副乾酪乳酸桿菌食品級表現載體之研究

副乾酪乳酸桿菌(Lactobacillus paracasei subsp paracasei BCRC14023，Lactobacillus casei ATCC27092)為食品級安全菌株，以遺傳工程技術表現功能性勝肽或蛋白質於副乾酪乳酸桿菌，可應用於腸道健康之增進或改善。傳統遺傳工程技術多以抗抗生素特性作為篩選標記。然而2003年聯合國食品標準委員會(Codex Alimentarius Commission，codex)公布基因改造食品安全評估指針，不允許使用抗抗生素選擇標記。本研究中以nisin免疫基因sisI及nisin抗性基因nsr，取代遺傳工程中使用之抗抗生素基因，作為食品級選擇標記。Nisin免疫基因nisI以nsr啓動子增進表現量。結果顯示，500至700 IU之nisin濃度為適當的nisin抗性基因nsr選擇濃度；而nisin免疫基因nisI則在500至900 IU之nisin濃度均可達選擇效果。本研究建立副乾酪乳酸桿菌之食品級表現載體，未來可提供安全的食品級基因工程改造副乾酪乳酸桿菌。

關鍵字

nisin免疫基因nisI ； nisin抗性基因nsr ；副乾酪乳酸桿菌；食品級載體

並列摘要

Lactobacillus paracasei subsp. paracasei BCRC14023 (Lactobacillus casei ATCC27092) is a food grad strain. Production of bioactive peptides or proteins in the strain enables the potent application as functional peptides delivery vehicle. Previous genetic expression systems used antibiotic resistant genes as selective marker. Codex Alimentarius Commission (Codex) announced a guideline for the safety evaluation of foods produced using recombinant-DNA microorganisms. In which an antibiotic resistant marker is not admitted. In this study, resistance or immunity toward a FDA approved food grade bacteriocin, nisin, by nisin immunity protein (NisI) gene nisI or nisin resistant protein (Nsr) gene nsr were evaluated to develop food grade vectors for L. paracasei. The chloramphenicol resistance gene was replaced and the expression level was improved by replacing the artificial promoter into more efficient nsr promoter. The nisin concentration of 500 to 700 IU is applicable in Nsr selection and a nisin concentration of 500 to 900 IU is applicable in NisI selection. The two plasmids are promised as potent food grade vectors for L. paracasei. This study provides the immediate possibility of constructing food grade genentic modified L. paracasei.

並列關鍵字

Nisin immunity gene nisI ； Nisin resistant gene nsr ； Lactobacillus paracasei ； Food grade vector

國際替代計量

副乾酪乳酸桿菌食品級表現載體之研究

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