BioVector? pSP108 Standard In Vitro Transcription Vector / pSP108 標(biāo)準(zhǔn)體外轉(zhuǎn)錄與克隆非表達(dá)型質(zhì)粒載體

一 產(chǎn)品基本信息與分子生物學(xué)背景

• 載體名稱：pSP108。

• 克隆質(zhì)粒分類：體外轉(zhuǎn)錄質(zhì)粒、克隆質(zhì)粒（無哺乳動(dòng)物或原核細(xì)胞表達(dá)啟動(dòng)子，屬于非表達(dá)型工程質(zhì)粒）。

• 質(zhì)粒大小：約 3.0 - 4.0 kb（具體因不同衍生亞型和多克隆位點(diǎn)微調(diào)而異）。

• 核心骨架源起與設(shè)計(jì)背景：pSP108 是分子生物學(xué)中非常經(jīng)典的體外轉(zhuǎn)錄（In vitro transcription, IVT）專用質(zhì)粒骨架。它的核心構(gòu)造是基于經(jīng)典的 pUC 或 pSP 系列質(zhì)粒優(yōu)化而來。該載體不具備用于在活細(xì)胞內(nèi)（如大腸桿菌、大鼠、人類細(xì)胞）轉(zhuǎn)錄翻譯蛋白質(zhì)的 CMV、SV40 或 lac 驅(qū)動(dòng)性表達(dá)啟動(dòng)子，而是專門為了高效率地在體外無細(xì)胞體系中合成高純度、高豐度的 RNA 片段而設(shè)計(jì)。

• 核心順式作用元件與圖譜特征：

  • 噬菌體 RNA 聚合酶啟動(dòng)子（Phage RNA Polymerase Promoter）：pSP108 骨架中緊鄰多克隆位點(diǎn)（MCS）的上方，核心配置了高特異性的 SP6 噬菌體啟動(dòng)子（部分雙向轉(zhuǎn)錄變亞型會(huì)在另一端配置 T7 或 T3 啟動(dòng)子）。SP6 RNA 聚合酶對(duì)該段順式元件的識(shí)別特異性接近 100%，能夠在體外酶促反應(yīng)體系中極快地啟動(dòng)順式單鏈 RNA 的級(jí)聯(lián)延伸。

  • 多克隆位點(diǎn)（Multiple Cloning Site, MCS）：緊隨 SP6 啟動(dòng)子下游，包含一系列獨(dú)特的內(nèi)切酶切位點(diǎn)（如 EcoRI, SmaI, BamHI, SalI, PstI, HindIII 等）。這些位點(diǎn)被致密而合理地排布，方便研究人員將目標(biāo)基因或非編碼 RNA 模板精確地定向克隆插入。

  • 原核復(fù)制子（Prokaryotic Replicon）：配置了高拷貝的 pUC ori，確保該質(zhì)粒在大腸桿菌（E. coli）感受態(tài)細(xì)胞中能夠?qū)崿F(xiàn)極其兇猛的自主增殖與擴(kuò)增，日常質(zhì)粒抽提產(chǎn)量極高。

  • 抗性選擇標(biāo)記（Selection Marker）：攜帶氨芐青霉素抗性基因（Ampicillin resistance, $Amp^R$ / bla），用于在原核平板篩選和液體擴(kuò)增中維持陽性克隆。

二 核心科研價(jià)值與體外實(shí)驗(yàn)應(yīng)用

pSP108 質(zhì)粒雖不能在活細(xì)胞內(nèi)表達(dá)蛋白，但在RNA生物學(xué)、結(jié)構(gòu)生物學(xué)及分子雜交等領(lǐng)域扮演著極核心的分子靶底角色：

1. 反義/正義單鏈 RNA 探針（RNA Probes）的高效體外制備：通過將特定基因片段克隆入 pSP108 的 MCS 中，利用單酶切將質(zhì)粒進(jìn)行末端線性化（Linearization），隨后以線性化 DNA 為模板，加入 SP6 RNA 聚合酶以及帶有同位素（如 $^{32}P$）或非同位素標(biāo)記（如地高辛 Digoxigenin, 生物素 Biotin）的 NTPs。可在體外穩(wěn)定、大量地合成出極高質(zhì)量的單鏈 RNA 探針。這些探針被廣泛用于原位雜交（In situ Hybridization, ISH）、Northern Blot 分子雜交以及核酸酶保護(hù)實(shí)驗(yàn)（RPA）。

