BioVector® pARA13 阿拉伯糖诱导高表达载体 BioVector® pARA13 Arabinose-Inducible Expression Vector

第一部分：中文说明

一、 产品基本信息与详细特征描述

• 产品名称：BioVector® pARA13 阿拉伯糖诱导高表达载体

• 载体名称：BioVector® pARA13

• 质粒类型：原核转录诱导表达载体 (Prokaryotic Inducible Expression Vector)

• 抗性基因：氨苄青霉素抗性基因 (bla / Ampicillin)

• 启动子区域：araBAD 启动子（L-阿拉伯糖启动调控系统）

• 调控基因：araC 转录调节因子基因

• 生物安全级别：1级 (BSL-1)

• 详细特征描述：BioVector® pARA13 是一种基于 L-阿拉伯糖（L-arabinose）诱导系统设计的高效原核细胞表达载体。该载体整合了经典的 araBAD 强启动子及紧邻的 araC 调控基因，构成了极其严密的“开关”式转录调控网络。在缺乏 L-阿拉伯糖的常规培养环境中，调控蛋白 AraC 以二聚体形式结合在操作子位点上，使下游启动子处于高度关闭（Repressed）状态，基础漏表达（Leaky expression）水平极低。这一极其严密的调控特性使其成为克隆、维持和表达那些对大肠杆菌宿主具有高度细胞毒性或可能导致生长抑制的“困难蛋白”的理想选择。当向培养体系中添加 L-阿拉伯糖后，AraC 的构象发生特异性改变，转变为转录激活因子，驱动下游目标异源蛋白发生高效、快速的转录与高水平级联表达。载体还配备了标准的大肠杆菌高拷贝复制子和氨苄青霉素抗性筛选标记，能够保障重组分子在克隆阶段的高效制备。

二、 细胞培养与质粒转录克隆条件

• 克隆与常规扩增条件：进行外源基因片段连接、重组子构建或质粒大量制备时，应选用常规大肠杆菌克隆宿主（如 DH5α 或 Top10）。使用无菌配制的 BioVector® LB 液体培养基或 LB 固体琼脂平板，培养基中必须添加终浓度为 100 微克每毫升的 BioVector® 氨苄青霉素。置于 37°C 恒温振荡培养箱中，以每分钟 200 到 220 转的转速孵育过夜（约 14 至 16 小时）。

• 蛋白诱导表达宿主选择：由于 araBAD 系统的调控特性受细胞内阿拉伯糖转运和代谢的影响，为了获得均匀、可控且呈线性剂量效应的表达活性，强烈建议选用内源阿拉伯糖转运/代谢途径被改造缺陷的大肠杆菌作为表达宿主菌株（例如：araBAD 缺陷型的宿主株如 Top10，或能够实现单细胞精密调控的表达宿主株）。

三、 目标蛋白的诱导表达操作步骤

1. 种子液扩增：挑取经测序验证正确的 BioVector® pARA13 重组表达菌落，接种至含有 100 微克每毫升氨苄青霉素的 BioVector® LB 液体培养基中，在 37°C 下振荡培养过夜，作为种子菌液。

2. 扩大培养：按照 1 比 100 的无菌体积比例将种子菌液转接至新鲜的、含有同等浓度氨苄青霉素的完全培养基中。置于 37°C、每分钟 220 转的振荡培养箱中进行扩大培养，定期测量菌液的吸光度。

3. 诱导前测定：当培养物中的菌体生长至对数生长中期、即 OD600 的数值达到 0.5 到 0.7 之间时，即可开启诱导。在加入诱导剂之前，无菌吸取 1 毫升培养物离心收集菌体，标记为“诱导前对照组”。

4. 添加阿拉伯糖诱导：向剩余的培养体系中加入无菌的 BioVector® L-阿拉伯糖溶液。标准的起始诱导终浓度通常设定在 0.002% 到 0.2% 之间。需要注意的是，通过改变 L-阿拉伯糖的添加浓度，可在极广的范围内精确且按比例调节目标蛋白的最终表达产率。

