BioVector® pET28 His6-mEGFP-D4H 质粒 / pET28 His6-mEGFP-D4H Plasmid

一、 背景与来源说明 / Background & Origin

pET28 His6-mEGFP-D4H 是一种基于经典 pET28 表达骨架构建的细菌高水平蛋白表达与细胞学探针质粒。该载体融合了 N 端 His6 六组氨酸标签、mEGFP（单体增强型绿色荧光蛋白）以及 D4H 结构域（来源于产气荚膜梭菌灌注蛋白 Perfringolysin O 的 C 端 D4 结构域突变体）。D4H 能以极高的亲和力和特异性结合质膜及细胞膜上的游离胆固醇（Free Cholesterol）。利用该质粒在 E. coli 中重组表达并纯化出的 His6-mEGFP-D4H 重组蛋白，可作为检测活细胞或固定细胞膜脂质分布、胆固醇转运及细胞器脂质质控的经典荧光探针。

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

• 质粒类型 / Vector Type：原核蛋白表达质粒 / 膜胆固醇检测探针构建体 (Bacterial Expression Vector / Membrane Cholesterol Probe)

• 宿主表达体系 / Expression Host：E. coli 表达宿主 (如 BL21(DE3) 或 Rigami/Rosetta)

• 细菌筛选标记 / Bacterial Resistance：卡那霉素 (Kanamycin, 50 ug/mL)

• 核心元件构成 / Key Functional Components：

  • T7 启动子 (T7 Promoter)：受 IPTG 强诱导表达

  • N-His6 Tag：用于 Ni-NTA 亲和层析重组蛋白纯化

  • mEGFP (Monomeric EGFP)：单体绿色荧光蛋白，提供高亮度的绿色荧光信号 (激发光 ~488 nm, 发射光 ~507 nm)

  • D4H Domain：胆固醇特异性结合结构域，专一性识别与结合膜上的游离胆固醇

  • pBR322 ori & lacI：高拷贝/可调控复制起点及 lac 阻遏蛋白编码基因

• 应用领域 / Applications：重组 His6-mEGFP-D4H 探针蛋白的大肠杆菌表达与 Ni-NTA 纯化、细胞膜及细胞器游离胆固醇体外/荧光显微成像分析、脂质代谢与动脉粥样硬化机制研究

三、 培养条件与转化/表达指南 / Culture & Handling Protocols

1. 质粒构建与克隆增殖 / Cloning & Propagation

• 克隆宿主 / Cloning Host：E. coli DH5alpha 或 TOP10

• 培养基 / Broth & Agar：LB 液体/固体培养基 + 50 ug/mL 卡那霉素 (Kanamycin)

• 转化步骤 / Transformation：

  1. 取 50 uL DH5alpha 感受态细胞于冰上融化，加入 1 uL 质粒 DNA。

  2. 冰浴 30 分钟，42 摄氏度热休克 45 秒，立即置于冰上 2 分钟。

  3. 加入无抗 LB，37 摄氏度复苏 1 小时，涂布于 Kan 培养皿。

2. 重组蛋白表达与诱导 / Protein Expression

• 表达宿主 / Expression Host：E. coli BL21(DE3)

• 诱导条件 / Induction Conditions：

  1. 将质粒转化入 BL21(DE3) 菌株，挑取单菌落接种于含 50 ug/mL 卡那霉素的 LB 中过夜培养。

  2. 按 1:100 转接大容量 LB，37 摄氏度培养至 OD600 达到 0.6 至 0.8。

  3. 加入 0.2 至 0.5 mM IPTG，降温至 16 至 20 摄氏度诱导表达 12 至 16 小时（低诱导温度有助于消除蛋白包涵体，促进 His6-mEGFP-D4H 可溶性表达）。

  4. 离心收集菌体，利用 Ni-NTA 柱层析进行 His 标签亲和纯化。

四、 技术指标简表 / Technical Data Summary

BioVector® pET28 His6-mEGFP-D4H Plasmid

1. Background and Origin

pET28 His6-mEGFP-D4H is a bacterial expression vector engineered for high-yield recombinant protein production of a cholesterol-specific biosensor. Constructed on the classical pET28 backbone, it features an N-terminal His6 affinity tag, a monomeric green fluorescent protein (mEGFP), and the D4H domain (a mutated D4 domain derived from Clostridium perfringens Perfringolysin O). The D4H moiety specifically binds to unesterified/free cholesterol on cellular membranes with high affinity. Recombinant His6-mEGFP-D4H protein purified from E. coli serves as a widely used fluorescent probe for visualizing membrane cholesterol distribution and lipid trafficking.

2. Biological Characteristics

• Vector Type: Bacterial Expression Vector / Membrane Cholesterol Biosensor Probe

• Expression Host: E. coli expression strains (e.g., BL21(DE3), Rosetta)

• Bacterial Selection: Kanamycin (50 ug/mL)

• Key Functional Components:

  • T7 Promoter: Strong, IPTG-inducible promoter

  • N-His6 Tag: Facilitates Ni-NTA affinity chromatography purification

  • mEGFP: Monomeric fluorescent reporter (Ex ~488 nm, Em ~507 nm) for visualization

  • D4H Domain: Engineered toxin-derived domain for selective free cholesterol recognition

  • pBR322 ori & lacI: High-copy replication origin and lac repressor gene

• Applications: Production of recombinant cholesterol probes, confocal fluorescent staining of plasma/organellar membrane free cholesterol, and lipid trafficking assays

3. Culture and Handling Protocols

Cloning and Propagation

• Cloning Host: E. coli DH5alpha or TOP10

• Growth Medium: LB Broth / LB Agar supplemented with 50 ug/mL Kanamycin

• Transformation Protocol:

  1. Thaw DH5alpha competent cells on ice and add 1 uL of plasmid DNA.

  2. Incubate on ice for 30 minutes, heat-shock at 42 degrees Celsius for 45 seconds, then return to ice for 2 minutes.

  3. Outgrow in antibiotic-free LB broth for 1 hour at 37 degrees Celsius, then spread onto Kanamycin plates.

Recombinant Protein Expression

• Expression Host: E. coli BL21(DE3)

• Induction Protocol:

  1. Transform into BL21(DE3), pick a single colony, and grow an overnight starter culture in selective LB.

  2. Inoculate 1:100 into fresh LB with Kanamycin and shake at 37 degrees Celsius until OD600 reaches 0.6 to 0.8.

  3. Induce with 0.2 to 0.5 mM IPTG at 16 to 20 degrees Celsius for 12 to 16 hours to promote soluble expression.

  4. Harvest bacteria by centrifugation and purify using standard Ni-NTA affinity chromatography.

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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