BioVector® pMECS 纳米抗体噬菌体展示载体质粒 / pMECS Vector

一、 背景与来源说明 / Background & Origin

pMECS 是一种专为纳米抗体（Nanobody / VHH，来自驼类单域抗体）文库构建、噬菌体展示（Phage Display）以及周质腔分泌表达设计的经典噬菌体表达载体（Phagemid Vector）。pMECS 载体结合了 PelB 信号肽、lac 启动子、双融合标签（如 HA-tag 和 6xHis-tag）以及与 M13 噬菌体 g3p 外壳蛋白相连的 amber 终止密码子（TAG）。该结构设计允许研究人员利用同一载体在抑制型宿主菌（SupE，如 TG1）中进行纳米抗体的噬菌体展示筛选，并在非抑制型宿主菌（如 WK6）中直接实现可溶性纳米抗体向大肠杆菌周质腔的分泌表达与纯化。

二、 质粒主要结构与生物学特征 / Biological Characteristics

• 载体类型 / Vector Type：噬菌体展示质粒 / 纳米抗体分泌表达载体 (Phagemid Vector / Nanobody Expression Vector)

• 表达宿主 / Hosts：

  • 文库筛选/展示宿主：E. coli TG1 或 XL1-Blue（SupE 抑制型菌株，将 TAG 翻译为谷氨酰胺 Q，表达 VHH-g3p 融合蛋白并展示于噬菌体表面）

  • 可溶性表达宿主：E. coli WK6 或 HB2101（非抑制型菌株，识别 TAG 为终止信号，直接分泌表达可溶性 VHH-HA-His 蛋白）

• 抗生素筛选标记 / Selection Marker：氨苄青霉素 (Ampicillin, 100 ug/mL)

• 核心元件构成 / Key Functional Components：

  • lac 启动子 (lac Promoter)：受 IPTG 强诱导，可被葡萄糖 (Glucose) 阻遏抑制

  • PelB 信号肽 (PelB Leader Sequence)：引导目的 VHH 蛋白定向分泌至大肠杆菌周质腔（Periplasm），促进正确二硫键形成

  • 多克隆位点 (MCS)：通常包含 PstI 与 BstEII (或 NotI) 限制性内切酶切位点，专为驼类 VHH 基因定位克隆设计

  • 融合标签 (Tags)：C 端 HA-tag（用于 ELISA/Western Blot 检测）与 6xHis-tag（用于 Ni-NTA 亲和纯化）

  • Amber 终止密码子 (TAG)：介导噬菌体展示与可溶性蛋白表达模式的无缝切换

  • g3p 基因片段：编码 M13 噬菌体 minor coat protein pIII，用于噬菌体展示

  • f1 ori & pBR322 ori：单链噬菌体打包复制起点及高拷贝质粒复制起点

• 应用领域 / Applications：驼类（羊驼/骆驼）纳米抗体（VHH）免疫文库/合成文库构建、噬菌体展示靶向淘筛（Biopanning）、高亲和力纳米抗体在大肠杆菌周质腔中的可溶性表达与纯化

三、 培养条件与操作指南 / Culture & Handling Protocols

1. 质粒构建与文库扩增 / Cloning & Library Construction

• 文库克隆宿主 / Library Host：E. coli TG1 感受态细胞（要求高电转效率，大于 1 x 10^9 cfu/ug）

• 培养基 / Broth & Agar：2xTY 培养基 + 100 ug/mL 氨苄青霉素 + 2% 葡萄糖 (Glucose, 用于完全阻遏 lac 启动子以防止泄露表达)

