BioVector? lpB-CMV-MCS-EF1a-GFP-Puro 重組慢病毒/轉(zhuǎn)座子表達(dá)質(zhì)粒載體

BioVector? lpB-CMV-MCS-EF1a-GFP-Puro Recombinant Lentiviral/Transposon Expression Plasmid Vector

第一部分 中文說明

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 載體名稱：BioVector? lpB-CMV-MCS-EF1a-GFP-Puro

• 產(chǎn)品類型：哺乳動物雙啟動子高效重組慢病毒（Lentiviral）/轉(zhuǎn)座子（Transposon）雙功能表達(dá)質(zhì)粒載體。

• 復(fù)制子與骨架基礎(chǔ)：

  • 大腸桿菌復(fù)制子：pUC origin（高拷貝復(fù)制子，用于在大腸桿菌中的質(zhì)粒高效擴(kuò)增與分子克?。?。

  • 病毒/轉(zhuǎn)座子骨架：基于改良型第三代HIV慢病毒載體或特定非病毒轉(zhuǎn)座子集成系統(tǒng)構(gòu)建，兩端攜帶關(guān)鍵的順式作用元件，用以介導(dǎo)目的片段精準(zhǔn)整合至宿主細(xì)胞基因組中。

• 雙標(biāo)記選擇系統(tǒng)（Dual Reporter & Selection Markers）：

  • 熒光報告標(biāo)記：綠色熒光蛋白 GFP（Green Fluorescent Protein）?？捎糜谵D(zhuǎn)化/轉(zhuǎn)染后通過熒光顯微鏡或流式細(xì)胞儀（FACS）實時、定量地監(jiān)測細(xì)胞的轉(zhuǎn)導(dǎo)效率。

  • 哺乳動物篩選抗性：嘌呤霉素抗性基因 Puro（Puromycin resistance, pac）。用于在轉(zhuǎn)染/感染后加入嘌呤霉素，快速殺死未成功整合的陰性細(xì)胞，篩選建立高純度的穩(wěn)定細(xì)胞株（Stable cell line）。

• 分子大小：約 8.5 kb 到 9.5 kb（依骨架細(xì)節(jié)變動）。

• 生物安全級別：1級（BSL-1）。注：當(dāng)該質(zhì)粒與慢病毒包裝輔助質(zhì)粒（如 pMD2.G 和 psPAX2）混合共轉(zhuǎn)染 293T 細(xì)胞制備活體慢病毒顆粒時，生成的病毒液操作級別必須提升至 2 級（BSL-2）。

二 核心功能元件與雙啟動子轉(zhuǎn)錄調(diào)控圖譜

lpB-CMV-MCS-EF1a-GFP-Puro 載體采用先進(jìn)的獨立雙啟動子（Dual-Promoter Architecture）級聯(lián)設(shè)計。這種結(jié)構(gòu)能夠有效避免多順反子系統(tǒng)（如 IRES 或 2A 肽）中后位基因翻譯效率低下或切除不完全的問題。其核心功能元件排列如下：

1. CMV 啟動子驅(qū)動核心轉(zhuǎn)錄軸（第一轉(zhuǎn)錄單元）：

   • CMV 啟動子（CMV Promoter）：源自人類巨細(xì)胞病毒的極強(qiáng)、廣譜型組成性啟動子，在絕大多數(shù)常見的哺乳動物細(xì)胞系（如 HeLa, 293T, CHO 等）和原代細(xì)胞中具備極高的轉(zhuǎn)錄活性。

   • 多克隆位點（MCS）：緊跟在 CMV 啟動子下方，提供豐富的限制性內(nèi)切酶單一切點，用于高效克隆和插入實驗人員的目的基因（Gene of Interest, GOI）。

2. EF1a 啟動子驅(qū)動標(biāo)記物軸（第二轉(zhuǎn)錄單元）：

   • EF1a 啟動子（EF1a Promoter）：人類延伸因子 1 alpha 啟動子。這是一個在哺乳動物體內(nèi)高度穩(wěn)定、不易受表觀遺傳學(xué)修飾干擾而發(fā)生基因沉默（Gene silencing）的強(qiáng)啟動子。即使在 CMV 啟動子容易發(fā)生沉默的干細(xì)胞（如 iPSC、ES 細(xì)胞）或免疫細(xì)胞（如 T 細(xì)胞）中，EF1a 依然能維持強(qiáng)勁、持久的驅(qū)動效能。

   • GFP-Puro 融合或級聯(lián)表達(dá)：由 EF1a 啟動子協(xié)同驅(qū)動 GFP 熒光標(biāo)記與 Puromycin 抗性標(biāo)記的聯(lián)合表達(dá)，確保只要綠色熒光亮起或在嘌呤霉素壓力下存活的細(xì)胞，其內(nèi)部的第二轉(zhuǎn)錄單元均在處于活躍工作狀態(tài)。

