BioVector? pARA13 阿拉伯糖誘導高表達載體 BioVector? pARA13 Arabinose-Inducible Expression Vector

第一部分：中文說明

一、 產(chǎn)品基本信息與詳細特征描述

• 產(chǎn)品名稱：BioVector? pARA13 阿拉伯糖誘導高表達載體

• 載體名稱：BioVector? pARA13

• 質(zhì)粒類型：原核轉(zhuǎn)錄誘導表達載體 (Prokaryotic Inducible Expression Vector)

• 抗性基因：氨芐青霉素抗性基因 (bla / Ampicillin)

• 啟動子區(qū)域：araBAD 啟動子（L-阿拉伯糖啟動調(diào)控系統(tǒng)）

• 調(diào)控基因：araC 轉(zhuǎn)錄調(diào)節(jié)因子基因

• 生物安全級別：1級 (BSL-1)

• 詳細特征描述：BioVector? pARA13 是一種基于 L-阿拉伯糖（L-arabinose）誘導系統(tǒng)設(shè)計的高效原核細胞表達載體。該載體整合了經(jīng)典的 araBAD 強啟動子及緊鄰的 araC 調(diào)控基因，構(gòu)成了極其嚴密的“開關(guān)”式轉(zhuǎn)錄調(diào)控網(wǎng)絡(luò)。在缺乏 L-阿拉伯糖的常規(guī)培養(yǎng)環(huán)境中，調(diào)控蛋白 AraC 以二聚體形式結(jié)合在操作子位點上，使下游啟動子處于高度關(guān)閉（Repressed）狀態(tài)，基礎(chǔ)漏表達（Leaky expression）水平極低。這一極其嚴密的調(diào)控特性使其成為克隆、維持和表達那些對大腸桿菌宿主具有高度細胞毒性或可能導致生長抑制的“困難蛋白”的理想選擇。當向培養(yǎng)體系中添加 L-阿拉伯糖后，AraC 的構(gòu)象發(fā)生特異性改變，轉(zhuǎn)變?yōu)檗D(zhuǎn)錄激活因子，驅(qū)動下游目標異源蛋白發(fā)生高效、快速的轉(zhuǎn)錄與高水平級聯(lián)表達。載體還配備了標準的大腸桿菌高拷貝復制子和氨芐青霉素抗性篩選標記，能夠保障重組分子在克隆階段的高效制備。

二、 細胞培養(yǎng)與質(zhì)粒轉(zhuǎn)錄克隆條件

• 克隆與常規(guī)擴增條件：進行外源基因片段連接、重組子構(gòu)建或質(zhì)粒大量制備時，應選用常規(guī)大腸桿菌克隆宿主（如 DH5α 或 Top10）。使用無菌配制的 BioVector? LB 液體培養(yǎng)基或 LB 固體瓊脂平板，培養(yǎng)基中必須添加終濃度為 100 微克每毫升的 BioVector? 氨芐青霉素。置于 37°C 恒溫振蕩培養(yǎng)箱中，以每分鐘 200 到 220 轉(zhuǎn)的轉(zhuǎn)速孵育過夜（約 14 至 16 小時）。

• 蛋白誘導表達宿主選擇：由于 araBAD 系統(tǒng)的調(diào)控特性受細胞內(nèi)阿拉伯糖轉(zhuǎn)運和代謝的影響，為了獲得均勻、可控且呈線性劑量效應的表達活性，強烈建議選用內(nèi)源阿拉伯糖轉(zhuǎn)運/代謝途徑被改造缺陷的大腸桿菌作為表達宿主菌株（例如：araBAD 缺陷型的宿主株如 Top10，或能夠?qū)崿F(xiàn)單細胞精密調(diào)控的表達宿主株）。

三、 目標蛋白的誘導表達操作步驟

1. 種子液擴增：挑取經(jīng)測序驗證正確的 BioVector? pARA13 重組表達菌落，接種至含有 100 微克每毫升氨芐青霉素的 BioVector? LB 液體培養(yǎng)基中，在 37°C 下振蕩培養(yǎng)過夜，作為種子菌液。

