High-throughput generation of P. falciparum functional molecules by recombinational cloning
High-throughput generation of P. falciparum functional molecules by recombinational cloning
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DOI：
10.1101/gr.2416604
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发表时间：
2004-10-01
期刊：
影响因子：
7
通讯作者：
Carucci, DJ
中科院分区：
文献类型：
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作者：
Aguiar, JC;LaBaer, J;Carucci, DJ
疟疾疫苗和药物开发的大规模功能基因组学研究将取决于研究蛋白质表达的分子工具的产生。我们研究了使用 Gateway 系统创建大量编码恶性疟原虫单外显子基因的表达克隆的高通量克隆方法的可行性。主克隆及其 ORF 被集体转移到多个表达载体中。使用特定的标准集选择目标基因（n = 303），包括阶段表达和二级结构。每次捕获反应筛选 4 个菌落后，我们实现了 84% 的克隆效率。这些基因被平行亚克隆到三个表达载体中：一个DNA疫苗载体和两个蛋白质表达载体。基于单菌落筛选，这些转移取得了 100% 的成功率，没有观察到任何重组。在使用 DNA 疫苗构建体的小鼠中评估了 95 个基因的功能表达，以产生针对寄生虫各个阶段的抗体。其中，19 个诱导了针对红细胞阶段的抗体滴度，3 个诱导了针对子孢子阶段的抗体滴度。我们克服了在多个表达载体中产生大量恶性疟原虫克隆集的潜在限制。这种方法代表了一种强大的技术，用于生产用于恶性疟原虫基因组全基因组功能分析的分子试剂，并将为通过公共存储库分发的疟疾资源社区提供资源。
Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P, falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.