3. 基因組整合與終止元件（Integration & Termination）：

   • 載體兩側(cè)包含 5' WRE/LTR 及 3' Delta-LTR（或相應(yīng)的轉(zhuǎn)座子左右端反向重復(fù)序列 ITR），以及 WPRE（土撥鼠肝炎病毒 post-transcriptional 調(diào)控元件），用以大幅增強(qiáng) mRNA 在細(xì)胞質(zhì)內(nèi)的穩(wěn)定性和重組蛋白的翻譯豐度。

三 質(zhì)粒擴(kuò)增、轉(zhuǎn)染、慢病毒包裝與穩(wěn)株篩選標(biāo)準(zhǔn)操作步驟

1. 大腸桿菌中的質(zhì)粒擴(kuò)增（Plasmid Propagation）：

   • 推薦宿主：由于慢病毒/轉(zhuǎn)座子骨架兩端含有高度重復(fù)的長末端重復(fù)序列（LTR/ITR），在常規(guī)大腸桿菌中極易發(fā)生自發(fā)性同源重組導(dǎo)致骨架塌陷。強(qiáng)烈強(qiáng)制使用 Stbl3、SURE 或 Epi300 等抗重組的特殊大腸桿菌感受態(tài)菌株。嚴(yán)禁使用普通 DH5alpha 或 TOP10 進(jìn)行大規(guī)模擴(kuò)增。

   • 篩選培養(yǎng)基：標(biāo)準(zhǔn) LB 固體/液體培養(yǎng)基，加入 100 μg/ml 的氨芐青霉素（Ampicillin）。

   • 培養(yǎng)參數(shù)：建議在 30 攝氏度下低速振蕩（200 rpm）孵育 16 到 24 小時。低溫擴(kuò)增能夠大幅度降低兩端重復(fù)序列發(fā)生突變或重組脫落的幾率。

2. 慢病毒包裝制備（Lentiviral Packaging Protocol，若作為慢病毒使用）：

   • 轉(zhuǎn)染準(zhǔn)備：使用高純度無內(nèi)毒素的質(zhì)粒中提試劑盒提取質(zhì)粒。將本表達(dá)質(zhì)粒與第二代或第三代慢病毒包裝混合質(zhì)粒按比例混合，使用轉(zhuǎn)染試劑（如 Lipofectamine 3000 或 PEI）共轉(zhuǎn)染至處于對數(shù)生長期的 293T 細(xì)胞中。

   • 病毒收集：轉(zhuǎn)染后 48 小時及 72 小時分別收集細(xì)胞上清液。通過低速離心（3000 rpm，10分鐘）及 0.45 μm 聚醚砜（PES）低蛋白結(jié)合濾器過濾以去除細(xì)胞碎片。所得濾液即為包含 lpB-CMV-MCS-EF1a-GFP-Puro 整合盒的活體慢病毒液，可直接用于轉(zhuǎn)導(dǎo)或通過超速離心進(jìn)行濃縮后存于 -80 攝氏度。

3. 宿主細(xì)胞轉(zhuǎn)導(dǎo)與嘌呤霉素穩(wěn)株篩選（Stable Cell Line Generation）：

   • 病毒轉(zhuǎn)導(dǎo)：將目標(biāo)宿主細(xì)胞接種于培養(yǎng)皿中。當(dāng)細(xì)胞融合度達(dá)到 50% 左右時，加入適量慢病毒液，并可加入 6-8 μg/ml 的聚布雷線（Polybrene）以增強(qiáng)病毒吸附轉(zhuǎn)導(dǎo)效率。

   • 熒光觀測：轉(zhuǎn)導(dǎo) 24 至 48 小時后，在熒光顯微鏡下觀察綠色熒光（GFP），評估初始感染效率。

   • 加壓篩選：轉(zhuǎn)導(dǎo) 48 小時后，吸除含病毒舊基，加入含有適當(dāng)濃度嘌呤霉素（Puromycin，工作濃度通常需提前通過死時間曲線 Kill Curve 測定，一般細(xì)胞系為 1 μg/ml 至 5 μg/ml）的新鮮完全培養(yǎng)基。每 2 到 3 天更換一次含有 Puro 的選擇性培養(yǎng)基。持續(xù)篩選 7 到 10 天，直至陰性對照孔細(xì)胞完全死亡，而實驗孔中的存活細(xì)胞全部發(fā)出強(qiáng)烈的綠色熒光，即成功建立穩(wěn)定表達(dá)目的基因的單克隆或多克隆細(xì)胞株。

