BioVector® Helicobacter pylori G27 Strain / G27 幽门螺杆菌标准模式菌株

一 产品基本信息与遗传学背景

• 菌株名称：G27 幽门螺杆菌（Helicobacter pylori G27）。

• 物种分类：细菌界（Bacteria），变形菌门（Pseudomonadota），Epsilon-变形菌纲（Epsilonproteobacteria），弯曲菌目（Campylobacterales），螺杆菌科（Helicobacteraceae），螺杆菌属（Helicobacter）。

• 菌株背景与来源：G27 是全球幽门螺杆菌研究领域最经典、应用最广泛的标准模式参考株（Type Strain）之一。最初于 20 世纪 80 年代末至 90 年代初，由意大利科学家从一名患有严重胃溃疡（Gastric Ulcer）的意大利内镜患者的胃黏膜活检标本中分离获得。

• 基因组与关键毒力因子特征（核心背景）：

  • CagA 阳性（CagA+）：G27 菌株拥有一条完整的、功能活跃的 cag 致病岛（cag Pathogenicity Island, cagPAI），能够高效表达细胞毒素相关基因 A（CagA）蛋白。CagA 被称为 细菌癌蛋白，是引发宿主胃上皮细胞恶性转化的核心因子。

  • VacA 活性（VacA-s1/m1 型）：携带高毒力的空泡毒素 A（VacA）基因型（通常为 s1/m1 型）。该毒素可在宿主细胞内诱导大量的细胞质空泡化，破坏线粒体膜电位并诱导上皮细胞凋亡。

  • IV 型分泌系统（T4SS）：G27 具备完全组装的 T4SS 分泌针状结构。当细菌与胃上皮细胞接触时，T4SS 像微型注射器一样穿透宿主细胞膜，将 CagA 蛋白以及肽聚糖等物理注入宿主胞质内。

• 电生理与操作优势：相比于另一株经典高毒力株位点（如 26695 株），G27 最大的核心遗传学优势在于其具备极高且稳定的自然转化能力（Highly Naturally Transformable）。这使得实验室对其进行基因敲除、等位基因更换（Allelism Exchange）、插入突变以及荧光标记质粒导入的操作极为简便且成功率极高。

• 生物安全级别：2级（BSL-2）。幽门螺杆菌是 I 类人类致癌物，活菌操作必须在二级生物安全柜内进行。

二 核心科研价值与转化医学应用

G27 菌株将高毒力因子的病理特征与极佳的基因工程可塑性完美结合，是解析胃壁发病机制的黄金模型：

1. 宿主-病原体相互作用与细胞骨架重塑（Hummingbird Phenotype）：在体外侵染人胃癌上皮细胞（如 AGS、MKN45）时，G27 通过 T4SS 将 CagA 注入细胞。CagA 随后被宿主激酶（如 Src、Abl）磷酸化，与 SHP-2 磷酸酶结合，引发细胞出现特征性的 蜂鸟表型（Hummingbird Phenotype） 也就是细胞拉长、伪足伸展、运动性剧增。这一过程是研究胃癌细胞上皮-间充质转化（EMT）和癌变初始机制的标准通路。

2. IV 型分泌系统（T4SS）动力学与超微结构研究：G27 常被用作解构 T4SS 针状复合体组装、CagL 蛋白与宿主细胞表面整合素（alpha v beta 1 integrin）识别受体结合机制的分子底盘。通过冷冻电镜（Cryo-EM）或超分辨荧光显微镜，可以实时追踪 G27 毒力因子在菌体与胃黏膜接触面上的定向极化转运。

3. 新型根除药物与新型疫苗靶点高通量筛选：鉴于全球临床幽门螺杆菌对克拉霉素、甲硝唑的耐药性剧增，G27 作为敏感/耐药演变的标准对照株，被广泛用于筛选新型天然抗菌小分子、特异性尿素酶（Urease）抑制剂，以及开发针对其外膜蛋白（如 BabA、SabA）的靶向黏附阻断剂。

