小鼠CD40L重组腺病毒载体的构建

Construction of the Recombinant Adenovirus Vector Expressing Mouse CD40 Ligand Gene

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。

Objective： To construct a recombinant adenovirus vector expressing mCIMOL 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, final/y, 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.