2. 體外翻譯（In vitro Translation）模板構(gòu)建：轉(zhuǎn)錄出的高純度 RNA，在體外經(jīng)過加帽（Capping）和加尾（Poly-A tailing）酶促修飾后，可直接投入麥胚提取物（Wheat Germ Extract）或兔網(wǎng)織紅細(xì)胞裂解液（Rabbit Reticulocyte Lysate）等無細(xì)胞翻譯體系中。這被用于在完全不依賴活細(xì)胞的環(huán)境下，精準(zhǔn)解構(gòu)蛋白質(zhì)的早期折疊、翻譯起始調(diào)控及新型小分子藥物對(duì)翻譯鏈延伸的干預(yù)阻斷。

3. RNA 二級(jí)結(jié)構(gòu)、RNA-蛋白相互作用（RBP）探討：用于在體外轉(zhuǎn)錄出特定的非編碼 RNA（ncRNA）、微小 RNA（miRNA）前體或特定具有空間構(gòu)象的 RNA 片段。這些高質(zhì)量 RNA 可直接用于 RNA 電泳遷移率變動(dòng)分析（REMSA/EMSA）、RNA Pull-down 實(shí)驗(yàn)或冷凍電鏡（Cryo-EM）結(jié)構(gòu)解構(gòu)，用以捕捉并鑒定與之結(jié)合的各種靶標(biāo)RNA結(jié)合蛋白（RBPs）。

三 實(shí)驗(yàn)室大腸桿菌轉(zhuǎn)化、擴(kuò)增純化與線性化操作標(biāo)準(zhǔn)步驟

pSP108 骨架極小且屬于高拷貝質(zhì)粒，克隆與擴(kuò)增極其簡便、產(chǎn)量極大。操作中最核心的控制點(diǎn)是：在進(jìn)行體外轉(zhuǎn)錄前，必須保證質(zhì)粒完全線性化，并且全流程執(zhí)行無 RNA 酶（RNase-Free）的嚴(yán)苛規(guī)范。

1. 大腸桿菌感受態(tài)轉(zhuǎn)化與平板篩選

• 推薦原核宿主菌株：DH5$\alpha$、JM109 或 TOP10 等通用型大腸桿菌克隆菌株。

• 轉(zhuǎn)化標(biāo)準(zhǔn)流程：

  1. 從零下 80 攝氏度冰箱中取出一管 50 $\mu$L 的大腸桿菌感受態(tài)細(xì)胞，放置于冰上讓其緩慢融化（嚴(yán)禁使用水浴快速融化）。

  2. 向融化的感受態(tài)細(xì)胞中加入 10 - 50 ng 的 pSP108 質(zhì)粒（或連接產(chǎn)物），用手指輕彈管底混勻，置于冰上靜置 25 - 30 分鐘。

  3. 將離心管立刻投入 42 攝氏度恒溫水浴箱中，不加搖晃地?zé)釗簦℉eat-shock）整 90 秒，隨后迅速插回冰上靜置 2 - 3 分鐘。該步驟利用熱脈沖改變細(xì)菌膜通透性，促使質(zhì)粒內(nèi)吞。

  4. 在無菌超凈臺(tái)內(nèi)，向管中加入 200 - 400 $\mu$L 不含抗生素的常規(guī) LB 液體培養(yǎng)基（或 SOC 培養(yǎng)基）。

  5. 置于 37 攝氏度搖床中，以 150 rpm 的低速溫和復(fù)蘇搖育 45 - 60 分鐘，使細(xì)菌完全表達(dá)質(zhì)粒攜帶的氨芐青霉素抗性蛋白。

  6. 收集復(fù)蘇后的菌液，均勻涂布于含有 100 $\mu$g/mL 氨芐青霉素（Ampicillin）的 LB 固體瓊脂糖平板上。

  7. 倒置平板，置于 37 攝氏度恒溫培養(yǎng)箱中，暗培養(yǎng) 12 - 16 小時(shí)，直至平板表面生長出清晰、飽滿的單克隆菌落。

2. 液體菌種擴(kuò)增與高質(zhì)質(zhì)粒抽提（Maxi/Mini-prep）

• 擴(kuò)增操作：

  1. 挑取平板上通過 PCR 驗(yàn)證陽性的 pSP108 單菌落，接種至盛有 3 - 5 mL 含氨芐青霉素（100 $\mu$g/mL）的 LB 液體培養(yǎng)基中（若進(jìn)行大抽，可按 1:1000 比例放大接種至 100 - 200 mL 培養(yǎng)基中）。

