BioVector? 胞內(nèi)勞森菌標(biāo)準(zhǔn)菌株說明書

BioVector? Lawsonia intracellularis Standard Strain Manual

第一部分 中文說明

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 菌株名稱：胞內(nèi)勞森菌（Lawsonia intracellularis）。

• 物種分類：細(xì)菌界（Bacteria），變形菌門（Pseudomonadota），$\delta$-變形菌綱（Deltaproteobacteria），脫硫振子菌目（Desulfovibrionales），脫硫振子菌科（Desulfovibrionaceae），勞森菌屬（Lawsonia）。

• 核心生物學(xué)與生理學(xué)特征：

  • 專性胞內(nèi)寄生菌（Obligate Intracellular Bacterium）：胞內(nèi)勞森菌是一種革蘭氏陰性（Gram-negative）、彎曲或微彎的微需氧彎曲桿菌。該菌具有極度苛刻的寄生特性，在宿主細(xì)胞外完全無法生長和繁殖。在體內(nèi)，它特異性地寄生在哺乳動(dòng)物（尤其是豬和馬）的小腸（回腸）隱窩上皮細(xì)胞的細(xì)胞質(zhì)內(nèi)。

  • 增殖性腸?。≒PE）的唯一病原體：它是引發(fā)豬增殖性腸病（Porcine Proliferative Enteropathy, PPE，俗稱“回腸炎”）的特異性病原體，臨床表現(xiàn)為腸壁上皮細(xì)胞惡性增生、增厚，導(dǎo)致嚴(yán)重的出血性或壞死性腸炎。

• 來源歷史：通常分離自患有急性或慢性回腸炎的病豬回腸黏膜病變組織。通過將其接種于體外培養(yǎng)的大鼠小腸上皮細(xì)胞（IEC-18）或人類結(jié)腸癌細(xì)胞（McCoy細(xì)胞）中，在微需氧條件下進(jìn)行特殊的分離與傳代維持。

• 生物安全級(jí)別：2級(jí)（BSL-2）。涉及活菌擴(kuò)增及受感染細(xì)胞系的操作，必須在二級(jí)生物安全柜內(nèi)嚴(yán)格按照厭氧/微需氧胞內(nèi)菌規(guī)范執(zhí)行。

二 核心科研價(jià)值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

胞內(nèi)勞森菌是獸醫(yī)學(xué)、比較醫(yī)學(xué)及腸道黏膜免疫學(xué)研究中公認(rèn)極難培養(yǎng)但危害巨大的標(biāo)桿性病原體：

1. 豬回腸炎發(fā)病機(jī)制與腸道增生研究：用于深入探究該菌如何通過特定分泌系統(tǒng)（如三型分泌系統(tǒng) T3SS 或相關(guān)效應(yīng)蛋白）注入宿主細(xì)胞，如何激活 Wnt/beta-catenin 等信號(hào)通路進(jìn)而阻斷小腸隱窩上皮細(xì)胞的分化，并異常驅(qū)動(dòng)其進(jìn)行不受控制的惡性增殖。

2. 獸用疫苗與抗生素藥物篩選（Vaccine & Drug Screening）：是評(píng)價(jià)針對(duì) PPE 的新型滅活疫苗、亞單位疫苗及活疫苗效能的關(guān)鍵抗原株；同時(shí)用于體外篩選對(duì)胞內(nèi)菌敏感的抗生素（如泰樂菌素、泰萬菌素、渥尼妙林等）的胞內(nèi)殺菌動(dòng)力學(xué)。

3. 分子診斷質(zhì)控物（Diagnostics & Quality Control）：作為官方標(biāo)準(zhǔn)的參考品，廣泛用于開發(fā)和校準(zhǔn)檢測豬/馬回腸炎的常規(guī) PCR、實(shí)時(shí)熒光定量 qPCR 試劑盒、原位雜交（ISH）探針以及血清學(xué) ELISA 診斷芯片。