5. 条件优化与终止收获：加入诱导剂后，通常需将培养箱的温度下调至 20°C 至 30°C（降低温度有助于提高部分异源蛋白的折叠效率，减少包涵体形成），并在低速（如每分钟 160 转）下持续震荡孵育 4 至 6 小时或过夜。诱导结束后，以每分钟 4000 转的转速离心 15 分钟，彻底收获菌体沉淀，随后通过 SDS-PAGE 电泳分析来评估目的蛋白在总细胞、上清或包涵体中的高产率分布。

四、 质粒及工程菌株的保藏技术

• 表达重组菌种冻存：将通过验证的重组大肠杆菌工程菌株扩增培养至对数生长旺盛期（OD600 达到 0.6 左右且尚未加入诱导剂）。无菌吸取 700 微里菌液与 300 微里无菌的 BioVector® 细胞级甘油（使最终甘油体积百分比浓度为 30%）在无菌冷冻管中充分颠倒混匀。将其直接移入零下 80°C 超低温冰箱中进行长期、稳定的冷冻保藏。

• 高纯度质粒 DNA 储存：使用 BioVector® 高纯度无内毒素质粒提取试剂盒回收重组质粒 DNA。将最终洗脱的 DNA 溶解于无菌的 BioVector® TE 缓冲液（pH 8.0）中。准确测定其吸光度与物理浓度后进行分装，保存在零下 20°C 环境中。严禁反复、频繁地进行冻融循环。

五、 质量控制标准与科研应用指南

• 质量控制标准：BioVector® pARA13 载体经过极其严苛的纯化与全长序列验证。通过高通量 Sanger 测序确认 araBAD 启动子区、araC 基因序列及多克隆位点无任何自发性错义突变；经酶切鉴定物理电泳图谱与设计图谱完全契合；产品经检测不含宿主基因组 DNA 残留、无外源 DNase/RNase 酶污染，且其细菌内毒素含量控制在极低水平。

• 核心实验应用方向：该诱导载体主要用于大肠杆菌高密度发酵中异源重组蛋白的工业级制备、对宿主细胞具有强毒性的致死性毒素或小分子肽类的受控克隆表达、多亚基复杂蛋白质复合体的多质粒协同微调共表达，以及细菌代谢通路中速率限制酶活性的体内定量调控和滴定实验。

PART 2: ENGLISH SECTION

I. General Information and Detailed Product Characterization

• Product Name: BioVector® pARA13 Arabinose-Inducible Expression Vector

• Vector Name: BioVector® pARA13

• Plasmid Type: Prokaryotic Inducible Expression Vector

• Selection Marker: Ampicillin resistance gene (bla / Ampicillin)

• Promoter System: araBAD Promoter (L-arabinose induction regulatory mechanism)

• Regulatory Element: araC transcriptional regulator gene

• Biosafety Level: BSL-1

• Detailed Description: BioVector® pNV18 is an exceptional and highly strict prokaryotic expression vector utilizing the L-arabinose-inducible system for high-yield recombinant protein synthesis in E. coli. The foundational architecture of this plasmid comprises the robust araBAD promoter nested directly downstream of the araC regulatory gene, establishing an incredibly tight regulatory feedback loop. In the absence of exogenous L-arabinose, the AraC regulatory protein forms a homodimer that binds tightly to operator sequences, placing the downstream expression cassette under maximum repression and ensuring virtually undetectable basal leaky expression. This remarkable stringency makes the vector an unparalleled option for cloning, maintaining, and expressing highly toxic or growth-inhibitory "difficult" proteins that would otherwise jeopardize host viability. Upon the addition of L-arabinose to the culture system, a conformational change switches AraC into a transcriptional activator, initiating robust, synchronized transcription and continuous high-level cascade translation of the heterologous gene. Complete with a reliable high-copy E. coli replicon and an Ampicillin selection marker, pARA13 ensures high efficiency during standard molecular cloning and propagation assays.

II. Culture Conditions and Cloning Parameters

• Propagation and Routine Cloning Parameters: For standard plasmid expansion, insert ligations, or large-scale plasmid purifications, the recombinant molecule must be transformed into standard E. coli cloning strains (such as DH5α or Top10). Cells are selected and propagated using sterile BioVector® LB Liquid Medium or LB Agar plates supplemented with BioVector® Ampicillin at a final working concentration of 100 micrograms per milliliter. Cultivate overnight (approximately 14 to 16 hours) inside a temperature-controlled shaking incubator at 37°C with an agitation speed of 200 to 220 RPM.