  2. 置于 37 攝氏度恒溫?fù)u床中，以 220 - 250 rpm 的高速劇烈振蕩培養(yǎng) 12 - 14 小時(shí)，至菌液呈現(xiàn)濃稠的乳白色。（注：高拷貝質(zhì)粒切勿過度培養(yǎng)超過 16 小時(shí)，以防細(xì)菌裂解老化導(dǎo)致質(zhì)粒質(zhì)量下降）。

• 純化與質(zhì)量控制：

  1. 采用經(jīng)典的堿裂解法質(zhì)粒抽提試劑盒（如 Qiagen 系列）回收質(zhì)粒 DNA。

  2. 關(guān)鍵質(zhì)控指標(biāo)：

     • 使用納米分光光度計(jì)（NanoDrop）測(cè)定，要求 OD260/OD280 純度比值嚴(yán)格處于 1.80 - 1.90 之間（若有蛋白質(zhì)或苯酚殘留會(huì)導(dǎo)致比值偏低或偏高）。

     • 使用 1% 瓊脂糖凝膠電泳檢測(cè)，質(zhì)粒應(yīng)呈現(xiàn)清晰、明亮、以超螺旋（Supercoiled）狀態(tài)為主的條帶。

3. 體外轉(zhuǎn)錄前的關(guān)鍵準(zhǔn)備：模板完全線性化（Linearization）

為了防止 RNA 聚合酶在體外轉(zhuǎn)錄時(shí)順著環(huán)狀質(zhì)粒進(jìn)行無休止的“滾環(huán)轉(zhuǎn)錄”，轉(zhuǎn)錄前必須將質(zhì)粒進(jìn)行完全的單酶切線性化。

• 酶切位點(diǎn)選擇原則：必須選擇多克隆位點(diǎn)（MCS）中、位于目標(biāo)插入片段下游（即遠(yuǎn)離 SP6 啟動(dòng)子的一端）的獨(dú)特單酶切位點(diǎn)，且該酶切位點(diǎn)絕對(duì)不能在目標(biāo)基因片段內(nèi)部出現(xiàn)。

• 推薦酶切體系（50 $\mu$L）：

  • pSP108 重組質(zhì)粒 DNA：2 - 5 $\mu$g

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

  • 相應(yīng)的限制性內(nèi)切酶（高濃度型）：2 - 3 $\mu$L（約 20 - 30 U，確保過量酶切）

  • 無菌無無核酸酶水（Nuclease-Free $H_2O$）：補(bǔ)至 50 $\mu$L

• 反應(yīng)參數(shù)與終止：置于該內(nèi)切酶對(duì)應(yīng)的最佳溫度（通常為 37 攝氏度）下徹底酶切 2 - 3 小時(shí)。酶切結(jié)束后，取出 2 $\mu$L 進(jìn)行凝膠電泳，必須看到原本快帶的超螺旋質(zhì)粒徹底消失，100% 轉(zhuǎn)化變?yōu)閱螚l緩慢移動(dòng)的線性化 DNA 長條帶。

• 線性化 DNA 純化與回收（嚴(yán)防 RNase 污染）：

  1. 確認(rèn)完全線性化后，向體系中加入等體積的酚:氯仿:異戊醇（25:24:1，pH 8.0），劇烈抽提一次以徹底失活并沉淀所有的限制性內(nèi)切酶。

  2. 12000 rpm 離心 5 分鐘，吸取上清，加入 2.5 倍體積的無水乙醇及 1/10 體積的 3M 醋酸鈉（NaAc, pH 5.2），置于 零下 20 攝氏度沉淀 30 分鐘。

  3. 4 攝氏度下 12000 rpm 高速離心 15 分鐘，棄上清，使用 75% 乙醇（必須使用 Nuclease-Free 水配制）洗滌沉淀 1 - 2 次。

  4. 晾干沉淀，最終使用 DEPC 處理水（無 RNA 酶水）重懸溶解線性化 DNA 模板，置于 零下 20 攝氏度保存，作為下游體外轉(zhuǎn)錄體系的標(biāo)準(zhǔn)投入底盤。

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.

BioVector NTCC質(zhì)粒載體菌株細(xì)胞蛋白抗體基因保藏中心

電話:400-800-2947

工作QQ/微信同號(hào):1843439339

網(wǎng)址http://www.nedfriskphoto.com