BioVector® pSP108 Standard In Vitro Transcription Vector / pSP108 标准体外转录与克隆非表达型质粒载体

一 产品基本信息与分子生物学背景

• 载体名称：pSP108。

• 克隆质粒分类：体外转录质粒、克隆质粒（无哺乳动物或原核细胞表达启动子，属于非表达型工程质粒）。

• 质粒大小：约 3.0 - 4.0 kb（具体因不同衍生亚型和多克隆位点微调而异）。

• 核心骨架源起与设计背景：pSP108 是分子生物学中非常经典的体外转录（In vitro transcription, IVT）专用质粒骨架。它的核心构造是基于经典的 pUC 或 pSP 系列质粒优化而来。该载体不具备用于在活细胞内（如大肠杆菌、大鼠、人类细胞）转录翻译蛋白质的 CMV、SV40 或 lac 驱动性表达启动子，而是专门为了高效率地在体外无细胞体系中合成高纯度、高丰度的 RNA 片段而设计。

• 核心顺式作用元件与图谱特征：

  • 噬菌体 RNA 聚合酶启动子（Phage RNA Polymerase Promoter）：pSP108 骨架中紧邻多克隆位点（MCS）的上方，核心配置了高特异性的 SP6 噬菌体启动子（部分双向转录变亚型会在另一端配置 T7 或 T3 启动子）。SP6 RNA 聚合酶对该段顺式元件的识别特异性接近 100%，能够在体外酶促反应体系中极快地启动顺式单链 RNA 的级联延伸。

  • 多克隆位点（Multiple Cloning Site, MCS）：紧随 SP6 启动子下游，包含一系列独特的内切酶切位点（如 EcoRI, SmaI, BamHI, SalI, PstI, HindIII 等）。这些位点被致密而合理地排布，方便研究人员将目标基因或非编码 RNA 模板精确地定向克隆插入。

  • 原核复制子（Prokaryotic Replicon）：配置了高拷贝的 pUC ori，确保该质粒在大肠杆菌（E. coli）感受态细胞中能够实现极其凶猛的自主增殖与扩增，日常质粒抽提产量极高。

  • 抗性选择标记（Selection Marker）：携带氨苄青霉素抗性基因（Ampicillin resistance, $Amp^R$ / bla），用于在原核平板筛选和液体扩增中维持阳性克隆。

二 核心科研价值与体外实验应用

pSP108 质粒虽不能在活细胞内表达蛋白，但在RNA生物学、结构生物学及分子杂交等领域扮演着极核心的分子靶底角色：

1. 反义/正义单链 RNA 探针（RNA Probes）的高效体外制备：通过将特定基因片段克隆入 pSP108 的 MCS 中，利用单酶切将质粒进行末端线性化（Linearization），随后以线性化 DNA 为模板，加入 SP6 RNA 聚合酶以及带有同位素（如 $^{32}P$）或非同位素标记（如地高辛 Digoxigenin, 生物素 Biotin）的 NTPs。可在体外稳定、大量地合成出极高质量的单链 RNA 探针。这些探针被广泛用于原位杂交（In situ Hybridization, ISH）、Northern Blot 分子杂交以及核酸酶保护实验（RPA）。

2. 体外翻译（In vitro Translation）模板构建：转录出的高纯度 RNA，在体外经过加帽（Capping）和加尾（Poly-A tailing）酶促修饰后，可直接投入麦胚提取物（Wheat Germ Extract）或兔网织红细胞裂解液（Rabbit Reticulocyte Lysate）等无细胞翻译体系中。这被用于在完全不依赖活细胞的环境下，精准解构蛋白质的早期折叠、翻译起始调控及新型小分子药物对翻译链延伸的干预阻断。

