BioVector® pNV18 诺卡氏菌-大肠杆菌穿梭克隆载体 BioVector® pNV18 Nocardia-E. coli Shuttle Cloning Vector

第一部分：中文说明

一、 产品基本信息与详细特征描述

• 产品名称：BioVector® pNV18 诺卡氏菌-大肠杆菌穿梭克隆载体

  
  

• 载体名称：BioVector® pNV18

• 质粒大小：4411 bp

• 载体类型：广宿主穿梭克隆载体 (Broad Host-Range Shuttle Vector)

• 抗性基因：卡那霉素 (Kanamycin) / 新霉素 (Neomycin) 抗性基因 (aph)

• 复制子：大肠杆菌高拷贝复制子 (ori / pUC来源) + 分枝杆菌/诺卡氏菌质粒复制起点 (pAL5000 ori)

• 生物安全级别：1级 (BSL-1)

• 详细特征描述：BioVector® pNV18 是一种专为诺卡氏菌属 (Nocardia spp.) 遗传操作系统开发的经典、高效的穿梭克隆载体。该载体分子量小（约 4.4 kb），是由分枝杆菌质粒 pAL5000 的 1.8 kb 复制起点片段精准插入到大肠杆菌经典载体 pK18 的 NheI 独特位点中构建而成。这种设计赋予了它独特的“穿梭”功能：既包含大肠杆菌复制子，能在大肠杆菌宿主中进行高拷贝扩增；又包含广宿主分枝杆菌复制起点，使其在星形诺卡氏菌 (N. asteroides)、皮生诺卡氏菌 (N. farcinica)、巴西诺卡氏菌 (N. brasiliensis) 以及多种分枝杆菌中表现出卓越的复制与维持能力。载体带有源自转座子 Tn5 的 aph 基因，该基因表达的氨基糖苷磷酸转移酶能够同时对大肠杆菌和诺卡氏菌提供高水平的卡那霉素和新霉素抗性选择表型。此外，pNV18 完整保留了 lacZ 基因（α 片段）和多克隆位点 (MCS)，支持在大肠杆菌中进行极为便利的蓝白斑筛选，是诺卡氏菌基因克隆、异源表达及基因功能研究的核心分子生物学工具。

  
  

二、 细胞培养与质粒克隆条件

• 大肠杆菌克隆与扩增条件：进行重组克隆或质粒大量提取时，应将质粒转化至大肠杆菌常规感受态细胞（如 Top10 或 DH5α）。使用标准的 BioVector® LB 液体培养基或 LB 固体琼脂培养基，培养基中需添加终浓度为 50 微克每毫升的 BioVector® 卡那霉素。于 37°C 恒温振荡培养箱中以每分钟 200 到 220 转的转速孵育过夜。若需进行蓝白斑筛选，平板表面应提前涂布适量的 IPTG 和 X-gal。

  
  

• 诺卡氏菌转化与筛选条件：纯化后的重组质粒通过电转化法导入诺卡氏菌感受态细胞。转化后的细菌接种于 BioVector® 脑心浸液 (BHI) 琼脂平板或营养琼脂平板上。为了确保质粒在诺卡氏菌中的高稳定性，筛选培养基中通常添加终浓度为 25 微克每毫升的 BioVector® 新霉素或 50 微克每毫升的卡那霉素。培养温度根据宿主菌株设定（通常为 30°C 至 37°C）。

  
  

三、 多克隆位点 (MCS) 与蓝白斑筛选

• 多克隆位点酶切位点：BioVector® pNV18 拥有丰富的独特限制性内切酶位点，顺序排列包括：HindIII, SphI, PstI, SalI, HincII, XbaI, BamHI, KpnI, 以及 EcoRI。

  
  

• 蓝白斑筛选机制：MCS 位于 lacZ 的 α 片段编码区内。当无外源片段插入时，载体能在大肠杆菌表达宿主中通过 α 互补作用产生有活性的 β-半乳糖苷酶，水解 X-gal 使菌落呈现蓝色。当外源目的基因精准克隆至 MCS 后，会破坏 α 片段的阅读框或完整性，导致互补作用丧失，转化子菌落呈现白色，从而实现重组子的快速肉眼鉴定。