三 實(shí)驗(yàn)室菌株復(fù)蘇、細(xì)胞協(xié)同培養(yǎng)與冷凍保存標(biāo)準(zhǔn)步驟

由于胞內(nèi)勞森菌是專性胞內(nèi)寄生菌，嚴(yán)禁將其接種于任何普通細(xì)菌培養(yǎng)基（如血平板、LB肉湯等）上。復(fù)蘇和傳代必須依賴活的宿主細(xì)胞系。

1. 宿主細(xì)胞準(zhǔn)備與微需氧環(huán)境配置

• 推薦宿主細(xì)胞：大鼠小腸上皮細(xì)胞（IEC-18, ATCC CRL-1589）或 McCoy 細(xì)胞（ATCC CRL-1696）。

• 細(xì)胞培養(yǎng)基：含 10% 優(yōu)質(zhì)胎牛血清（FBS）的高糖 DMEM 或 McCoy's 5A 培養(yǎng)基。

• 核心微需氧參數(shù)（關(guān)鍵）：胞內(nèi)勞森菌的生長對(duì)氧氣濃度極度敏感?；旌吓囵B(yǎng)必須置于特殊的微需氧環(huán)境（通常為：8.0% - 10.0% $O_2$、8.0% - 10.0% $CO_2$，其余為 $N_2$）中進(jìn)行，普通 5% $CO_2$ 三氣孵箱或完全嚴(yán)格厭氧環(huán)境均會(huì)導(dǎo)致細(xì)菌死亡。

2. 凍存菌種的復(fù)蘇與細(xì)胞接種（Thawing & Inoculation）

1. 提前一天將 IEC-18 或 McCoy 細(xì)胞接種于 T25 培養(yǎng)瓶中，使其在復(fù)蘇當(dāng)天達(dá)到約 30% - 50% 的低融合度（此時(shí)細(xì)胞處于旺盛分裂期，最有利于勞森菌的入侵與體內(nèi)繁殖）。

2. 從液氮中取出胞內(nèi)勞森菌的裂解凍存管，立即投入 37 攝氏度水浴中急速融化。

3. 消毒凍存管外部，移入生物安全柜。將菌液吸出，直接加入到長有低融合度宿主細(xì)胞的培養(yǎng)瓶中。

4. 迅速轉(zhuǎn)入 37 攝氏度專用微需氧孵箱中孵育 3 至 5 小時(shí)（或過夜），以利于細(xì)菌充分吸附并侵入細(xì)胞。

5. 次日輕輕吸除含有殘余凍存液的舊培養(yǎng)基，更換為新鮮的細(xì)胞培養(yǎng)基，重新送回微需氧孵箱中連續(xù)培養(yǎng) 5 至 7 天。

3. 菌株傳代與細(xì)菌釋放（Passaging & Harvesting）

胞內(nèi)勞森菌在胞內(nèi)過度繁殖會(huì)導(dǎo)致宿主細(xì)胞裂解并釋放。日常傳代需要人工輔助將其從宿主細(xì)胞中釋放出來：

• 細(xì)胞裂解釋放法：

  1. 當(dāng)培養(yǎng)至第 6-7 天時(shí)，吸除舊培養(yǎng)基。加入無菌去離子水或低滲 KCl 溶液（0.1%），在 37 攝氏度下孵育 10 分鐘使宿主細(xì)胞溶脹。

  2. 使用移液管對(duì)瓶壁進(jìn)行劇烈吹打，或者通過無菌注射器針頭（如 21G 針頭）來回抽吸 3-5 次，依靠機(jī)械剪切力徹底破碎宿主細(xì)胞，釋放出胞內(nèi)的勞森菌。

  3. 將裂解液在 500 × g 離心 3 分鐘以沉淀未破碎的細(xì)胞碎片；收集含有細(xì)菌的上清液。

• 接種傳代：將收集到的細(xì)菌上清液，按 1:3 至 1:5 的比例直接加到新準(zhǔn)備好的低融合度（30%）宿主細(xì)胞平板/瓶中，繼續(xù)置于微需氧環(huán)境培養(yǎng)。

