犬2型腺病毒通用载体的构建及鉴定

Construction of a Transfer Vector Based on Canine Adenovirus Type-2

摘要:

为了获得能够携带较大外源基因的犬2型腺病毒E3区缺失性载体，以犬2型腺病毒全基因组质粒pPolyⅡ-CAV-2及E3区重组质粒pVAX-E3为基础，缺失1381bp的E3区片段(92.6%的E3区全序列)，插入Linker-NF(内含NotⅠ、ClaⅠ、FseⅠ多克隆位点)，获得重组载体质粒pPolyⅡ-CAV-2-ΔE3（NF）(31.9kb)。以AscⅠ和PmeⅠ双酶切，游离重组基因组，在脂质体Lipofectamine^TM 2000介导下，转染MDCK细胞系，获得了E3区缺失的重组病毒CAV-2-ΔE3（NF）。通过病毒的形态学观察，血凝性、生长特性、感染性实验证明，该重组病毒与母源病毒没有差异。重组病毒CAV-2-ΔE3（NF）可以作为载体表达外源基因，其外源基因插入片段不小于3.3kb。

Abstract:

Canine adenovirus type 2 (CAV-2) has been proposed as a vector for recombinant vaccine. Alternatively, it may be an attractive tool for gene transfer due to lack of pre-existing immunity in humans. In this study, a transfer vector based on CAV-2, in which the 1381bp fragment of the E3 region was deleted, and a linker containing the NotⅠ,ClaⅠ,FseⅠ restriction enzyme sites were cloned into the deleted region. The recombinant CAV-2 genome was released from the plasmids enzyme digestion and transfected into MDCK cells by lipofectamine to obtain the recombinant virus. No significant difference in morphology, hemagglutination and replication between the recombinant and the wide type CAV-2 was found. These results indicated that this recombinant virus CAV-2-ΔE3（NF）may be an efficient vector for gene transfer and the capacity of the vector for inserted foreign gene was up to 3.3kb.