三 实验室菌株复苏、固体/液体微需氧培养与保存标准步骤

幽门螺杆菌是严格的微需氧菌（Microaerophilic），对氧气浓度、湿度和培养基质的营养及新鲜度有着极其严苛的要求。普通有氧或严格厌氧环境均会导致其迅速死亡或转化为无法转化的球形菌（Coccoid form，不具备感染力的代谢休眠态）。

1. 培养基与微需氧环境配置

• 固体培养基（最常用）：哥伦比亚琼脂（Columbia Agar base） 或 脑心浸液琼脂（BHI Agar）。

  • 必须添加剂：培养基灭菌冷却至 50 摄氏度时，必须添加 5% 到 7% 的脱纤维裂解绵羊血（Defibrinated Sheep Blood） 或 马血，或添加 10% 胎牛血清（FBS）。血清能有效清除培养基中对幽门螺杆菌致命的自由基氧化物。

  • 选择性双抗/多抗（针对复苏活检或防污染）：通常添加 幽门螺杆菌专属选择性添加剂（如含有万古霉素 Vancomycin、多粘菌素 Polymyxin B、Trimethoprim、两性霉素 B 的添加剂包），以抑制杂菌生长。

• 液体培养基（需特定摇摆条件）：含 10% FBS 的 BHI 肉汤。

• 微需氧物理环境（核心关键）：

  • 气体标准成分：5% 氧气、10% 二氧化碳、85% 氮气。

  • 实验室常规实现方式：使用专业的微需氧培养箱、或者将平板置于密封专用的抽气罐/产气袋（如 CampyGen 产气包）中。

  • 培养温度：37 摄氏度（绝对禁止 30 度或 42 度培养）。

  • 相对湿度：大于 95% 高湿环境（常在抽气罐底部放一块无菌湿透的纱布）。

2. 冻存菌种复苏与接种步骤

1. 将配置好的优质血平板提前放入微需氧环境内平衡预热（保证平板表面没有冷凝水水滴）。

2. 从液氮罐或 零下 80 摄氏度超低温冰箱中取出 G27 冻存管，由于幽门螺杆菌对温度剧烈波动极度敏感，必须立刻投入 37 摄氏度水浴中快速摇晃融化（1分钟内）。

3. 融化后迅速移入生物安全柜，用无菌移液枪吸取全量菌液（通常含有甘油保护剂），直接倾倒或滴加到血平板浓集区。

4. 轻柔操作：使用无菌涂布棒或无菌接种环，极其轻柔地在平板上进行三区或四区划线（切勿用力过猛划破培养基表面）。

5. 迅速将平板倒置放入微需氧产气罐中，投入新鲜的微需氧产气袋，密封。置于 37 摄氏度恒温培养箱中。

6. 耐心孵育：幽门螺杆菌生长缓慢。复苏第一代通常需要 3 至 5 天（72 到 120 小时）。在满 72 小时前，尽量不要频繁打开密封罐查看，否则大量空气逸入会导致脆弱的对数初期复苏菌株直接氧化死亡。次日或第三天起，可见平板上出现细小的、针尖大（0.5 到 1 毫米）、半透明、水滴状、不溶血的健康微小菌落。

3. 日常传代与活化维持

• 传代时机：当菌落长成清晰可见的水滴状、且尚未融合成大片干枯菌斑时（通常在接种后 48 到 72 小时内），必须进行传代。如果任由菌落老化超过 96 小时，细菌会大量由健康的螺旋状（Spiral/Rod form）自发转变为球状（Coccoid form）。球状菌在镜下呈细碎的小圆点，失去定殖和促使宿主细胞病变的能力，且无法再次传代复苏。