四 核心科研應(yīng)用方向

1. 頑固或難轉(zhuǎn)染細(xì)胞系中的外源基因穩(wěn)定超表達(dá)：本載體作為慢病毒/轉(zhuǎn)座子雙功功能系，能夠極高效率地感染常規(guī)脂質(zhì)體極難轉(zhuǎn)染的細(xì)胞（如懸浮淋巴細(xì)胞、原代內(nèi)皮細(xì)胞、原代神經(jīng)元及各類干細(xì)胞）。通過將外源目的基因（GOI）整合至宿主基因組中，實現(xiàn)基因的永久性、高豐度、不隨傳代而丟失的穩(wěn)定超表達(dá)。

2. 雙標(biāo)記系統(tǒng)用于實時細(xì)胞示蹤與體內(nèi)成像研究（Cell Tracking）：利用內(nèi)源集成的強(qiáng)熒光 GFP 標(biāo)記，重組構(gòu)建的穩(wěn)定細(xì)胞株可直接用于共聚焦顯微鏡下的動態(tài)形態(tài)學(xué)觀察、流式細(xì)胞術(shù)定量分析，或者將細(xì)胞注射入免疫缺陷小鼠體內(nèi)建立異種移植瘤模型（Xenograft），通過活體熒光成像系統(tǒng)（IVIS）實時追蹤腫瘤的體內(nèi)生長、遷移與骨轉(zhuǎn)移規(guī)律。

3. 大規(guī)模功能基因篩選、RNAi 及突變體庫構(gòu)建的反應(yīng)骨架：由于該載體帶有 MCS 和強(qiáng)力的 CMV 啟動子，可用于批量克隆各種不同的致癌基因、抑癌基因突變體或特定非編碼 RNA（如 lncRNA）。通過慢病毒大規(guī)模感染靶細(xì)胞并結(jié)合 Puromycin 快速篩選，能夠極高通量地構(gòu)建高一致性的細(xì)胞表型篩選平臺，用以評估小分子靶向藥物的敏感性及耐藥基因篩查。

PART 2 ENGLISH SECTION

I General Information and Genetic Background

• Vector Name: BioVector? lpB-CMV-MCS-EF1a-GFP-Puro

• Product Type: Dual-functional recombinant mammalian lentiviral/transposon expression plasmid vector.

• Replicon & Structural Backbone:

  • Prokaryotic Replicon: pUC origin (High-copy framework engineered for high-yield propagation and restriction cloning inside standard bacterial selection systems).

  • Viral/Transposon Architecture: Constructed based on an optimized third-generation HIV-1 lentiviral vector matrix or integrated non-viral transposon cassette systems. It carries essential cis-acting elements flanking the core to coordinate precise chromosomal integration into the host genome.

• Dual Reporter & Selection Marker Framework:

  • Fluorescent Reporter: Green Fluorescent Protein (GFP). Permits rapid, real-time visual tracking of transduction efficiencies via standard fluorescence microscopy or Quantitative Flow Cytometry (FACS).

  • Mammalian Selection Marker: Puromycin resistance gene (Puro, pac). Enables rapid clearing of un-integrated negative cellular fractions post-transduction, establishing high-purity stable lineages.

• Molecular Size: Approximately 8.5 kb to 9.5 kb (subject to custom modifications of the core cloned parameters).

• Biosafety Level: BSL-1. Note: When this specific transfer plasmid is combined with companion packaging vectors (e.g., pMD2.G and psPAX2) via co-transfection into 293T factories to manufacture functional lentiviral particles, the operational bio-risk rating scales up to Biosafety Level 2 (BSL-2).

II Core Structural Elements and Dual-Promoter Transcriptional Map

The lpB-CMV-MCS-EF1a-GFP-Puro vector implements an advanced dual-promoter級聯(lián) topology. This architecture effectively circumvents the translational attenuation and incomplete proteolytic cleavage profiles frequently associated with polycistronic designs such as IRES or 2A-peptide linkers. The sequential configuration of its elements is outlined below:

1. CMV Promoter-Driven Primary Transcription Unit:

   • CMV Promoter: A strong, broad-spectrum constitutive promoter derived from human cytomegalovirus, demonstrating extreme transcriptional velocities across nearly all mainstream mammalian immortalized cell lines (e.g., HeLa, 293T, CHO) and primary somatic isolates.

   • Multiple Cloning Site (MCS): Positioned directly downstream of the CMV promoter, hosting an array of unique restriction endonuclease slots to streamline the precise cloning of the Gene of Interest (GOI).

2. EF1a Promoter-Driven Marker Selection Unit:

   • EF1a Promoter: Human elongation factor 1 alpha promoter. Known for its strong constitutive resilience against epigenetic silencing mechanisms. It maintains sustained, robust transcription inside demanding environments like human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), and primary T-lymphocytes where viral promoters like CMV often undergo methylation and inactivation.