• 淘筛步骤 / Biopanning：

  1. 将含有 pMECS-VHH 文库的 TG1 菌株接种，辅以 M13K07 救援噬菌体（Helper Phage）进行超感染打包。

  2. 收集表达有 VHH-g3p 融合蛋白的重组噬菌体颗粒，针对包被靶蛋白进行 3 至 4 轮固相/液相淘筛。

2. 纳米抗体可溶性表达与纯化 / Soluble Expression & Purification

• 表达宿主 / Expression Host：E. coli WK6

• 表达与周质腔提取步骤 / Protocol：

  1. 将淘筛获得的阳性 pMECS-VHH 质粒转化入 E. coli WK6 菌株。

  2. 接种于含 100 ug/mL 氨苄青霉素的 2xTY 培养基中，37 摄氏度培养至 OD600 达到 0.6 至 0.9。

  3. 加入终浓度 1.0 mM IPTG，降温至 28 摄氏度或 16 摄氏度诱导表达 16 小时。

  4. 离心收集菌体，通过高渗/低渗缓冲液渗透休克法（Osmotic Shock）破裂外膜提取周质腔蛋白（Periplasmic Fraction）。

  5. 利用 Ni-NTA 亲和层析柱结合 C 端 6xHis 标签纯化纳米抗体。

四、 技术指标简表 / Technical Data Summary

BioVector® pMECS Vector

1. Background and Origin

pMECS is a specialized phagemid vector designed for nanobody (VHH / single-domain antibody) library construction, phage display selection, and periplasmic expression. It incorporates a PelB signal peptide, an inducible lac promoter, dual detection/purification tags (HA-tag and 6xHis-tag), and an amber stop codon (TAG) positioned upstream of the M13 phage g3p coat protein gene. This architecture enables seamless switching between phage display in suppressor E. coli strains (e.g., TG1) and soluble VHH secretion into the bacterial periplasm in non-suppressor strains (e.g., WK6).

2. Biological Characteristics

• Vector Type: Phagemid Vector / Nanobody Expression Vector

• Expression Hosts:

  • Phage Display Host: E. coli TG1 or XL1-Blue (SupE suppressor strain; translates TAG as glutamine Q to yield VHH-g3p fusion for phage display)

  • Soluble Production Host: E. coli WK6 or HB2101 (Non-suppressor strain; recognizes TAG as a stop codon to produce soluble VHH-HA-His)

• Bacterial Selection: Ampicillin (100 ug/mL)

• Key Functional Components:

  • lac Promoter: IPTG-inducible promoter tightly repressed by 2 percent glucose

  • PelB Leader Sequence: Directs the recombinant VHH protein into the E. coli periplasmic space for proper disulfide bond formation

  • Multiple Cloning Site (MCS): Tailored for camelid VHH insertion using conserved restriction sites (PstI and BstEII / NotI)

  • Fusion Tags: C-terminal HA-tag (detection) and 6xHis-tag (Ni-NTA affinity purification)

  • Amber Codon (TAG): Enables dual-mode display/expression switching

  • g3p Fragment: Encodes the M13 minor coat protein pIII for surface display

  • f1 & pBR322 Origins: Single-stranded phage packaging and high-copy plasmid replication

• Applications: Camelid (alpaca/llama) nanobody immune/synthetic library construction, biopanning via phage display, and high-yield periplasmic soluble VHH production

3. Culture and Handling Protocols

Cloning and Library Propagation

• Library Host: Electrocompetent E. coli TG1 cells (transformation efficiency > 1 x 10^9 cfu/ug)

• Growth Medium: 2xTY medium containing 100 ug/mL Ampicillin and 2 percent Glucose (glucose prevents leaky expression during propagation)

• Phage Display & Biopanning:

  1. Superinfect pMECS-VHH transformed TG1 cells with M13K07 helper phage to produce recombinant phage particles.

  2. Harvest VHH-displaying phages and perform 3 to 4 rounds of biopanning against target antigens.

Soluble Nanobody Expression and Purification

• Production Host: E. coli WK6

• Periplasmic Extraction Protocol:

  1. Transform isolated positive pMECS-VHH clones into E. coli WK6.

  2. Inoculate into 2xTY medium with 100 ug/mL Ampicillin and culture at 37 degrees Celsius until OD600 reaches 0.6 to 0.9.

  3. Induce expression with 1.0 mM IPTG at 28 degrees Celsius or 16 degrees Celsius for 16 hours.

  4. Harvest cells and extract the periplasmic fraction via osmotic shock using TES/cold water treatment.

  5. Purify soluble nanobodies from the periplasmic extract using Ni-NTA immobilised metal affinity chromatography (IMAC).

4. Technical Data Summary

BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

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