BioVector? pNV18 諾卡氏菌-大腸桿菌穿梭克隆載體 BioVector? pNV18 Nocardia-E. coli Shuttle Cloning Vector

第一部分：中文說明

一、 產(chǎn)品基本信息與詳細(xì)特征描述

• 產(chǎn)品名稱：BioVector? pNV18 諾卡氏菌-大腸桿菌穿梭克隆載體

  
  

• 載體名稱：BioVector? pNV18

• 質(zhì)粒大小：4411 bp

• 載體類型：廣宿主穿梭克隆載體 (Broad Host-Range Shuttle Vector)

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

• 復(fù)制子：大腸桿菌高拷貝復(fù)制子 (ori / pUC來源) + 分枝桿菌/諾卡氏菌質(zhì)粒復(fù)制起點(diǎn) (pAL5000 ori)

• 生物安全級(jí)別：1級(jí) (BSL-1)

• 詳細(xì)特征描述：BioVector? pNV18 是一種專為諾卡氏菌屬 (Nocardia spp.) 遺傳操作系統(tǒng)開發(fā)的經(jīng)典、高效的穿梭克隆載體。該載體分子量小（約 4.4 kb），是由分枝桿菌質(zhì)粒 pAL5000 的 1.8 kb 復(fù)制起點(diǎn)片段精準(zhǔn)插入到大腸桿菌經(jīng)典載體 pK18 的 NheI 獨(dú)特位點(diǎn)中構(gòu)建而成。這種設(shè)計(jì)賦予了它獨(dú)特的“穿梭”功能：既包含大腸桿菌復(fù)制子，能在大腸桿菌宿主中進(jìn)行高拷貝擴(kuò)增；又包含廣宿主分枝桿菌復(fù)制起點(diǎn)，使其在星形諾卡氏菌 (N. asteroides)、皮生諾卡氏菌 (N. farcinica)、巴西諾卡氏菌 (N. brasiliensis) 以及多種分枝桿菌中表現(xiàn)出卓越的復(fù)制與維持能力。載體帶有源自轉(zhuǎn)座子 Tn5 的 aph 基因，該基因表達(dá)的氨基糖苷磷酸轉(zhuǎn)移酶能夠同時(shí)對(duì)大腸桿菌和諾卡氏菌提供高水平的卡那霉素和新霉素抗性選擇表型。此外，pNV18 完整保留了 lacZ 基因（α 片段）和多克隆位點(diǎn) (MCS)，支持在大腸桿菌中進(jìn)行極為便利的藍(lán)白斑篩選，是諾卡氏菌基因克隆、異源表達(dá)及基因功能研究的核心分子生物學(xué)工具。

  
  

二、 細(xì)胞培養(yǎng)與質(zhì)?？寺l件

• 大腸桿菌克隆與擴(kuò)增條件：進(jìn)行重組克隆或質(zhì)粒大量提取時(shí)，應(yīng)將質(zhì)粒轉(zhuǎn)化至大腸桿菌常規(guī)感受態(tài)細(xì)胞（如 Top10 或 DH5α）。使用標(biāo)準(zhǔn)的 BioVector? LB 液體培養(yǎng)基或 LB 固體瓊脂培養(yǎng)基，培養(yǎng)基中需添加終濃度為 50 微克每毫升的 BioVector? 卡那霉素。于 37°C 恒溫振蕩培養(yǎng)箱中以每分鐘 200 到 220 轉(zhuǎn)的轉(zhuǎn)速孵育過夜。若需進(jìn)行藍(lán)白斑篩選，平板表面應(yīng)提前涂布適量的 IPTG 和 X-gal。

  
  

• 諾卡氏菌轉(zhuǎn)化與篩選條件：純化后的重組質(zhì)粒通過電轉(zhuǎn)化法導(dǎo)入諾卡氏菌感受態(tài)細(xì)胞。轉(zhuǎn)化后的細(xì)菌接種于 BioVector? 腦心浸液 (BHI) 瓊脂平板或營養(yǎng)瓊脂平板上。為了確保質(zhì)粒在諾卡氏菌中的高穩(wěn)定性，篩選培養(yǎng)基中通常添加終濃度為 25 微克每毫升的 BioVector? 新霉素或 50 微克每毫升的卡那霉素。培養(yǎng)溫度根據(jù)宿主菌株設(shè)定（通常為 30°C 至 37°C）。