   • GFP-Puro Fusion Expression: Coordinates the simultaneous translation of the fluorescent reporter and selection marker under a single transcript, ensuring that any cell displaying green fluorescence or surviving puromycin pressure is actively running the selection cassette.

3. Genomic Integration and Processing Elements:

   • Flanked by 5' WRE/LTR and 3' Delta-LTR segments (or respective transposon Left/Right Inverted Terminal Repeats - ITRs) alongside a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) to heavily augment mRNA stability and target protein expression levels.

III Plasmid Propagation, Lentiviral Packaging, and Stable Line Selection

1. Bacterial Plasmid Propagation:

   • Mandatory Host Strains: Due to the presence of highly repetitive LTR/ITR palindromic architectures flanking the transfer core, this plasmid is structurally prone to spontaneous homologous recombination resulting in backbone deletions inside standard cloning bacteria. Investigators must exclusively deploy recombination-deficient strains such as Stbl3, SURE, or Epi300 competent cells. Standard DH5alpha or TOP10 variants are strictly contraindicated.

   • Selection Agar/Broth: Standard Luria-Bertani (LB) media supplemented with 100 μg/ml Ampicillin.

   • Incubation Parameters: Highly recommended to culture cultures at 30 degrees Celsius under low-speed agitation (200 rpm) for 16 to 24 hours. Lowered incubation thermal inputs drastically suppress recombination rates and stabilize the repeat components.

2. Lentiviral Particle Generation and Harvesting (If deployed as a viral delivery tool):

   • Transfection Formulation: Extract high-purity, endotoxin-free plasmid stocks. Co-transfect the lpB transfer plasmid together with 2nd or 3rd generation packaging plasmid systems into active, log-phase 293T cells using liposomal transfection reagents (e.g., Lipofectamine 3000 or polyethylenimine - PEI).

   • Harvest Routine: Harvest the media supernatants at 48 hours and 72 hours post-transfection. Pellet out detached cellular components via low-speed centrifugation (3000 rpm for 10 minutes) and pass the fluid through a 0.45 μm low protein-binding Polyethersulfone (PES) filter. The cleared viral filtrate can be deployed immediately for target infection or concentrated via ultracentrifugation and locked down at -80 degrees Celsius.

3. Target Transduction and Puromycin Selection Tracking:

   • Viral Transduction: Seed out target mammalian cells. At approximately 50% surface confluence, introduce calculated volumes of the harvested lentiviral supernatant, optionally supplemented with 6-8 μg/ml Polybrene to maximize membrane attachment.

   • Fluorescence Analysis: Evaluate active GFP expression profiles under an inverted fluorescence microscope 24 to 48 hours post-infection to compute baseline transduction efficiency.

   • Selection Pressure Execution: At 48 hours post-transduction, replace media with fresh growth broth supplemented with appropriate working concentrations of Puromycin (derived empirically via a pre-determined Kill Curve, typically spanning 1 μg/ml to 5 μg/ml for standard cell lines). Refresh selection media every 2 to 3 days. Continue the selection pressure for 7 to 10 days until all non-transduced cells in the negative control vessel are fully cleared and 100% of the surviving experimental mass shows uniform GFP tracking.

IV Strategic Research Applications

1. Sustained Ectopic Overexpression inside Refractory and Primary Cell Types: This vector acts as a highly effective system for driving permanent overexpression inside difficult-to-transfect cell populations (e.g., suspension lymphocytes, primary endothelial networks, primary cortical neurons, and varied stem cell niches). By permanently embedding the GOI cassette into the host chromosomal framework, it bypasses transient dilution effects.

2. Dual-Marker Tracing for Live-Cell Confocal and In Vivo Tumor Imaging: Utilizing the built-in highly active GFP reporter, engineered stable cell lines can be deployed directly in live-cell confocal tracking assays, flow cytometry characterizations, or injected into immunodeficient mice (e.g., BALB/c Nude or NSG) to generate xenograft models. Tumor propagation, systemic metastasis, and tissue tropism can then be mapped using In Vivo Imaging Systems (IVIS).

3. High-Throughput Functional Screenings, Mutant Library Generation, and RNAi Overexpression Blprints: The dual-promoter template with an expansive MCS serves as an ideal vector platform to construct multiplexed mutant validation screens, evaluate panel sets of oncogene/tumor-suppressor variants, or overexpress structural non-coding RNAs (lncRNAs). Following high-efficiency viral library delivery and rapid puromycin-mediated enrichment, investigators can systematically profile candidate lines against small-molecule targeted inhibitor compounds or map therapeutic resistance loops.

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:1843439339

網(wǎng)址http://www.nedfriskphoto.com