• 传代操作：

  1. 用无菌无抗 PBS 或液体 BHI 肉汤温润血平板。

  2. 使用无菌刮铲或接种环轻轻将平板上的菌落刮下，收集至无菌管内。

  3. 按照 1 比 3 至 1 比 5 的稀释比例，重新接种涂布到新鲜配置的血平板上。

  4. 重新密封放入微需氧环境，后续常规传代通常仅需 48 小时即可长满。

4. 菌株长期冷冻保存

• 冻存液配方：脑心浸液（BHI）肉汤加 20% 到 25% 优质无菌甘油加 10% 胎牛血清（FBS）。

• 冷冻操作：

  1. 收集培养了 48 小时左右、正处于对数生长最旺盛期、镜下观察 95% 以上均为标准短杆状/螺旋状的健康 G27 菌体。

  2. 用无菌刮铲将菌体洗脱并高密度悬浮于上述配置好的专属冻存液中（菌液需要达到极高密度，呈现明显的乳白色浑浊浊度）。

  3. 分装至冷冻管中，禁止梯度降温。为了防止液体中慢速结冰产生的晶体刺破细菌脆弱的双层膜，应将冻存管直接投入 零下 80 摄氏度超低温冰箱中急速深冻，或者直接没入液氮（零下 196 摄氏度）中长期保存。在此温度下，活菌的毒力因子及活性可稳定维持数年以上。

Part 2 English Section

I General Information and Genetic Architecture

• Organism Name: Helicobacter pylori G27 Standard Paradigm Reference Strain.

• Taxonomic Classification: Domain Bacteria, Phylum Pseudomonadota, Class Epsilonproteobacteria, Order Campylobacterales, Family Helicobacteraceae, Genus Helicobacter, Species Helicobacter pylori.

• Strain Background and Origin:G27 is recognized globally as one of the most vital paradigm standard type strains within the Helicobacter pylori clinical research ecosystem. It was originally recovered in the late 1980s to early 1990s by Italian investigators from a gastric mucosal antral biopsy obtained from an endoscopy patient presenting with severe Gastric Ulcer pathology in Italy.

• Genomic Architecture and Virulence Profiles (Critical Background):

  • CagA Positive Matrix (CagA positive): The G27 genome harbors a completely intact, functionally hyper-active cag Pathogenicity Island (cagPAI) matrix. It drives robust transcription of the Cytotoxin-Associated Gene A (CagA) effector protein. CagA functions as a designated bacterial oncoprotein, acting as the primary catalyst driving host gastric epithelial cell oncogenic transformation.

  • VacA Activity (VacA-s1/m1 Allelic Mosaic): Expresses the high-toxicity Vacuolating Cytotoxin A (VacA) genotype (classically mapped to the s1/m1 mosaic structural permutation). This secreted toxin creates vast intracellular vacuoles inside target gastric epithelial sheets, down-regulates mitochondrial membrane potential, and induces host cellular apoptosis cascades.

  • Type IV Secretion System (T4SS): G27 features a fully assembled, rigid T4SS macromolecular syringe structure. Upon physical alignment with host target membranes, the T4SS system penetrates epithelial boundaries to physically translocate CagA, alongside specific cell wall peptidoglycans, directly into the host cell cytoplasm.

• Electrophysiological and Genetic Engineering Advantages:When benchmarked against alternative clinical reference isolates (such as strain 26695), G27 premier evolutionary advantage is its highly robust, stable endogenous capacity for natural transformation (Highly Naturally Transformable). This biological baseline streamlines laboratory gene targeting protocols, including allelic exchange, insertional mutagenesis, targeted locus knockouts, and the introduction of custom fluorescent reporting replicons.

• Biosafety Matrix: Classified under Biosafety Level 2 (BSL-2) containment parameters. Helicobacter pylori is indexed as a Class I Definitive Human Carcinogen; consequently, all continuous vegetative workflows must be handled inside validated Class II Biosafety Cabinets.