  
  

四、 质粒冻存与长期保藏技术

• 含质粒工程菌株保藏：将处于对数生长旺盛期（OD600 约为 0.6 至 0.8）的含质粒大肠杆菌菌液，按照 7 比 3 的体积比与无菌的 BioVector® 细胞级甘油充分混合（最终甘油浓度为 30%）。分装至冻存管中，直接置于零下 80°C 超低温冰箱中冷冻保藏。

• 质粒 DNA 长期储存：使用 BioVector® 高纯度质粒无内毒素提取试剂盒纯化质粒 DNA。将纯化后的 DNA 溶解于 BioVector® TE 缓冲液（无菌，pH 8.0）或无核酸酶的超纯水中。测量准确浓度后，建议分装成小体系（如每管 5 微克），置于零下 20°C 冰箱或零下 80°C 超低温冰箱中保存。严格避免反复冻融以防 DNA 链发生物理剪切或降解。

  
  

五、 质量控制与科研应用指南

• 质量控制标准：BioVector® pNV18 载体经过极其严格的全面质量检测。通过全长高通量测序确认 4411 bp 序列无自发性突变，确保 MCS 序列、aph 选择标记及复制子的绝对准确性；酶切电泳图谱与官方理论质粒图谱 100% 一致；产品不含外源宿主核酸、DNase/RNase（核酸酶）污染及细菌内毒素。

  
  

• 核心实验应用：该穿梭载体广泛用于皮生诺卡氏菌等致病性/非致病性诺卡氏菌的基因功能敲除补救实验、环境微生物降解基因的异源表达改造、诺卡氏菌独特次级代谢产物（如抗生素及活性物质）的生物合成途径重组、放线菌门复杂启动子活性的体内定量分析，以及分枝杆菌属与诺卡氏菌属的通用型基础克隆研究。

  
  

PART 2: ENGLISH SECTION

I. General Information and Detailed Product Characterization

• Product Name: BioVector® pNV18 Nocardia-E. coli Shuttle Cloning Vector

  
  

• Vector Name: BioVector® pNV18

• Plasmid Size: 4411 bp

• Vector Type: Broad Host-Range Shuttle Cloning Vector

• Selection Marker: Kanamycin / Neomycin resistance gene (aph)

• Origins of Replication: E. coli high-copy replicon (ori / pUC-derived) + Mycobacterial/Nocardial plasmid origin (pAL5000 ori)

  
  

• Biosafety Level: BSL-1

• Detailed Description: BioVector® pNV18 is a classic and highly robust shuttle cloning vector purposefully designed for genetic engineering and manipulation within the genus Nocardia. Characterized by its compact size (approximately 4.4 kb), this plasmid was engineered by integrating a 1.8 kb functional DNA fragment containing the minimal replication origin of the mycobacterial plasmid pAL5000 directly into the unique NheI restriction site of the E. coli vector pK18. This structural configuration grants the plasmid dual replication capabilities: it behaves as a high-copy cloning vector in E. coli hosts, and maintains reliable replication stability as a shuttle vector inside Nocardia farcinica, Nocardia asteroides, Nocardia brasiliensis, and various Mycobacteria species. The plasmid carries the aph gene derived from transposon Tn5, which encodes an aminoglycoside phosphotransferase conferring high-level resistance to both Kanamycin and Neomycin, working effectively in both E. coli and Nocardia expression systems. Retaining the full lacZ alpha-peptide sequence and a comprehensive multiple cloning site (MCS), pNV18 supports convenient blue-white colony screening in E. coli, making it an indispensable molecular biology tool for gene cloning, heterologous pathway expression, and functional genomics in actinomycetes.

