BioVector? 減毒單核細(xì)胞增生李斯特氏菌 / BioVector? Attenuated Listeria monocytogenes (Lm)

通用定義 / General Definition:

BioVector? 減毒李斯特菌（Listeria monocytogenes, Lm）是通過基因工程手段敲除關(guān)鍵毒力基因而獲得的安全性改良菌株。作為一種革蘭氏陽性、兼性胞內(nèi)寄生菌，李斯特菌具有獨(dú)特的胞內(nèi)生活史：它能誘導(dǎo)非吞噬細(xì)胞將其內(nèi)吞，隨后通過分泌李斯特菌溶血素 O (LLO) 逃逸出吞噬泡，進(jìn)入宿主細(xì)胞質(zhì)中復(fù)制，并利用宿主肌動(dòng)蛋白（Actin）進(jìn)行細(xì)胞間擴(kuò)散。減毒菌株（如經(jīng)典的 $\Delta actA / \Delta inlB$ 雙缺失株）保留了進(jìn)入抗原提呈細(xì)胞（APC）并在細(xì)胞質(zhì)內(nèi)表達(dá)抗原的能力，但失去了在組織間的擴(kuò)散能力和致病性。這使其成為理想的腫瘤免疫治療載體和疫苗研發(fā)平臺，能夠強(qiáng)力誘導(dǎo) $CD8^+$ T 細(xì)胞免疫反應(yīng)。

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? 減毒李斯特菌技術(shù)說明書 (Technical Datasheet)

中文版說明書 (Chinese Datasheet)

1. 常用菌株背景與基因型

• 經(jīng)典雙缺失株 (LADD):$\Delta actA$（缺失肌動(dòng)蛋白聚合蛋白，防止細(xì)胞間擴(kuò)散）+ $\Delta inlB$（缺失內(nèi)化素 B，降低對肝、脾實(shí)質(zhì)細(xì)胞的感染，提高安全性）。

• 代謝缺陷型 (Lmdd): 敲除 dal (丙氨酸消旋酶) 和 dat (D-氨基酸轉(zhuǎn)氨酶)，需在培養(yǎng)基中添加 D-丙氨酸才能生長，常作為質(zhì)粒平衡致死系統(tǒng)的宿主。

• 親本株: 通?；?EGD-e 或 10403S 模式菌株構(gòu)建。

2. 培養(yǎng)條件

• 培養(yǎng)基: BioVector? BHI (腦心浸液肉湯) 或 LB 培養(yǎng)基。

• 特殊添加: 對于 Lmdd 菌株，需額外添加 100 $\mu$g/mL D-Alanine。

• 環(huán)境: 37 攝氏度，好氧搖床培養(yǎng) (200 rpm)。

• 抗性篩選: 根據(jù)整合的抗性標(biāo)記（如紅霉素、氯霉素或卡那霉素）選擇性添加。

3. 主要應(yīng)用

• 腫瘤免疫治療: 攜帶腫瘤相關(guān)抗原（TAAs，如 PSA、HPV-E7），利用其胞內(nèi)寄生特性激活強(qiáng)烈的抗腫瘤 CTL 反應(yīng)。

• 傳染病疫苗載體: 開發(fā)針對 HIV、結(jié)核桿菌及病毒感染的新型活載體疫苗。

• 胞內(nèi)生物學(xué)研究: 研究細(xì)菌逃逸吞噬泡、胞內(nèi)代謝及宿主天然免疫識別機(jī)制。

4. 注意事項(xiàng)

• 生物安全: 雖然已減毒，但仍屬于 BSL-2 級別，操作時(shí)需穿戴防護(hù)裝備，防止意外刺傷或攝入。

• 抗生素敏感性: 該菌對萬古霉素、氨芐青霉素敏感。若實(shí)驗(yàn)中發(fā)生污染或感染，可用此類抗生素處理。

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: 減毒李斯特菌在 4 攝氏度下仍具有代謝活性（嗜冷性）。BioVector? 建議在實(shí)驗(yàn)結(jié)束后徹底使用 121 攝氏度高壓滅菌處理所有廢棄物，確保生物安全性。

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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