  
  

三、 多克隆位點(diǎn) (MCS) 與藍(lán)白斑篩選

• 多克隆位點(diǎn)酶切位點(diǎn)：BioVector? pNV18 擁有豐富的獨(dú)特限制性內(nèi)切酶位點(diǎn)，順序排列包括：HindIII, SphI, PstI, SalI, HincII, XbaI, BamHI, KpnI, 以及 EcoRI。

  
  

• 藍(lán)白斑篩選機(jī)制：MCS 位于 lacZ 的 α 片段編碼區(qū)內(nèi)。當(dāng)無外源片段插入時(shí)，載體能在大腸桿菌表達(dá)宿主中通過 α 互補(bǔ)作用產(chǎn)生有活性的 β-半乳糖苷酶，水解 X-gal 使菌落呈現(xiàn)藍(lán)色。當(dāng)外源目的基因精準(zhǔn)克隆至 MCS 后，會(huì)破壞 α 片段的閱讀框或完整性，導(dǎo)致互補(bǔ)作用喪失，轉(zhuǎn)化子菌落呈現(xiàn)白色，從而實(shí)現(xiàn)重組子的快速肉眼鑒定。

  
  

四、 質(zhì)粒凍存與長期保藏技術(shù)

• 含質(zhì)粒工程菌株保藏：將處于對(duì)數(shù)生長旺盛期（OD600 約為 0.6 至 0.8）的含質(zhì)粒大腸桿菌菌液，按照 7 比 3 的體積比與無菌的 BioVector? 細(xì)胞級(jí)甘油充分混合（最終甘油濃度為 30%）。分裝至凍存管中，直接置于零下 80°C 超低溫冰箱中冷凍保藏。

• 質(zhì)粒 DNA 長期儲(chǔ)存：使用 BioVector? 高純度質(zhì)粒無內(nèi)毒素提取試劑盒純化質(zhì)粒 DNA。將純化后的 DNA 溶解于 BioVector? TE 緩沖液（無菌，pH 8.0）或無核酸酶的超純水中。測量準(zhǔn)確濃度后，建議分裝成小體系（如每管 5 微克），置于零下 20°C 冰箱或零下 80°C 超低溫冰箱中保存。嚴(yán)格避免反復(fù)凍融以防 DNA 鏈發(fā)生物理剪切或降解。

  
  

五、 質(zhì)量控制與科研應(yīng)用指南

• 質(zhì)量控制標(biāo)準(zhǔn)：BioVector? pNV18 載體經(jīng)過極其嚴(yán)格的全面質(zhì)量檢測。通過全長高通量測序確認(rèn) 4411 bp 序列無自發(fā)性突變，確保 MCS 序列、aph 選擇標(biāo)記及復(fù)制子的絕對(duì)準(zhǔn)確性；酶切電泳圖譜與官方理論質(zhì)粒圖譜 100% 一致；產(chǎn)品不含外源宿主核酸、DNase/RNase（核酸酶）污染及細(xì)菌內(nèi)毒素。

  
  

• 核心實(shí)驗(yàn)應(yīng)用：該穿梭載體廣泛用于皮生諾卡氏菌等致病性/非致病性諾卡氏菌的基因功能敲除補(bǔ)救實(shí)驗(yàn)、環(huán)境微生物降解基因的異源表達(dá)改造、諾卡氏菌獨(dú)特次級(jí)代謝產(chǎn)物（如抗生素及活性物質(zhì)）的生物合成途徑重組、放線菌門復(fù)雜啟動(dòng)子活性的體內(nèi)定量分析，以及分枝桿菌屬與諾卡氏菌屬的通用型基礎(chǔ)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.

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