3. RNA 二级结构、RNA-蛋白相互作用（RBP）探讨：用于在体外转录出特定的非编码 RNA（ncRNA）、微小 RNA（miRNA）前体或特定具有空间构象的 RNA 片段。这些高质量 RNA 可直接用于 RNA 电泳迁移率变动分析（REMSA/EMSA）、RNA Pull-down 实验或冷冻电镜（Cryo-EM）结构解构，用以捕捉并鉴定与之结合的各种靶标RNA结合蛋白（RBPs）。

三 实验室大肠杆菌转化、扩增纯化与线性化操作标准步骤

pSP108 骨架极小且属于高拷贝质粒，克隆与扩增极其简便、产量极大。操作中最核心的控制点是：在进行体外转录前，必须保证质粒完全线性化，并且全流程执行无 RNA 酶（RNase-Free）的严苛规范。

1. 大肠杆菌感受态转化与平板筛选

• 推荐原核宿主菌株：DH5$\alpha$、JM109 或 TOP10 等通用型大肠杆菌克隆菌株。

• 转化标准流程：

  1. 从零下 80 摄氏度冰箱中取出一管 50 $\mu$L 的大肠杆菌感受态细胞，放置于冰上让其缓慢融化（严禁使用水浴快速融化）。

  2. 向融化的感受态细胞中加入 10 - 50 ng 的 pSP108 质粒（或连接产物），用手指轻弹管底混匀，置于冰上静置 25 - 30 分钟。

  3. 将离心管立刻投入 42 摄氏度恒温水浴箱中，不加摇晃地热击（Heat-shock）整 90 秒，随后迅速插回冰上静置 2 - 3 分钟。该步骤利用热脉冲改变细菌膜通透性，促使质粒内吞。

  4. 在无菌超净台内，向管中加入 200 - 400 $\mu$L 不含抗生素的常规 LB 液体培养基（或 SOC 培养基）。

  5. 置于 37 摄氏度摇床中，以 150 rpm 的低速温和复苏摇育 45 - 60 分钟，使细菌完全表达质粒携带的氨苄青霉素抗性蛋白。

  6. 收集复苏后的菌液，均匀涂布于含有 100 $\mu$g/mL 氨苄青霉素（Ampicillin）的 LB 固体琼脂糖平板上。

  7. 倒置平板，置于 37 摄氏度恒温培养箱中，暗培养 12 - 16 小时，直至平板表面生长出清晰、饱满的单克隆菌落。

2. 液体菌种扩增与高质质粒抽提（Maxi/Mini-prep）

• 扩增操作：

  1. 挑取平板上通过 PCR 验证阳性的 pSP108 单菌落，接种至盛有 3 - 5 mL 含氨苄青霉素（100 $\mu$g/mL）的 LB 液体培养基中（若进行大抽，可按 1:1000 比例放大接种至 100 - 200 mL 培养基中）。

  2. 置于 37 摄氏度恒温摇床中，以 220 - 250 rpm 的高速剧烈振荡培养 12 - 14 小时，至菌液呈现浓稠的乳白色。（注：高拷贝质粒切勿过度培养超过 16 小时，以防细菌裂解老化导致质粒质量下降）。

• 纯化与质量控制：

  1. 采用经典的碱裂解法质粒抽提试剂盒（如 Qiagen 系列）回收质粒 DNA。

  2. 关键质控指标：

     • 使用纳米分光光度计（NanoDrop）测定，要求 OD260/OD280 纯度比值严格处于 1.80 - 1.90 之间（若有蛋白质或苯酚残留会导致比值偏低或偏高）。

     • 使用 1% 琼脂糖凝胶电泳检测，质粒应呈现清晰、明亮、以超螺旋（Supercoiled）状态为主的条带。

3. 体外转录前的关键准备：模板完全线性化（Linearization）

为了防止 RNA 聚合酶在体外转录时顺着环状质粒进行无休止的“滚环转录”，转录前必须将质粒进行完全的单酶切线性化。

• 酶切位点选择原则：必须选择多克隆位点（MCS）中、位于目标插入片段下游（即远离 SP6 启动子的一端）的独特单酶切位点，且该酶切位点绝对不能在目标基因片段内部出现。

