。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。14天左右观察细胞病变及PCR鉴定重组的腺病毒。结果酶切分析、PCR验证mCD40L基因克隆到腺病毒pAdEasy-1载体中。结论成功构建表达mCD40L基因的重组腺病毒载体,为进一步研究其功能和基因治疗提供了基础。

Construction of the Recombinant Adenovirus Vector Expressing Mouse CD40 Ligand Gene

Objective:To construct a recombinant adenovirus vector expressing mCD40L gene and explore it in the use of antitumor gene therapy.Methods:1955bp gene fragment was obtained from plasmid pORF-mCD40L by XhoI/SwaI cutting and then cloned directionally into the pShuttle plasmid,finally,the resultant plasmid was digested by PmeI and cotransformed into BJ5183 cells with pAdEasy-1 to obtain the homologous recombinant and was packaged in the 293 cells.PCR and PacI digestion were employed to identify the recombinant adenovirus.Results:The evidences of endonulease digestion and PCR analysis confirmed that recombinant mCD40L gene was correctly inserted into adenovirus vector.Conclusion:The adenoviral vector which expressed mCD40L gene was constructed and can be used for further reserch in tumor gene therapy.