BioVector® pHS19 具核梭杆菌自杀质粒 / BioVector® pHS19 Fusobacterium nucleatum Suicide Plasmid

通用定义 / General Definition:BioVector® pHS19 是一种专门用于具核梭杆菌 (Fusobacterium nucleatum) 基因组定向修饰的自杀质粒 (Suicide Plasmid)。在分子遗传学研究中，该质粒被广泛应用于具核梭杆菌的基因敲除（Targeted Mutagenesis）。由于 pHS19 包含在大肠杆菌 (E. coli) 中复制的起点，但在具核梭杆菌中无法自主复制，因此它可以通过同源重组的方式整合到具核梭杆菌的染色体上，从而实现特定基因的失活或缺失。它是研究具核梭杆菌毒力因子、定殖机制及其与结直肠癌 (CRC) 关联的核心遗传工具。

BioVector® pHS19 is a specialized suicide plasmid used for targeted genome modification in Fusobacterium nucleatum. In molecular genetic research, it is the primary tool for targeted mutagenesis and gene knockouts within this species. Because pHS19 contains an origin of replication functional in E. coli but lacks one for F. nucleatum, it cannot replicate autonomously in the target host. Instead, it must integrate into the F. nucleatum chromosome via homologous recombination to achieve gene disruption. It is a fundamental genetic tool for studying virulence factors, colonization mechanisms, and the association of F. nucleatum with colorectal cancer (CRC).

BioVector® pHS19 技术说明书 (Technical Datasheet)

中文版说明书 (Chinese Datasheet)

1. 质粒基本信息

• 载体类型： 自杀质粒 / 自杀载体 (Suicide Vector)

• 大小： 约 4.1 kb

• 构建背景： 基于 pBluescript 骨架构建。

• 筛选抗性 (细菌/E. coli)： 初始构建包含红霉素/克林霉素抗性，同时删除了原有的氨苄青霉素抗性基因。

• 宿主筛选抗性： 红霉素 (Erythromycin, erm) / 克林霉素 (Clindamycin, clin) 盒 (ermF-ermB)。

• 应用领域： 具核梭杆菌的等位基因置换、基因定向敲除、靶向诱变。

2. 核心生物学功能

• 不可复制性： 在具核梭杆菌中属于非复制载体，强制发生染色体整合。

• 高效同源重组： 配合具核梭杆菌特定的转化技术（如电转化），用于打断染色体上的目标 ORF。

• 里程碑意义： pHS19 的成功应用实现了具核梭杆菌历史上首例靶向诱变（如敲除 rnr 基因）。

3. 实验应用方案

• 克隆： 将目标基因的内部片段或同源臂克隆至 pHS19。

• 转化： 使用高浓度质粒 DNA 电转化入具核梭杆菌感受态细胞（需严格厌氧操作）。

• 筛选： 在含有克林霉素或红霉素的厌氧培养基上筛选整合株。

• 验证： 通过 PCR 和测序验证目标基因是否被成功阻断。

4. 注意事项

• 厌氧操作： 具核梭杆菌为严格厌氧菌，转化后的复苏和筛选必须在厌氧箱中完成。

• 抗生素浓度： 具核梭杆菌对不同抗生素的敏感性差异较大，需根据具体菌株确定筛选浓度。

English Datasheet

1. Basic Plasmid Information

• Vector Type: Suicide Plasmid / Non-replicative Vector

• Size: Approximately 4.1 kb

• Backbone: Derived from pBluescript.

• Marker: Erythromycin/Clindamycin resistance cassette (ermF-ermB); ampicillin resistance gene deleted.

• Host Selection: Clindamycin/Erythromycin for F. nucleatum integration selection.

• Primary Purpose: Targeted mutagenesis, gene disruption, and allelic exchange in F. nucleatum.

2. Key Biological Functions

• Non-replicative Nature: Lack of a functional fusobacterial origin of replication ensures that any antibiotic-resistant clones have undergone chromosomal integration.

• Site-Specific Disruption: Ideal for inactivating specific open reading frames (ORFs) to study phenotypic changes.

• Historical Significance: pHS19 was instrumental in the first demonstration of targeted mutagenesis in F. nucleatum (e.g., disruption of the rnr gene).

3. Experimental Applications

• Insert Cloning: Cloning of internal gene fragments or flanking homologous arms into the pHS19 MCS.

• Electroporation: High-efficiency gene transfer into F. nucleatum under strictly anaerobic conditions.

• Mutant Selection: Screening for erythromycin/clindamycin-resistant colonies on anaerobic agar plates.

• Phenotypic Screening: Utilizing the generated mutants to study virulence, biofilm formation, or host-cell interaction.

4. Key Usage Notes

• Anaerobic Handling: Maintain a strict anaerobic environment throughout the transformation and recovery phases.

• Selection Pressure: Standard clindamycin concentrations may need optimization depending on the specific F. nucleatum subspecies (e.g., polymorphum vs nucleatum).

注意 / Note: pHS19 应储存在 -20 摄氏度。在进行敲除实验时，建议先通过 PCR 确认载体插入的方向和同源重组的准确性。Store at -20°C. For gene knockout experiments, BioVector® recommends verifying the orientation of the insert and the precision of the homologous recombination event via junction PCR and sequencing.

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