• 推荐酶切体系（50 $\mu$L）：

  • pSP108 重组质粒 DNA：2 - 5 $\mu$g

  • 10× Restriction Enzyme Buffer：5 $\mu$L

  • 相应的限制性内切酶（高浓度型）：2 - 3 $\mu$L（约 20 - 30 U，确保过量酶切）

  • 无菌无无核酸酶水（Nuclease-Free $H_2O$）：补至 50 $\mu$L

• 反应参数与终止：置于该内切酶对应的最佳温度（通常为 37 摄氏度）下彻底酶切 2 - 3 小时。酶切结束后，取出 2 $\mu$L 进行凝胶电泳，必须看到原本快带的超螺旋质粒彻底消失，100% 转化变为单条缓慢移动的线性化 DNA 长条带。

• 线性化 DNA 纯化与回收（严防 RNase 污染）：

  1. 确认完全线性化后，向体系中加入等体积的酚:氯仿:异戊醇（25:24:1，pH 8.0），剧烈抽提一次以彻底失活并沉淀所有的限制性内切酶。

  2. 12000 rpm 离心 5 分钟，吸取上清，加入 2.5 倍体积的无水乙醇及 1/10 体积的 3M 醋酸钠（NaAc, pH 5.2），置于 零下 20 摄氏度沉淀 30 分钟。

  3. 4 摄氏度下 12000 rpm 高速离心 15 分钟，弃上清，使用 75% 乙醇（必须使用 Nuclease-Free 水配制）洗涤沉淀 1 - 2 次。

  4. 晾干沉淀，最终使用 DEPC 处理水（无 RNA 酶水）重悬溶解线性化 DNA 模板，置于 零下 20 摄氏度保存，作为下游体外转录体系的标准投入底盘。

Part 2 English Section

I General Information and Molecular Biological Background

• Vector Name: pSP108.

• Cloning Plasmid Classification: In vitro transcription (IVT) vector, standard cloning engineering plasmid (lacks mammalian or prokaryotic expression promoters; non-expression profile design).

• Plasmid Size Scale: Approximately 3.0 - 4.0 kb (subject to minor structural variances based on distinct downstream sub-cloning multi-cloning sites modifications).

• Core Backbone Origin and Engineering Background:The pSP108 plasmid template serves as a classic, benchmark tool meticulously engineered for high-efficiency In vitro transcription (IVT) applications. Its baseline architecture was optimized from legacy pUC and pSP series structural platforms. Crucially, pSP108 does not contain conventional cell-based expression promoters (such as CMV, SV40, or active lac induction engines) configured for protein synthesis inside living prokaryotic or eukaryotic host systems. Instead, it is strictly streamlined as a high-yield, cell-free synthetic chassis engineered to generate dense, ultra-pure RNA single-strand polymers in a test-tube enzymatic environment.

• Core Cis-Acting Elements and Map Characterization:

  • Phage RNA Polymerase Promoter Matrix: Embedded directly upstream of the Multiple Cloning Site (MCS), the backbone features a high-affinity SP6 bacteriophage promoter. (Select dual-transcription variants may feature a T7 or T3 flanking promoter configuration at the opposite terminus). SP6 RNA polymerase recognizes this cis-element with nearly 100% fidelity, initiating robust enzymatic RNA chain propagation without transcript background stuttering.

  • Multiple Cloning Site (MCS): Arranged directly downstream of the SP6 promoter node, this region embeds a dense, strategic array of unique restriction endonuclease recognition sites (e.g., EcoRI, SmaI, BamHI, SalI, PstI, HindIII). These loci permit seamless directional directional cloning of target genes or non-coding RNA structural matrices.

  • Prokaryotic Replicon: Outfitted with a high-copy-number pUC ori, forcing robust, continuous replication inside competent Escherichia coli hosts, yielding exceptional plasmid concentrations during regular mini/maxi extraction protocols.