• Expression Host Strain Recommendations: Because the regulatory behavior of the araBAD promoter system is linked to intracellular arabinose transport and accumulation, achieving linear, dosage-dependent, and homogenous single-cell expression profiles requires precise host selection. It is highly recommended to perform protein induction assays within E. coli host backgrounds that are genetically deficient in the native arabinose degradation pathways (e.g., araBAD deficient strains like Top10, or customized expression strains configured for linear rheostat-like induction control).

III. Standardized Protein Induction and Harvesting Protocol

1. Starter Culture Expansion: Inoculate a sequence-verified single colony of E. coli harboring the BioVector® pARA13 recombinant vector into sterile BioVector® LB Liquid Medium supplemented with 100 micrograms per milliliter of ampicillin. Incubate at 37°C overnight with vigorous shaking to prepare the starter culture.

2. Large-Scale Subculturing: Dilute the overnight starter culture 1:100 into fresh complete medium containing the same selection pressure. Place the vessels inside a 37°C incubator shaking at 220 RPM and closely monitor cellular proliferation via optical density tracking.

3. Pre-Induction Assessment: When the growing culture reaches its mid-logarithmic developmental window—specifically spanning an OD600 value between 0.5 and 0.7—the culture is optimized for transcript induction. Prior to adding the inducer, harvest a 1-milliliter aliquot of the culture, centrifuge down the cells, and archive it as the "uninduced control sample."

4. L-Arabinose Transcript Induction: Supplement the remaining culture with sterile BioVector® L-Arabinose Solution. The standard starting final concentration can be adjusted dynamically from 0.002% up to 0.2%. Notably, altering the concentration of L-arabinose added enables precise, rheostat-like modulation of target protein output over a wide range.

5. Condition Optimization and Biomass Harvesting: Following inducer addition, it is frequently advantageous to decrease the cultivation temperature down to 20°C–30°C (cooler temperatures frequently optimize correct tertiary protein folding and reduce the formation of insoluble inclusion bodies) while agitating at a reduced speed of 160 RPM for 4 to 6 hours or overnight. Terminate incubation by executing a centrifugation step at 4000 RPM for 15 minutes to gather the cell pellet. Evaluate protein yield and distribution (soluble fraction vs. inclusion bodies) via standard SDS-PAGE analysis.

IV. Plasmid Preservation and Long-Term Storage Methodology

• Glycerol Stock Preservation of Host Strains: To preserve validated recombinant expression strains, cultivate the host cells into their active logarithmic growth phase (OD600 around 0.6, making sure no inducer is present). Thoroughly blend 700 microliters of the fresh bacterial culture with 300 microliters of sterile, BioVector® Cell-Grade Glycerol to achieve a final concentration of 30% glycerol inside a sterile cryovial. Store the archived specimens directly inside a minus 80°C ultra-low temperature freezer.

• Purified Plasmid DNA Archiving: Extract the engineered plasmid construct using a BioVector® High-Purity Endotoxin-Free Plasmid Extraction Kit. Elute the final DNA product in sterile BioVector® TE Buffer (pH 8.0). Measure the exact concentration via spectrophotometry, fractionate into single-use aliquots, and store them securely at minus 20°C. Strictly avoid subjecting the DNA to repeated freeze-thaw cycles.

V. Quality Control and Research Application Guidelines

• Quality Control Standards: The BioVector® pARA13 vector undergoes rigorous biochemical validation. Full-length Sanger sequencing certifies zero spontaneous mutations across the araBAD regulatory locus, the araC repressor sequence, and the multiple cloning site (MCS). Structural fingerprinting via restriction digestion shows 100% alignment with theoretical plasmid maps. The reagent is certified free from host genomic DNA background, active nucleases (DNase/RNase), and contains negligible bacterial endotoxin levels.

• Core Experimental Applications: This inducible vector system is heavily utilized for industrial-scale recombinant protein manufacturing during high-density fermentation, controlled expression of lethal proteins or small toxic peptides that undermine host cell viability, multi-plasmid fine-tuning for the stoichiometric co-expression of multi-subunit protein complexes, and the in vivo quantitative titration of rate-limiting enzymes within engineered metabolic pathways.

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