  
  

II. Culture Conditions and Cloning Parameters

• E. coli Maintenance and Amplification: For common cloning assays, insert ligations, or large-scale plasmid extractions, the vector should be transformed into traditional E. coli competent cells (such as Top10 or DH5α).Transformed cells must be propagated in standardized BioVector® LB Liquid Medium or LB Agar Medium containing a final concentration of 50 micrograms per milliliter of BioVector® Kanamycin.Incubate overnight in a temperature-controlled shaking incubator at 37°C with an agitation rate of 200 to 220 RPM.For blue-white screening assays, ensure selective plates are pre-coated with proper amounts of IPTG and X-gal.

  
  

• Nocardia Transformation and Selection Parameters: Purified recombinant plasmids are introduced into Nocardia competent cells primarily via electroporation protocols. Following pulse recovery, cells are spread onto BioVector® Brain Heart Infusion (BHI) Agar or Nutrient Agar plates. To ensure long-term stability and prevent segregational loss of the plasmid within Nocardia populations, selective media must be supplemented with either 25 micrograms per milliliter of BioVector® Neomycin or 50 micrograms per milliliter of Kanamycin.Incubation temperatures should be optimized based on the specific host strain requirements, typically spanning 30°C to 37°C.

  
  

III. Multiple Cloning Site (MCS) and Blue-White Screening Mechanics

• MCS Restriction Endonuclease Sites: The multiple cloning site region inside BioVector® pNV18 comprises a versatile array of unique restriction sites ordered as follows: HindIII, SphI, PstI, SalI, HincII, XbaI, BamHI, KpnI, and EcoRI.

  
  

• Blue-White Selection System: The MCS is nested precisely within the coding sequence of the lacZ alpha-peptide. In the absence of an integrated insert, alpha-complementation occurs normally within appropriate E. coli hosts, producing active β-galactosidase which cleaves X-gal and stains the colony distinctively blue.Successful ligation of a target gene fragment into any MCS site disrupts the reading frame or structural integrity of the alpha-peptide, abolishing complementation activity.Consequently, recombinant transformants stand out as white colonies, allowing rapid visual identification.

  
  

IV. Plasmid Preservation and Long-Term Storage Methodology

• Glycerol Stock Preservation of Host Strains: Collect host E. coli strains carrying the validated plasmid during their active logarithmic growth phase (OD600 around 0.6–0.8). Mix 700 microliters of the bacterial suspension thoroughly with 300 microliters of sterile, BioVector® Cell-Grade Glycerol to yield a final concentration of 30% glycerol inside a sterile cryovial. Store the vials directly in a minus 80°C ultra-low temperature freezer.

• Purified Plasmid DNA Archiving: Extract the plasmid DNA using a BioVector® High-Purity Endotoxin-Free Plasmid Extraction Kit.Dissolve the clean DNA pellet in sterile BioVector® TE Buffer (pH 8.0) or nuclease-free ultra-pure water.After executing a precise concentration measurement, aliquot the DNA into small single-use batches (e.g., 5 micrograms per tube) and store at minus 20°C or minus 80°C.Avoid repeating freeze-thaw cycles, as this degrades the structural integrity of circular plasmid DNA strands.

  
  

V. Quality Control and Research Application Guidelines

• Quality Control Standards: The BioVector® pNV18 vector undergoes exhaustive quality testing protocols. High-throughput sequencing verifies the absolute correctness of the 4411 bp full-length sequence, confirming zero point mutations across critical features including the MCS, aph gene, and replication machinery. Restriction digestion profiles show a 100% alignment with theoretical map expectations. The final formulation is certified free from contaminating genomic DNA, active nucleases (DNase/RNase), and bacterial endotoxins.

  
  

• Core Experimental Applications: This versatile shuttle vector is heavily utilized for genetic complementation assays following gene-knockouts in pathogenic and non-pathogenic Nocardia, heterologous expression and engineering of bioremediation pathways, reconstitution of complex biosynthetic gene clusters (BGCs) for novel secondary metabolites/antibiotics, in vivo quantitative profiling of actinomycetal promoter strengths, and general cloning applications bridging the genera Mycobacterium and Nocardia.

BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

电话:400-800-2947

工作QQ/微信同号:1843439339

网址

http://www.biovector.net