DNA vaccines require a vector to replicate genes and express encoding antigens. Antibiotic resistance genes are often used as selection markers, which must not be released to the environment upon final product commercialization. For this reason, generation of antibiotic resistance-free vectors is imperative. The pPAL vector contains the cytomegalovirus enhancer and promoter for expression in mammalian cells and the E. coli fabI chromosomal gene as a selectable marker. The fabI gene encodes the enoyl-ACP reductase (FabI). The bacteriostatic compound triclosan is an inhibitor of this enzyme. Therefore, the selection of positive clones depends on the enzyme:inhibitor molar ratio. According to western blot analysis, the pPAL vector is functional for expression of the Leishmania infantum (Kinetoplastid: Trypanosomatidae) gene encoding for the protein kinase C receptor analog (LACK/p36) in the HEK293T human cell line transfected with pPAL-LACK. The fabI gene sequence contains a 210 bp CpG island, suggesting a potential role as an adjuvant of the antibiotic resistance-free pPAL vector. In fact, Th1 response induction levels against canine leishmaniasis only using pPAL-LACK was shown to be as strong as in previous strategies using a recombinant vaccinia virus in combination with standard mammalian expression plasmid vectors. In summary, the pPAL plasmid contains the essential elements for manipulation and expression of any cloned DNA sequence in prokaryotic and mammalian cells using an E. coli endogenous gene as a selectable marker, which also provides a long CpG island. This element enhances Th1 immune response against L. infantum infection in dogs using the gene encoding for the LACK antigen. Therefore, this antibiotic resistance-free plasmid is a vaccine vector actively participating in protection against canine leishmaniasis and may be potentially tested as a vaccine vector with other antigens against different pathogens.

中文翻译：

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用于开发第三代疫苗的无抗生素抗性哺乳动物表达质粒载体pPAL。

DNA疫苗需要载体来复制基因并表达编码抗原。抗生素抗性基因通常用作选择标记，在最终产品商业化后，不得将其释放到环境中。因此，必须产生无抗生素抗性的载体。pPAL载体包含巨细胞病毒增强剂和启动子，用于在哺乳动物细胞中表达，并且大肠杆菌fabI染色体基因作为选择标记。fabI基因编码烯酰ACP还原酶（FabI）。抑菌化合物三氯生是该酶的抑制剂。因此，阳性克隆的选择取决于酶：抑制剂的摩尔比。根据蛋白质印迹分析，pPAL载体可用于婴儿利什曼原虫的表达（动质体：锥虫科（Trypanosomatidae）基因编码转染pPAL-LACK的HEK293T人细胞系中的蛋白激酶C受体类似物（LACK / p36）。fabI基因序列包含一个210 bp的CpG岛，表明它是无抗生素抗性pPAL载体佐剂的潜在作用。实际上，仅使用pPAL-LACK显示的针对犬利什曼病的Th1应答诱导水平与使用重组牛痘病毒结合标准哺乳动物表达质粒载体的先前策略一样强。总而言之，pPAL质粒包含使用大肠杆菌内源基因作为选择标记来操纵和表达任何克隆的DNA序列在原核和哺乳动物细胞中的基本元件，该标记也提供了一个长CpG岛。该元素增强了针对L的Th1免疫反应。使用编码LACK抗原的基因在犬中进行婴儿感染。因此，该无抗生素抗性质粒是积极参与针对犬利什曼病的保护的疫苗载体，并且可能被作为具有针对不同病原体的其他抗原的疫苗载体进行测试。