  • Selective Antibiotic Marker: Carries the functional Ampicillin resistance gene ($Amp^R$ / bla), allowing for precise positive colony selection and cultural maintenance across agar plates and liquid growth media.

II Strategic Research Value and In Vitro Analytical Fields

Although incapable of driving in vivo protein translation within cellular models, pSP108 serves as an essential tool in RNA biochemistry, structural biology, and hybridization networks:

1. High-Yield Synthesis of Single-Stranded Antisense/Sense RNA Probes:By cloning a target genetic segment into the MCS of pSP108 and subsequently linearizing the vector, investigators deploy the template in an in vitro reaction alongside SP6 RNA Polymerase and labeled NTPs (utilizing isotopic labels like $^{32}P$ or non-isotopic haptens such as Digoxigenin or Biotin). This generates uniform, high-specific-activity single-stranded RNA probes, which are standardly utilized in In Situ Hybridization (ISH), Northern Blotting setups, and Ribonuclease Protection Assays (RPA).

2. Generating Substrates for Cell-Free In Vitro Translation Studies:The pure RNA transcripts harvested from pSP108 can be enzymatically processed with 5'-capping and 3'-polyadenylation reactions. These functional transcripts are directly added into cell-free translation lysates (such as Wheat Germ Extract or Rabbit Reticulocyte Lysate systems), enabling investigators to resolve early protein folding mechanics, translation initiation configurations, and screen small-molecules engineered to interrupt peptide chain elongation without the confounding variables of live-cell homeostasis.

3. Deconstructing RNA Secondary Structures and RNA-Protein Interactions (RBPs):pSP108 is utilized to synthesize high-purity non-coding RNAs (ncRNAs), precursor microRNAs (miRNAs), or custom structural ribozyme segments. These in vitro transcripts are directly applied in RNA Electrophoresis Mobility Shift Assays (REMSA/EMSA), RNA Pull-down chromatography arrays, or Cryo-Electron Microscopy (Cryo-EM) imaging setups to trap, isolate, and characterize specific RNA-Binding Proteins (RBPs).

III Laboratory E. coli Transformation, Expansion, and DNA Linearization Protocols

As a streamlined, high-copy plasmid, pSP108 offers highly efficient primary cloning and extraction workflows.The critical parameters of this protocol are ensuring absolute vector linearization before beginning transcription, and implementing strict RNase-free environment handling protocols throughout the process.

1. Competent E. coli Transformation and Selection Routines

• Recommended Prokaryotic Host Strains: Standard cloning strains including DH5$\alpha$, JM109, or TOP10.

• Standard Transformation Protocol Sequence:

  1. Retrieve a 50 $\mu$L aliquot of chemically competent E. coli cells from minus 80 degrees Celsius storage and place immediately on ice to thaw slowly.Never submerge in water baths to accelerate thawing.

  2. Add 10 - 50 ng of pristine pSP108 plasmid (or ligation reaction mixture) directly into the thawed bacteria. Mix gently by tapping the base of the tube with a finger, and incubate on ice for 25 - 30 minutes.

  3. Transfer the tube instantly into a 42 degrees Celsius constant water bath and subject the mixture to a heat-shock for exactly 90 seconds without agitation. Immediately plunge back into ice for 2 - 3 minutes to close the bacterial pores.

  4. Working under aseptic conditions within a Class II Biosafety Cabinet, add 200 - 400 $\mu$L of antibiotic-free standard LB (or SOC) liquid growth medium to the cells.

  5. Place the tube into a shaking incubator at 37 degrees Celsius, running at a low speed of 150 rpm for 45 - 60 minutes to allow out-growth and structural expression of the ampicillin resistance baseline proteins.

  6. Harvest the recovered cell suspension and smoothly spread it across a solid LB agar plate enriched with 100 $\mu$g/mL Ampicillin.