II Strategic Research Value and Translational Fields

The G27 line cleanly unifies an aggressive native pathogenic profile with superb genetic tractability, serving as a golden tool to decipher the mechanical baseline of gastric barrier degradation:

1. Host-Pathogen Mechanical Cross-Talk and Morphological Cytoskeletal Remodeling (Hummingbird Phenotype):During in vitro infection screens targeting human gastric carcinoma lines (such as AGS, MKN45), G27 utilizes its T4SS apparatus to micro-inject CagA into the cell cytoplasm. Intracellular CagA is subsequently phosphorylated by host-derived oncogenic tyrosine kinases (such as Src and Abl family elements), setting off physical binding with SHP-2 tyrosine phosphatase. This triggers a dramatic, diagnostic cytoskeletal collapse known as the Hummingbird Phenotype, which is characterized by extreme cellular elongation, continuous filopodia extension, and hyper-activated cell motility. This framework models the genesis of Epithelial-Mesenchymal Transition (EMT) and early carcinogenetic pathways in real time.

2. Type IV Secretion System (T4SS) Structural Assembly and Transport Kinetics:G27 serves as a standard molecular chassis to resolve the fine-scale macromolecular building loops of the T4SS needle core, and to track how specialized loop proteins (such as CagL) align with host cell surface integrin receptors (alpha v beta 1 integrin). Leveraging Cryo-Electron Microscopy (Cryo-EM) or super-resolution fluorescence microscopy captures the structural polarization and transport kinetics of active virulence payloads along the bacteria-host cell junction plane.

3. High-Throughput Screening of Eradication Chemotypes and Target Vaccines:Driven by the steep global escalation in multi-drug resistant (MDR) clinical H. pylori isolates resistant to Clarithromycin and Metronidazole, G27 is integrated worldwide as a sensitive baseline control to discover novel small-molecule bactericidal compounds, identify potent Urease inhibitors, and validate blocking molecules engineered to interrupt essential outer membrane protein (OMP) attachment bridges (such as BabA, SabA adhesins).

III Microaerophilic Cultivation, Thawing, Passaging, and Cryopreservation Routines

Helicobacter pylori is a strict microaerophile. It maintains an exceptionally low tolerance for ambient oxygen tensions, moisture fluctuations, and stale nutrient conditions. Permitting exposure to standard atmospheric oxygen scales or absolute anaerobic conditions causes rapid cell death or forces cell sheets into an irreversible Coccoid Transformation (an encapsulated, morphologically spherical metabolic dormancy state completely stripped of infectious or proliferative capacities).

1. Formulating Growth Matrices and Microaerophilic Structural Calibration

• Solid Agar Matrices (Highly Standardized): Columbia Blood Agar Base or Brain Heart Infusion (BHI) Agar.

  • Mandatory Supplements: Once the post-autoclave basal agar drops to 50 degrees Celsius, investigators must supplement the matrix with 5% to 7% sterile, whole Defibrinated Sheep Blood (or Horse Blood equivalents), or incorporate 10% high-grade Fetal Bovine Serum (FBS). Blood/serum proteins act as critical metabolic scavengers, purging cytotoxic free-radical oxides from the media layout.

  • Selective Multi-Antibiotic Cocktails (Contamination Prevention): Supplement plates with specific H. pylori selective matrices (comprising calibrated volumes of Vancomycin, Polymyxin B, Trimethoprim, and Amphotericin B) to prevent overgrowth by secondary bacterial contaminants.

• Liquid Growth Matrix: Brain Heart Infusion (BHI) broth supplemented with 10% premium FBS (requires continuous orbital micro-agitation).

• Microaerophilic Gas Chemistry (Critical Operational Baseline):

  • Standard Certified Gas Formulation: 5% Oxygen, 10% Carbon Dioxide, and 85% Nitrogen.

  • Laboratory Implementation Routes: Deployed inside dedicated, gas-controlled Microaerophilic Incubators, or by sealing plates within custom gas-tight GasPak Jars/Canisters integrated with microaerophilic gas-generator sachets (such as CampyGen systems).

  • Thermal Window: Set strictly to 37 degrees Celsius (never cultivate at 30 degrees Celsius or 42 degrees Celsius).

  • Relative Environmental Humidity: Maintain greater than 95% continuous saturation (typically secured by placing a sterile, water-saturated gauze pad at the floor of the sealed jar).

2. Cryovial Thawing and Initial Inoculation Protocol

1. Pre-equilibrate premium blood agar plates inside the microaerophilic incubation workspace to ensure the agar matrix reaches 37 degrees Celsius and surface condensation fully desorbs.

