Ad5F35-LMP2重组腺病毒的构建及鉴定

Construction and identification of recombinant adenovirus Ad5F35-LMP2 containing LMP2 gene

目的构建Ad5F35-LMP2重组腺病毒。方法分别以EcoRⅠ单酶切pSH-LMP2和pDC316,将LMP2目的基因片段和线性化的pDC3l6连接,克隆构建pDC3l6-LMP2,以PCR和酶切的方法鉴定插入方向正确。pDC3l6-LMP2与腺病毒骨架pBHG-fiber5/35共转染293细胞构建重组腺病毒Ad5F35-LMP2,PCR及间接免疫荧光进行鉴定。结果经PCR和酶切鉴定证实pDC3l6-LMP2构建成功。pDC3l6-LMP2与腺病毒骨架共转染293细胞后见明显的毒斑,说明二者在293细胞中同源重组并包装成功。经PCR证实重组腺病毒Ad5F35-LMP2构建完成,间接免疫荧光鉴定LMP2蛋白能在细胞膜上有效表达。结论本实验成功构建了含EB病毒LMP2全序列的Ad5F35-LMP2重组腺病毒,为下一步进行该重组病毒生物安全性能和生物学功能研究及今后应用Ad5F35-LMP2重组腺病毒对鼻咽癌患者进行基因免疫治疗奠定了基础。

Objective： To construct recombinant adenovirus expressing Epstein-Barr Virus （EBV）-LMP2 in vector Ad5F35. Methods：Restriction fragment of LMP2 from pSH-LMP2 was inserted in the vector of pDC316. The insertion and the direction of the insert were confirmed by polymerase chain reaction （PCR） and restriction enzyme digestion. The constructed pDC316-LMP2 was co-transfected with adenovirus backbone pBHG-fiber5/35 into 293 cells to establish the recombinant adenovirus Ad5F35-LMP2, which was confirmed by PCR and indirect immune-fluorescence test. Results： The construction of pDC316-LMP2 was completed and confirmed with PCR and restriction enzyme digestion. Second, the significant virus plaques was observed in the 293 cells co-transfected with pDC316-LMP2 together with pBHG-fiber5/35, which means the successful homologue recombination and virus packaging of the interest fragment in 293 cells. Third, the successful construction of the recombinant adenovirus Ad5F35-LMP2 with PCR was confirmed, and the efficient expression of LMP2 protein was observed on the cell membrane with indirect immune-fluorescence test. Conclusion：The recombinant adenovirus of Ad5F35-LMP2 which contains the full-length coding region of EBV LMP2 protein has been established that could be used in the gene therapy for nasopharyngeal carcinoma patients and to test the bio-safety and biological function of Ad5F35- LMP2 in the future.