双片段survivin短发夹RNA腺病毒载体的构建与鉴定

Construction of the Recombinant Adenovirus Vector Bearing Dual-survivin shRNA

目的:构建负载双片段survivin短发夹RNA(shRNA)的腺病毒载体,为PCa的基因治疗提供实验基础。方法:分别设计2条靶向survivin基因的shRNA,克隆至穿梭质粒载体中,特异性酶切后亚克隆至腺病毒骨架,构建负载2条survivin shRNA的RNA干扰重组腺病毒载体。提取环状重组病毒质粒进行PCR、酶切双重鉴定后,大规模提取纯化正确的克隆。最后将线性化的重组腺病毒基因组转染至HEK293A包装细胞包装成病毒,并扩增到所需滴度。结果:负载双片段survivin shRNA的穿梭质粒载体pShuttle2经MluⅠ酶切鉴定,证实双片段survivinshRNA插入正确;完成目的片段至腺病毒骨架的亚克隆后,PCR及PI-SceⅠ/I-CeuⅠ双酶切鉴定均证实双片段sur-vivin shRNA插入正确。结论:负载双片段survivin shRNA腺病毒载体构建成功。

Objective： To construct a recombinant adenovirus vector bearing dual-survivin short hairpin RNA （shRNA）. Methods： Dual-survivin shRNA was designed and synthesized respectively, beth inserted into adenovirus DNA. The recombinant adenovirus vector was confirmed via beth sequencing and restriction digestion analysis, and then linearized and transfected into the HEK 293 cell line to generate recombinant adenoviruses. Results： The recombinant adenovirus vector was constructed and the target sequence was obtained. Conclusion ： The construction of the recombinant adenovirus vector provides a basis for the research of potential gene therapy for prostate cancer.