  7. Invert the plate and incubate at 37 degrees Celsius under dark parameters for 12 - 16 hours until well-defined, distinct single colonies become visible on the agar plane.

2. Liquid Culture Scaling and High-Quality Plasmid Extraction

• Expansion Mechanics:

  1. Inoculate a PCR-verified pSP108 positive single colony into 3 - 5 mL of LB liquid medium supplemented with 100 $\mu$g/mL Ampicillin. (For large-scale Maxi-preps, scale up by transferring at a 1:1000 ratio into 100 - 200 mL of identical selective broth).

  2. Secure the vessel inside a shaking incubator at 37 degrees Celsius and agitate vigorously at 220 - 250 rpm for 12 - 14 hours until the culture reaches a dense, opaque saturation phase.Caution: Do not exceed 16 hours of continuous incubation to prevent bacterial autolysis, which can degrade supercoiled plasmid yield.

• Purification and Analytical Quality Gates:

  1. Isolate the plasmid DNA using a standard alkaline-lysis purification framework (such as Qiagen Mini/Maxi-prep kits).

  2. Critical Quality Control Metrics:

     • Verify on a spectrophotometer (e.g., NanoDrop) that the $OD_{260}/OD_{280}$ purity coefficient rests securely between 1.80 and 1.90 (lower ratios reveal residual phenol/protein; elevated markers trace potential RNA carryover).

     • Resolve the product via 1% agarose gel electrophoresis; the resulting lanes must display a bright, sharp band representing predominantly supercoiled plasmid architecture.

3. Critical Pre-Transcription Step: Absolute Plasmid Linearization

To prevent RNA Polymerases from reading continuously around a circular plasmid track indefinitely via "rolling-circle transcription," the pSP108 vector must be completely linearized via single restriction enzyme cleavage before starting the transcription reaction.

• Enzyme Loci Selection Strategy: Investigators must choose a unique restriction site located within the MCS downstream of the target gene insertion insert (farthest from the SP6 promoter hub). Crucially, this enzyme must have zero cutting footprints inside the target gene sequence itself.

• Standard Linearization Digest Formulation (50 $\mu$L System):

  • pSP108 Recombinant Construct DNA: 2 - 5 $\mu$g

  • 10× Reaction Enzyme Buffer: 5 $\mu$L

  • Target Restriction Endonuclease (High-Concentration stock): 2 - 3 $\mu$L (~20 - 30 Units to secure excess digestion)

  • Nuclease-Free $H_2O$: Bring to a final volume of 50 $\mu$L

• Incubation Parameters and Validation: Run the digestion at the enzyme's optimal validated temperature setting (typically 37 degrees Celsius) for a minimum of 2 - 3 hours. Once complete, run a 2 $\mu$L sample on an agarose gel; the fast-migrating supercoiled plasmid profile must be completely absent, converting 100% into a single, clean, slow-migrating band representing linearized DNA.

• Phenol-Chloroform Extraction and Template Recovery (Strict RNase-Free Handling):

  1. Once complete linearization is confirmed, add an equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1, pH 8.0) directly to the digest to permanently denature and precipitate the restriction enzymes.

  2. Centrifuge at 12,000 rpm for 5 minutes, extract the clear aqueous upper layer, add 2.5 volumes of absolute ethanol alongside 1/10 volume of 3M Sodium Acetate (NaAc, pH 5.2), and store at minus 20 degrees Celsius for 30 minutes to precipitate the DNA.

  3. Spin down the precipitate at 12,000 rpm for 15 minutes at 4 degrees Celsius, decant the supernatant, and wash the pellet 1 - 2 times with 75% ethanol prepared with Nuclease-Free water.

  4. Air-dry the clean DNA pellet and resuspend it in DEPC-treated (RNase-Free) water. Store this linearized template DNA matrix at minus 20 degrees Celsius, ready as a validated substrate for downstream cell-free transcription setups.

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