Chromosomal Insertions in the Lactobacillus casei upp Gene That Are Useful for Vaccine Expression
Chromosomal Insertions in the Lactobacillus casei upp Gene That Are Useful for Vaccine Expression
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干酪乳杆菌 upp 基因中的染色体插入可用于疫苗表达
DOI：
10.1128/aem.00175-14
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发表时间：
2014-06-01
影响因子：
4.4
通讯作者：
Tang, Li-jie
中科院分区：
文献类型：
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作者：
Song, Bai-fen;Ju, Long-zhu;Tang, Li-jie
为了开发一种稳定且无标记的乳酸菌菌株用于疫苗的表达，我们开发了一种温度敏感的自杀质粒，其表达盒含有HCE启动子、PgsA锚点、α -毒素基因和使用5-氟尿嘧啶（5-FU）反选择标记的rrnB T1T2终止子（PPαT）。通过5-FU筛选的选择压力，获得了3株含有正确的PPαT表达带的菌株。我们通过基因组PCR扩增和测序证实了upp基因缺失，并将PPαT表达带插入到干酪乳杆菌ATCC 393的upp位点。重组菌对5-FU的耐药性在插入后可稳定遗传长达40代。然而，含有整合DNA的细菌比野生型干酪乳杆菌生长得更慢。间接酶联免疫吸附试验（ELISA）证实了α -毒素基因的表达。此外，我们还利用激光共聚焦显微镜观察了干酪乳杆菌细胞表面蛋白的表达情况。这些结果综合起来表明，这些重组细菌应该为有效的疫苗生产提供安全的工具。
ABSTRACT To develop a stable and marker-free Lactobacillus strain useful for the expression of vaccines, we developed a temperature-sensitive suicide plasmid with expression cassettes containing an HCE promoter, a PgsA anchor, the alpha-toxin gene, and an rrnB T1T2 terminator (PPαT) that uses a 5-fluorouracil (5-FU) counterselectable marker for Lactobacillus casei. Three strains containing the correct PPαT expression cassettes were produced via the selective pressure of 5-FU screening. We confirmed that the upp gene was deleted and that the PPαT expression cassettes were inserted into the upp site of L. casei ATCC 393 by genomic PCR amplification and sequencing. 5-FU resistance in recombinant bacteria could be stably inherited for as long as 40 generations following insertion. However, bacteria containing the integrated DNA grew more slowly than wild-type L. casei. An indirect enzyme-linked immunosorbent assay (ELISA) analysis demonstrated that the alpha-toxin gene was expressed. Also, we visualized expression of the protein on the surface of L. casei cells using laser confocal microscopy. These results taken together demonstrate that these recombinant bacteria should provide a safe tool for effective vaccine production.