BioVector® 减毒单核细胞增生李斯特氏菌 / BioVector® Attenuated Listeria monocytogenes (Lm)

通用定义 / General Definition:

BioVector® 减毒李斯特菌（Listeria monocytogenes, Lm）是通过基因工程手段敲除关键毒力基因而获得的安全性改良菌株。作为一种革兰氏阳性、兼性胞内寄生菌，李斯特菌具有独特的胞内生活史：它能诱导非吞噬细胞将其内吞，随后通过分泌李斯特菌溶血素 O (LLO) 逃逸出吞噬泡，进入宿主细胞质中复制，并利用宿主肌动蛋白（Actin）进行细胞间扩散。减毒菌株（如经典的 $\Delta actA / \Delta inlB$ 双缺失株）保留了进入抗原提呈细胞（APC）并在细胞质内表达抗原的能力，但失去了在组织间的扩散能力和致病性。这使其成为理想的肿瘤免疫治疗载体和疫苗研发平台，能够强力诱导 $CD8^+$ T 细胞免疫反应。

BioVector® Attenuated Listeria monocytogenes (Lm) refers to strains engineered by deleting key virulence genes to ensure safety for research and therapeutic use. As a Gram-positive, facultative intracellular pathogen, Lm possesses a unique lifecycle: it induces its own uptake into host cells, escapes the phagosome via Listeriolysin O (LLO), replicates in the cytosol, and spreads cell-to-cell using host actin. Attenuated strains (such as the classic $\Delta actA / \Delta inlB$ double-deletion mutant) retain the ability to infect Antigen Presenting Cells (APCs) and deliver antigens to the MHC class I pathway but lack pathogenicity and the ability to spread between tissues. This makes them premier cancer immunotherapy vectors and vaccine platforms capable of inducing potent $CD8^+$ T-cell mediated immunity.

BioVector® 减毒李斯特菌技术说明书 (Technical Datasheet)

中文版说明书 (Chinese Datasheet)

1. 常用菌株背景与基因型

• 经典双缺失株 (LADD):$\Delta actA$（缺失肌动蛋白聚合蛋白，防止细胞间扩散）+ $\Delta inlB$（缺失内化素 B，降低对肝、脾实质细胞的感染，提高安全性）。

• 代谢缺陷型 (Lmdd): 敲除 dal (丙氨酸消旋酶) 和 dat (D-氨基酸转氨酶)，需在培养基中添加 D-丙氨酸才能生长，常作为质粒平衡致死系统的宿主。

• 亲本株: 通常基于 EGD-e 或 10403S 模式菌株构建。

2. 培养条件

• 培养基: BioVector® BHI (脑心浸液肉汤) 或 LB 培养基。

• 特殊添加: 对于 Lmdd 菌株，需额外添加 100 $\mu$g/mL D-Alanine。

• 环境: 37 摄氏度，好氧摇床培养 (200 rpm)。

• 抗性筛选: 根据整合的抗性标记（如红霉素、氯霉素或卡那霉素）选择性添加。

3. 主要应用

• 肿瘤免疫治疗: 携带肿瘤相关抗原（TAAs，如 PSA、HPV-E7），利用其胞内寄生特性激活强烈的抗肿瘤 CTL 反应。

• 传染病疫苗载体: 开发针对 HIV、结核杆菌及病毒感染的新型活载体疫苗。

• 胞内生物学研究: 研究细菌逃逸吞噬泡、胞内代谢及宿主天然免疫识别机制。

4. 注意事项

• 生物安全: 虽然已减毒，但仍属于 BSL-2 级别，操作时需穿戴防护装备，防止意外刺伤或摄入。

• 抗生素敏感性: 该菌对万古霉素、氨苄青霉素敏感。若实验中发生污染或感染，可用此类抗生素处理。

English Datasheet

1. Common Strains & Genotypes

• LADD (Listeria ActA/InlB Deleted):$\Delta actA$ (prevents actin-based motility and cell-to-cell spread) + $\Delta inlB$ (reduces infection of hepatocytes/splenocytes), significantly enhancing the safety profile.

• Lmdd (Auxotrophic Strains):$\Delta dal \Delta dat$ double mutant. These strains require D-alanine for cell wall synthesis and are used in "balanced-lethal" plasmid systems to maintain expression vectors without antibiotics.

• Parental Background: Most commonly derived from EGD-e or 10403S serotypes.

2. Culture Conditions

• Media: BioVector® BHI (Brain Heart Infusion) Broth or LB Broth.

• Supplements: For Lmdd strains, the medium must be supplemented with 100 $\mu$g/mL D-Alanine.

• Environment: 37°C, aerobic shaking at 200 rpm.

• Selection: Antibiotics (e.g., Erythromycin, Chloramphenicol) should be added based on the specific integration markers used.

3. Key Applications

• Cancer Immunotherapy: Engineered to express Tumor-Associated Antigens (TAAs); leverages the Lm lifecycle to drive potent MHC-I presentation and $CD8^+$ T-cell activation.

• Vaccine Development: Acts as a vehicle for heterologous antigens from pathogens like HIV, M. tuberculosis, or influenza.

• Intracellular Biology: Serving as a model to study phagosome escape mechanisms, cytosolic metabolism, and host STING/interferon signaling pathways.

4. Key Usage Notes

• Biosafety Level: Handled under BSL-2 conditions. Despite attenuation, it remains a human-derived pathogen proxy; rigorous aseptic technique and proper waste disposal are mandatory.

• Antibiotic Sensitivity:Listeria species are generally sensitive to Penicillin, Ampicillin, and Vancomycin. These should be available for emergency decontamination or clinical intervention if necessary.

注意 / Note: 减毒李斯特菌在 4 摄氏度下仍具有代谢活性（嗜冷性）。BioVector® 建议在实验结束后彻底使用 121 摄氏度高压灭菌处理所有废弃物，确保生物安全性。

Listeria remains metabolically active even at 4°C (psychrotrophic). BioVector® recommends thorough autoclaving of all waste at 121°C to ensure complete biological inactivation after experiments.

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