4. 菌株長期冷凍保存（Cryopreservation）

• 凍存保護(hù)液：常規(guī)細(xì)胞凍存液（90% FBS + 10% DMSO）或者特殊的蔗糖-磷酸鹽-谷氨酸緩沖液（SPG）補(bǔ)充 10% DMSO。

• 凍存操作：

  1. 收集感染率極高（細(xì)胞內(nèi)充滿密密麻麻菌體）的混合培養(yǎng)物。

  2. 可選擇直接消化收集帶有細(xì)菌的完整宿主細(xì)胞，或者通過上述裂解法收集游離菌體。

  3. 加入凍存液重懸后，迅速分裝入凍存管，立即投入標(biāo)準(zhǔn)程序降溫盒中。

  4. 置于 -80 攝氏度過夜后，次日必須迅速轉(zhuǎn)移至 -196 攝氏度液氮罐 中進(jìn)行長期冷凍保存。

Part 2 English Section

I General Information and Genetic Architecture

• Organism Name: Lawsonia intracellularis Reference Strain.

• Taxonomic Classification: Domain Bacteria, Phylum Pseudomonadota, Class Deltaproteobacteria, Order Desulfovibrionales, Family Desulfovibrionaceae, Genus Lawsonia, Species Lawsonia intracellularis.

• Core Biological and Physiological Profiles:

  • Obligate Intracellular Bacterium: Lawsonia intracellularis is a Gram-negative, curved, or microaerophilic rod-shaped bacterium. This pathogen possesses a unique physiological property: it is completely unable to replicate outside of a supportive eukaryotic host cell. In vivo, it specifically targets and colonizes the cytoplasm of actively dividing intestinal (ileal) crypt epithelial cells of mammalian species, primarily pigs and horses.

  • Etiological Agent of PPE: It stands as the definitive causative pathogen of Porcine Proliferative Enteropathy (PPE, commonly referred to as ileitis). PPE is characterized by abnormal hyperplastic proliferation of infected enterocytes, resulting in a marked thickening of the intestinal wall, causing severe hemorrhagic or necrotic enteritis.

• Source Isolation Provenance: Typically recovered from the diseased ileal mucosa mucosa harvested from pigs exhibiting acute or chronic porcine proliferative enteropathy. Cultivation and maintenance are achieved by co-culturing the bacterium inside rat small intestinal epithelial cell matrices (IEC-18) or human colon cancer lines (McCoy cells) under custom microaerophilic conditions.

• Biosafety Matrix: Classified under Biosafety Level 2 (BSL-2) parameters. Any workflows involving active cellular infection matrices require certified Class II biosafety containment frameworks and microaerophilic atmospheric gas systems.

II Strategic Research Value and Translational Fields

Lawsonia intracellularis is universally recognized as one of the most fastidious yet economically devastating pathogens within veterinary medicine and mucosal gastroenterology:

1. Mechanisms of Proliferative Enteropathy & Cell-Cycle Subversion: Deployed to map how its dedicated secretion apparatus (such as Type III Secretion Systems [T3SS] or related effector loops) alters eukaryotic homeostatic machinery, specifically up-regulating pathways like Wnt/beta-catenin to arrest normal enterocyte differentiation and force host cell hyperproliferation.

2. Veterinary Vaccine Development and Antimicrobial Profiling: Serves as the index target for verifying the structural potency and protective parameters of novel inactivated, subunit, or modified live vaccines targeting PPE. It is also used to benchmark the intracellular bactericidal kinetics of targeted macrolides and pleuromutilins (e.g., tylosin, tylvalosin, valnemulin).

3. Molecular Diagnostic Standard Verification: Acts as the official positive metrology baseline to optimize and validate clinical qualitative/quantitative PCR assays (qPCR), in vitro immunohistochemistry setups, in situ hybridization (ISH) diagnostic probes, and serological enzyme-linked immunosorbent assay (ELISA) monitoring kits.

