表达人CD40配体的重组腺病毒载体的构建

Construction of the Recombinant Adenovirus Vector Expressing Human CD40 Ligand Gene

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 adenovims vector expressing hCD40L gene and explore it in the use of antitumor gene therapy.Methods 1 900 bp gene fragment was obtained form plasmid pORF-hCD40L by Xho Ⅰ / Swa Ⅰ cutting and then cloned directionaUy into the pShuttle plasmid, finally, the resultant plasmid was digested by restriction endonnuclease PmeI and subsequently cotransformtion into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant and then the recombinant was packaged in the 293 cells. Some methods such as PCR and endonulease digestion were employed to identify the recombinant adenovims. Results The evidences of endonulease digestion and PCR analysis confirmed that recombinant hCD10L gene was correctly inserted into adenovims vector. Conelusion The adenoviral vector which expressed hCD40L gene was constructed. It provides an experimental basis for studies on it expression in the mammalian ceils and in tumor gene therapy.