BioVector® Mouse PlexA1 过表达质粒 / Mouse PlexA1 Overexpression Plasmid

一、 背景与来源说明 / Background & Origin

Mouse PlexA1 过表达质粒是一种专为高水平表达小鼠丛状蛋白 A1（Plexin-A1, PlexA1）构建的重组表达载体。Plexin-A1 是 Semaphorin 3A (Sema3A) 及 Semaphorin 6A (Sema6A) 等信号分子的核心跨膜受体/共受体，在神经元轴突引导（Axon Guidance）、突触发育、心血管重塑及免疫细胞迁移调控中发挥关键作用。该过表达质粒整合了强启动子（如 CMV 或 EF1a）与小鼠 Plexa1 编码区（CDS），适用于在哺乳动物细胞系、原代神经元或动物模型中开展 PlexA1 的过表达、受体-配体相互作用、下游 Rho GTPase 信号转导及功能增益（Gain-of-Function）研究。

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

• 质粒类型 / Vector Type：哺乳动物表达质粒 / 基因过表达载体 (Mammalian Overexpression Vector)

• 靶基因与物种 / Target Gene & Species：小鼠 Plexin-A1 (Plexa1, Mus musculus)

• 宿主表达体系 / Expression Host：哺乳动物细胞 (Mammalian Cells, 如 HEK293T, HeLa, N2a, 原代神经元)

• 细菌筛选标记 / Bacterial Resistance：氨苄青霉素 (Ampicillin, 100 ug/mL) 或 卡那霉素 (Kanamycin, 50 ug/mL)

• 哺乳动物筛选标记 / Mammalian Marker：蒲洛霉素 (Puromycin)、新霉素 (G418/Neomycin) 或 Hygromycin

• 核心元件构成 / Key Functional Components：

  • CMV / EF1a 启动子：驱动小鼠 Plexa1 全长 CDS 的强劲转录

  • 多克隆位点 (MCS) / 融合标签：可选择带 C 端/N 端融合标签（如 Flag, Myc, HA 或 EGFP），便于 Western Blot 杂交与免疫共沉淀 (Co-IP)

  • SV40 polyA / bGH polyA：保障 mRNA 的稳定加工与高效翻译

• 应用领域 / Applications：Plexin-A1 介导的神经轴突塌陷与引导机制、Sema3A/Sema6A 受体复合物组装、细胞迁移与肿瘤微环境研究

三、 培养条件与转染指南 / Culture & Handling Protocols

1. 细菌增殖与提质粒 / Bacterial Propagation

• 克隆宿主 / E. coli Host：E. coli DH5alpha 或 Stbl3（针对大型膜蛋白质粒，Stbl3 可有效减少重组变异）

• 培养基 / Broth & Agar：LB 液体/固体培养基 + 对应抗生素

• 转化步骤 / Transformation：

  1. 取 50 uL DH5alpha 或 Stbl3 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，涂布于抗生素培养皿。

2. 细胞转染与表达检测 / Transfection & Expression Assays

• 转染试剂 / Transfection Reagents：Lipofectamine 3000、PEI 或电转法（如原代神经元转染）

• 转染与检测流程 / Protocol：

  1. 将 HEK293T 或 N2a 细胞铺于 6 孔板，待汇合度达到 70% 至 80% 时进行转染。

  2. 转染后 24 至 48 小时收集细胞裂解液。

  3. 利用 PlexA1 特异性抗体或融合标签抗体（如 Anti-Flag / Anti-Myc）通过 Western Blot 验证蛋白过表达效率。

四、 技术指标简表 / Technical Data Summary

BioVector® Mouse PlexA1 Overexpression Plasmid

1. Background and Origin

The Mouse PlexA1 Overexpression Plasmid is a mammalian expression vector engineered for robust ectopic expression of mouse Plexin-A1 (PlexA1). Plexin-A1 serves as a transmembrane receptor and coreceptor for Class 3 and Class 6 Semaphorins (e.g., Sema3A and Sema6A), playing indispensable roles in neuronal axon guidance, synaptic development, cardiovascular morphogenesis, and immune cell migration. Driven by a high-efficiency promoter (such as CMV or EF1a), this construct enables gain-of-function studies, receptor-ligand interaction assays, and downstream GTPase signaling analysis in mammalian cells and primary neurons.

2. Biological Characteristics

• Vector Type: Mammalian Expression Vector / Overexpression Construct

• Target Gene & Species: Mouse Plexin-A1 (Plexa1, Mus musculus)

• Expression Host: Mammalian cell lines (e.g., HEK293T, HeLa, N2a, primary cultured neurons)

• Bacterial Selection: Ampicillin (100 ug/mL) or Kanamycin (50 ug/mL)

• Mammalian Selection: Puromycin, G418 (Neomycin), or Hygromycin

• Key Functional Components:

  • CMV / EF1a Promoter: Ensures robust constitutive transcription of mouse Plexa1 CDS

  • Multiple Cloning Site (MCS) / Epitope Tags: Optional C-terminal or N-terminal tags (Flag, Myc, HA, or EGFP) for detection and co-immunoprecipitation (Co-IP)

  • polyA Signal: SV40 polyA or bGH polyA for mRNA stabilization

• Applications: Axon guidance and growth cone collapse assays, Sema3A/Sema6A receptor complex assembly, cell motility, and Rho GTPase signaling cascade analysis

3. Culture and Handling Protocols

Bacterial Propagation and Plasmid Isolation

• Cloning Host: E. coli DH5alpha or Stbl3 (Stbl3 is recommended for large transmembrane gene constructs)

• Growth Medium: LB Broth / LB Agar supplemented with appropriate selective antibiotics

• Transformation Protocol:

  1. Thaw competent E. coli 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 selective plates.

Cell Transfection and Expression Assays

• Transfection: Transfect mammalian hosts using standard lipid reagents (e.g., Lipofectamine 3000) or electroporation (for primary neurons).

• Expression Verification: Harvest cells 24 to 48 hours post-transfection. Verify ectopic expression via Western Blotting or immunofluorescence using anti-PlexA1 or tag-specific antibodies (e.g., Anti-Flag, Anti-Myc).

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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