III Thawing, Cell Co-Culture, Passaging, and Cryopreservation Routines

Because Lawsonia intracellularis is an obligate intracellular pathogen, never plate this bacterium onto standard cell-free agar formulas (such as blood agar, nutrient agar, or LB broth). Revitalization requires viable eukaryotic host cellular matrices.

1. Host Cell Configuration and Ambient Microaerophilic Parameters

• Recommended Host Cell Lines: Rat intestinal epithelial cells (IEC-18, ATCC CRL-1589) or McCoy cells (ATCC CRL-1696).

• Cultivation Media: High-glucose DMEM or McCoy's 5A matrix, supplemented with 10% premium Fetal Bovine Serum (FBS).

• Critical Atmospheric Gas Balance (Mandatory): Proliferation dictates narrow microaerophilic ranges. Co-cultures must be incubated within specialized microaerophilic environments (optimized at 8.0%–10.0% $O_2$, 8.0%–10.0% $CO_2$, with the balance configured as $N_2$). Standard 5% $CO_2$ incubators or absolute anaerobic configurations will rapidly cause irreversible loss of bacterial viability.

2. Cryovial Thawing and Cellular Infection Protocol

1. One day prior to thawing, seed the target host cells (IEC-18 or McCoy) into standard T25 flasks to establish a low confluency of approximately 30%–50% on the day of infection. (Dividing enterocytes provide the optimal targets for bacterial endocytosis and intracellular division).

2. Retrieve the Lawsonia intracellularis frozen lysate from liquid nitrogen storage and submerge it instantly into a 37 degree Celsius water bath. Thaw rapidly within 1 minute.

3. Swab the vial with 70% ethanol, transit to a biosafety cabinet, and transfer the thawed bacterial suspension directly into the flask containing the low-confluency host cell layer.

4. Promptly return the infected flask to the 37 degree Celsius microaerophilic incubator for 3 to 5 hours (or overnight) to optimize bacterial attachment and cell entry kinetics.

5. The following day, gently aspirate the media containing residual cryoprotectant elements, replenish with fresh cell culture media, and return to the microaerophilic workspace for 5 to 7 days of continuous incubation.

3. Subculturing and Mechanical Bacterial Release

Overwhelming intracellular bacterial loads eventually lead to spontaneous cell lysis. To achieve efficient passaging, manual harvesting is required to extract intact bacteria from intact cells:

• Hypotonic Mechanical Lysis Protocol:

  1. At day 6–7 post-infection, aspirate the spent growth medium. Introduce sterile deionized water or a hypotonic KCl solution (0.1%) and incubate at 37 degree Celsius for 10 minutes to induce host cell swelling.

  2. Aggressively pipette the fluid against the flask baseline or pass the slurry back and forth through a sterile 21-gauge syringe needle 3–5 times. The resulting mechanical shear forces disrupt the weakened host membranes, releasing intact bacteria.

  3. Centrifuge the crude lysate at 500 × g for 3 minutes to sediment large cellular debris; carefully collect the bacteria-rich supernatant fluid.

• Inoculation Routine: Blend the collected bacterial supernatant directly into fresh flasks pre-seeded with 30% confluent host cells, and store under microaerophilic parameters.

4. Cryopreservation Protocol

• Freezing Medium Matrix: Standard cell cryopreservation matrix (90% FBS + 10% DMSO) or a specialized sucrose-phosphate-glutamate (SPG) transport buffer fortified with 10% analytical-grade DMSO.

• Freezing Routine: Harvest heavily infected cultures showing peak intracellular densities. Researchers can freeze down the intact host cells carrying the bacteria, or execute the hypotonic mechanical lysis routine detailed above to freeze isolated bacterial fractions. Resuspend the pellet in freezing media, aliquot into cryovials, and place immediately inside an isopropyl alcohol controlled-rate freezing box. Keep at -80 degree Celsius overnight, and shift into the liquid nitrogen vapor phase (-196 degree Celsius) the next day for long-term preservation.

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