Dll4基因靶向RNAi重组腺相关病毒载体的构建和高滴度病毒制备

Construction of recombined adeno-associated virus vector expressing Dll4 short RNA interference and preparation of high-titer virus

目的:构建一个表达肿瘤血管生成调节基因Delta-like ligand4(Dll4)小干扰RNA的重组腺相关病毒载体,制备靶向性抑制Dll4表达的高滴度重组腺相关病毒。方法:将包含肿瘤血管生成调节基因Dll4特异性小干扰RNA片段的质粒用EcoRI+SalI双酶切,回收目的片段,将pSNAV2.0质粒用EcoRI+SalI双酶切后同目的片段连接,脂质体法转染BHK-2l细胞,G418筛选后以辅助病毒感染获取病毒。结果:PCR和测序证实,成功构建包含U6启动子和肿瘤血管生成调节基因Dll4特异性RNA干扰片段的重组腺相关病毒载体,并制备出滴度为2.4×l011vectorgenome/L高滴度腺相关病毒。结论:成功构建携带Dll4基因短发夹状干扰RNA的腺相关病毒载体。为下一步探索抗肿瘤血管生成基因治疗的新途径提供实验基础。

Objective： To construct the recombinant adeno-associated virus vector expressing tumor angiogenesis-related gene Dll4 short RNA interference（RNAi） and to use it for the preparation of high-titer virus. Methods： pDC316-Dll4-shRNA plasmid, constructed with Dll4 siRNA, was digested with EcoRI and SaIL A 1 500bp fragment, containing U6 promoter and Dll4 siRNA, was obtained and inserted into the adeno-associated virus vector plasmid pSNAV2.0 digested with EeoRI ＋SalI. Positive vectors were analyzed through enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into BHK-21 cells using LipofectamineTM^2000. G418-resistant cells were obtained and infected with HSVI-rc/△UL2, which have the function of packaging recombinant adeno-associated virus （rAAV）. After purification, target vector and virus were collected. Results： The target vector, pSNAV2.0-shRNA-Dll4, was successfully constructed, and the rAAV with a titer of 2.4 × 10^11 vg/L （ vg ： vectorgenome） was obtained. Conclusion： rAAV vector may be used in further investigation of Dll4 function in tumor angiogenesis and gene therapy.