BioVector? pHS19 具核梭桿菌自殺質(zhì)粒 / BioVector? pHS19 Fusobacterium nucleatum Suicide Plasmid

通用定義 / General Definition:BioVector? pHS19 是一種專(zhuān)門(mén)用于具核梭桿菌 (Fusobacterium nucleatum) 基因組定向修飾的自殺質(zhì)粒 (Suicide Plasmid)。在分子遺傳學(xué)研究中，該質(zhì)粒被廣泛應(yīng)用于具核梭桿菌的基因敲除（Targeted Mutagenesis）。由于 pHS19 包含在大腸桿菌 (E. coli) 中復(fù)制的起點(diǎn)，但在具核梭桿菌中無(wú)法自主復(fù)制，因此它可以通過(guò)同源重組的方式整合到具核梭桿菌的染色體上，從而實(shí)現(xiàn)特定基因的失活或缺失。它是研究具核梭桿菌毒力因子、定殖機(jī)制及其與結(jié)直腸癌 (CRC) 關(guān)聯(lián)的核心遺傳工具。

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 技術(shù)說(shuō)明書(shū) (Technical Datasheet)

中文版說(shuō)明書(shū) (Chinese Datasheet)

1. 質(zhì)?；拘畔?/strong>

• 載體類(lèi)型： 自殺質(zhì)粒 / 自殺載體 (Suicide Vector)

• 大?。?/strong> 約 4.1 kb

• 構(gòu)建背景： 基于 pBluescript 骨架構(gòu)建。

• 篩選抗性 (細(xì)菌/E. coli)： 初始構(gòu)建包含紅霉素/克林霉素抗性，同時(shí)刪除了原有的氨芐青霉素抗性基因。

• 宿主篩選抗性： 紅霉素 (Erythromycin, erm) / 克林霉素 (Clindamycin, clin) 盒 (ermF-ermB)。

• 應(yīng)用領(lǐng)域： 具核梭桿菌的等位基因置換、基因定向敲除、靶向誘變。

2. 核心生物學(xué)功能

• 不可復(fù)制性： 在具核梭桿菌中屬于非復(fù)制載體，強(qiáng)制發(fā)生染色體整合。

• 高效同源重組： 配合具核梭桿菌特定的轉(zhuǎn)化技術(shù)（如電轉(zhuǎn)化），用于打斷染色體上的目標(biāo) ORF。

• 里程碑意義： pHS19 的成功應(yīng)用實(shí)現(xiàn)了具核梭桿菌歷史上首例靶向誘變（如敲除 rnr 基因）。

3. 實(shí)驗(yàn)應(yīng)用方案

• 克?。?/strong> 將目標(biāo)基因的內(nèi)部片段或同源臂克隆至 pHS19。

• 轉(zhuǎn)化： 使用高濃度質(zhì)粒 DNA 電轉(zhuǎn)化入具核梭桿菌感受態(tài)細(xì)胞（需嚴(yán)格厭氧操作）。

• 篩選： 在含有克林霉素或紅霉素的厭氧培養(yǎng)基上篩選整合株。

• 驗(yàn)證： 通過(guò) PCR 和測(cè)序驗(yàn)證目標(biāo)基因是否被成功阻斷。

4. 注意事項(xiàng)

• 厭氧操作： 具核梭桿菌為嚴(yán)格厭氧菌，轉(zhuǎn)化后的復(fù)蘇和篩選必須在厭氧箱中完成。

• 抗生素濃度： 具核梭桿菌對(duì)不同抗生素的敏感性差異較大，需根據(jù)具體菌株確定篩選濃度。

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 應(yīng)儲(chǔ)存在 -20 攝氏度。在進(jìn)行敲除實(shí)驗(yàn)時(shí)，建議先通過(guò) PCR 確認(rèn)載體插入的方向和同源重組的準(zhǔn)確性。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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