2. Extract the G27 cryovial from ultra-low freezers or liquid nitrogen containment. Because H. pylori cells disintegrate under extended thermal transition pathways, submerge the vial instantly inside a 37 degrees Celsius water bath and shake rapidly to complete thawing within 60 seconds.

3. Spray the exterior with 75% ethanol and transfer into the Class II Biosafety Cabinet. Extract the entire slurry via a sterile pipette tip and drop it directly onto the central concentrated zone of the pre-warmed blood agar.

4. Gentle Mechanical Handling: Utilizing a sterile single-use cell spreader or wire loop, smoothly transition the fluid into a classical three- or four-quadrant streak orientation. Do not apply heavy mechanical friction, as tearing the delicate agar surface or shear-stressing the vegetative bacteria disrupts recovery.

5. Instantly invert the inoculated plates, drop them into the target airtight canister, activate a fresh microaerophilic sachet, and secure the sealing mechanisms. Transfer the assembly into the 37 degrees Celsius incubator.

6. Incubation Patience Window: G27 vegetative kinetics develop slowly. The initial post-thaw recovery generation (P1) requires a baseline of 3 to 5 days (72 to 120 hours). Avoid opening the canister to inspect plates prior to the 72-hour threshold, as influxes of atmospheric oxygen will oxidize and kill recovering cells. Tiny, pinpoint-sized (0.5 to 1.0 mm), translucent, water-drop-like non-hemolytic colonies will begin breaking through the agar matrix by day 3 or 4.

3. Subculturing and Continuous Line Passaging

• Confluency and Timing Mandate: Passaging must occur precisely when colonies show distinct dewdrop structures but prior to fusing into dry, flattened macro-crusts (typically within 48 to 72 hours post-inoculation). If allowed to age beyond 96 hours, the culture triggers a massive, autogenous shift from healthy, flagellated Spiral/Rod morphologies into aberrant, metabolic Coccoid spheres. Coccoid forms look like granular point-debris under oil-immersion microscopy, lose cell-adhesion and pathogenicity parameters, and cannot be subcultured or revived.

• Passaging Execution Steps:

  1. Moistened the donor agar plate surface by dripping 1 to 2 mL of sterile, antibiotic-free PBS or plain BHI broth over the colonies.

  2. Utilizing a sterile cell scraper or smooth loop, gently harvest the cell mass from the agar surface, pooling the slurry into a sterile microfuge tube.

  3. Dilute the slurry and inoculate fresh blood agar setups utilizing standard empirical split ratios ranging from 1 to 3 to 1 to 5.

  4. Seal the newly seeded plates back inside the microaerophilic workspace. Established continuous passages expand rapidly, routinely hitting full density within a crisp 48-hour window.

4. Cryopreservation Protocol

• Cryoprotectant Freezing Matrix: High-nutrient Brain Heart Infusion (BHI) broth supplemented with 20% to 25% analytical-grade sterile glycerol and 10% premium Fetal Bovine Serum (FBS).

• Cryovial Preservation Guide:

  1. Exclusively harvest active cultures incubated for approximately 48 hours (late-logarithmic phase), verifying via light microscopy that greater than 95% of the cell sheet retains standard, highly motile helical/short-rod phenotypes.

  2. Wash the vegetative mass from the plates using a sterile scraper and adjust the cell density inside the preservation matrix to a high density (the fluid must present a heavy, milky-white opaque turbidity profile).

  3. Aliquot the dense bacterial suspension into sterile cryovials. Do not implement slow controlled-rate gradient freezing box pathways. To prevent slow ice crystallization from tearing through the delicate dual-membrane actin envelope of the spiral bacteria, plunge the cryovials instantly into a minus 80 degrees Celsius ultra-low freezer or submerge directly within the vapor phase of a liquid nitrogen tank (minus 196 degrees Celsius). Under these deep cryogenic baselines, the viability, genomic integrity, and virulence cascades of G27 remain uncompromised for years.

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