Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors
Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors
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DOI：
10.3791/50833
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发表时间：
2013-12-01
影响因子：
1.2
通讯作者：
Klonjkowski, Bernard
中科院分区：
文献类型：
--
作者：
Szelechowski, Marion;Bergeron, Corinne;Klonjkowski, Bernard
腺病毒 (Ad) 衍生载体已广泛用于短期或长期基因转移，用于基因治疗和疫苗应用。由于针对经典使用的人类 5 型腺病毒的频繁预先存在的免疫力，犬腺病毒 2 型 (CAV2) 已被提议作为人类基因转移的替代载体。 CAV2 的良好生物学特征及其易于基因操作的特点提供了主要优势，特别是对于将基因转移到中枢神经系统，或诱导从体液免疫到细胞免疫的广泛保护性免疫反应。如今，CAV2 是用作疫苗载体的最有吸引力的非人类腺病毒之一。该协议描述了构建、生产和滴定重组 CAV2 载体的简单方法。将目的基因的表达盒克隆到穿梭质粒中后，在大肠杆菌BJ5183菌株中通过同源重组获得重组基因组质粒。然后将所得基因组质粒转染至表达互补 CAV2-E1 基因 (DK-E1) 的犬肾细胞中。病毒扩增能够产生大量病毒原液，通过氯化铯梯度超速离心纯化并通过透析脱盐。所得病毒悬浮液的滴度通常超过每毫升10（10）个感染性颗粒，并且可以直接在体内施用。
Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 10(10) infectious particles per ml and can be directly administrated in vivo.