2. 擴大培養(yǎng)：按照 1 比 100 的無菌體積比例將種子菌液轉(zhuǎn)接至新鮮的、含有同等濃度氨芐青霉素的完全培養(yǎng)基中。置于 37°C、每分鐘 220 轉(zhuǎn)的振蕩培養(yǎng)箱中進行擴大培養(yǎng)，定期測量菌液的吸光度。

3. 誘導前測定：當培養(yǎng)物中的菌體生長至對數(shù)生長中期、即 OD600 的數(shù)值達到 0.5 到 0.7 之間時，即可開啟誘導。在加入誘導劑之前，無菌吸取 1 毫升培養(yǎng)物離心收集菌體，標記為“誘導前對照組”。

4. 添加阿拉伯糖誘導：向剩余的培養(yǎng)體系中加入無菌的 BioVector? L-阿拉伯糖溶液。標準的起始誘導終濃度通常設(shè)定在 0.002% 到 0.2% 之間。需要注意的是，通過改變 L-阿拉伯糖的添加濃度，可在極廣的范圍內(nèi)精確且按比例調(diào)節(jié)目標蛋白的最終表達產(chǎn)率。

5. 條件優(yōu)化與終止收獲：加入誘導劑后，通常需將培養(yǎng)箱的溫度下調(diào)至 20°C 至 30°C（降低溫度有助于提高部分異源蛋白的折疊效率，減少包涵體形成），并在低速（如每分鐘 160 轉(zhuǎn)）下持續(xù)震蕩孵育 4 至 6 小時或過夜。誘導結(jié)束后，以每分鐘 4000 轉(zhuǎn)的轉(zhuǎn)速離心 15 分鐘，徹底收獲菌體沉淀，隨后通過 SDS-PAGE 電泳分析來評估目的蛋白在總細胞、上清或包涵體中的高產(chǎn)率分布。

四、 質(zhì)粒及工程菌株的保藏技術(shù)

• 表達重組菌種凍存：將通過驗證的重組大腸桿菌工程菌株擴增培養(yǎng)至對數(shù)生長旺盛期（OD600 達到 0.6 左右且尚未加入誘導劑）。無菌吸取 700 微里菌液與 300 微里無菌的 BioVector? 細胞級甘油（使最終甘油體積百分比濃度為 30%）在無菌冷凍管中充分顛倒混勻。將其直接移入零下 80°C 超低溫冰箱中進行長期、穩(wěn)定的冷凍保藏。

• 高純度質(zhì)粒 DNA 儲存：使用 BioVector? 高純度無內(nèi)毒素質(zhì)粒提取試劑盒回收重組質(zhì)粒 DNA。將最終洗脫的 DNA 溶解于無菌的 BioVector? TE 緩沖液（pH 8.0）中。準確測定其吸光度與物理濃度后進行分裝，保存在零下 20°C 環(huán)境中。嚴禁反復、頻繁地進行凍融循環(huán)。

五、 質(zhì)量控制標準與科研應用指南

• 質(zhì)量控制標準：BioVector? pARA13 載體經(jīng)過極其嚴苛的純化與全長序列驗證。通過高通量 Sanger 測序確認 araBAD 啟動子區(qū)、araC 基因序列及多克隆位點無任何自發(fā)性錯義突變；經(jīng)酶切鑒定物理電泳圖譜與設(shè)計圖譜完全契合；產(chǎn)品經(jīng)檢測不含宿主基因組 DNA 殘留、無外源 DNase/RNase 酶污染，且其細菌內(nèi)毒素含量控制在極低水平。

• 核心實驗應用方向：該誘導載體主要用于大腸桿菌高密度發(fā)酵中異源重組蛋白的工業(yè)級制備、對宿主細胞具有強毒性的致死性毒素或小分子肽類的受控克隆表達、多亞基復雜蛋白質(zhì)復合體的多質(zhì)粒協(xié)同微調(diào)共表達，以及細菌代謝通路中速率限制酶活性的體內(nèi)定量調(diào)控和滴定實驗。

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.

BioVector NTCC質(zhì)粒載體菌株細胞蛋白抗體基因保藏中心

電話:400-800-2947

工作QQ/微信同號:1843439339

網(wǎng)址

http://www.nedfriskphoto.com