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基本描述
| 规格或纯度 | 医药级 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 英文名称 | E. coli Poly(A) Polymerase | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 单位定义 | 在 37℃条件下，10 min 内将1 nmol AMP 掺入到 RNA 所需的酶量定义为 1 个活性单位（U） | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 储存温度 | -20°C储存 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 运输条件 | 超低温冰袋运输 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 产品介绍 |
产品描述 通过加尾酶在mRNA 3' 末端引入Poly A尾，可增加mRNA的稳定性，增加mRNA翻译效率。使用加尾酶引入的Poly A尾的优势是简便易行，避免在质粒构建时引入过多的连续T碱基造成的质粒不稳定等问题。 E. coli Poly (A) Polymerase 不依赖模板的存在，可以催化 ATP 以 AMP 的形式依次掺入到 RNA 的 3′末端，即在 RNA 的 3′末端加多聚 A 尾。Poly (A) 聚合酶具有很高的加尾效率，可以在 RNA 的 3′末端加入 20~200 个 A 碱基。Poly (A)结构有助于提高 mRNA 的翻译效率。? 本制品利用大肠杆菌大规模发酵表达，采用药用规格原辅料生产，并严格控制宿主蛋白质残留、核酸残留等，符合 GMP 规范的产品生产与质量管理规程保障生产过程及所有原辅料可追溯。 质量要求
遵循以下规范生产 1. ISO 9001:2015, certified facility。 2. 《GMP 附录-细胞治疗产品》国家药品监督管理局。 3. 《人用基因治疗总论-中国药典 2020》国家药典委。 4. USP Chapter <1043>, Ancillary Materials for Cell, Gene, and Tissue-Engineered Products 用于细胞治疗，基因治疗和组织工程产品中的辅料。 5. USP Chapter <92>, Growth Factors and Cytokines Used in Cell Therapy Manufacturing 细胞治疗产品生产过程中细胞因子和生长因子。 6.Ph. Eur. General Chapter 5.2.12, Raw Materials of Biological Origin for the Production of Cell-based and Gene Therapy Medicinal Products 用于生产细胞或基因治疗药物的生物来源原料。 产品用途 1.? 用于 RNA 3′末端标记。 2.? 为 RNA 添加 Poly(A)尾，用于克隆或亲和纯化。比如将 miRNA 添加 Poly (A)，为 cDNA 合成提供 oligo-dT 引物结合位点。 3.? 提高 RNA 在真核细胞中的翻译效率。 保存体系 20mM Tris-HCl; 300mM NaCl; 1mM DTT; 1mM EDTA; 0.1%TritonX-100; 50% (v/v) Glycerol, pH7.5。? 应用实例
完整mRNA在细胞内表达GFP蛋白，加帽酶与帽类似物对比 注意事项 1. 热失活条件：65℃，20 min。 2. 该酶只能以 RNA 为底物。 3. 该酶将 AMP 添加到 RNA 的3′末端具有较高的选择性，并不会在所有 RNA 分子中添加相同长度的 Poly (A)。 4. 该酶使用 M-MuLV 逆转录酶反应缓冲液，也可以进行反应。 5. 该酶需要 Mg2+等二价阳离子才具有活性。 6. RNA 加 A 尾长度受酶量、ATP 和反应时间等因素影响，不同的实验需要加 A 的量会有所不同，可通过减少反应时间来调整加 A 的长度，该酶在 37°C 反应 30 分钟时可加约 30 个 A 碱基，1 小时可加约 100 个 A 碱基。 7. EDTA 抑制该酶活性。若停止反应，可通过添加 EDTA 至终浓度 10mM，或直接对其进行纯化。 Product Description The introduction of Poly A tail at the 3'end of mRNA by tailing enzyme can increase the stability of mRNA and increase the efficiency of mRNA translation. The advantage of using the tailing enzyme to introduce the Poly A tail is that it is simple and easy to implement, avoiding the instability of the plasmid caused by the introduction of too many consecutive T bases during plasmid construction. E. coli Poly (A) Polymerase does not rely on the presence of a template, and can catalyze the incorporation of ATP into the 3′ end of RNA in the form of AMP, that is, a poly A tail is added to the 3′ end of RNA. Poly (A) polymerase has a high tailing efficiency and can add 20~200 A bases to the 3′ end of RNA. Poly (A) structure helps to improve the translation efficiency of mRNA. This product is expressed in large-scale fermentation using Escherichia coli, is produced with medicinal specifications of raw materials, and strictly controls host protein residues, nucleic acid residues, etc., and conforms to GMP standard product production and quality management procedures to ensure that the production process and all raw materials can be traced. Quality requirements
Follow the following specifications for production 1. ISO 9001:2015, certified facility. 2. "GMP Appendix-Cell Therapy Products" State Drug Administration. 3. "General Introduction to Human Gene Therapy-Chinese Pharmacopoeia 2020" National Pharmacopoeia Commission. 4. USP Chapter <1043>, Ancillary Materials for Cell, Gene, and Tissue-Engineered Products are used as excipients in cell therapy, gene therapy and tissue engineering products. 5. USP Chapter <92>, Growth Factors and Cytokines Used in Cell Therapy Manufacturing Cytokines and growth factors used in the production of cell therapy products. 6.Ph. Eur. General Chapter 5.2.12, Raw Materials of Biological Origin for the Production of Cell-based and Gene Therapy Medicinal Products. Product Usage 1. Used for RNA 3′ end labeling. 2. Add Poly(A) tail to RNA for cloning or affinity purification. For example, adding Poly (A) to miRNA provides an oligo-dT primer binding site for cDNA synthesis. 3. Improve the translation efficiency of RNA in eukaryotic cells. Preservation system 20mM Tris-HCl; 300mM NaCl; 1mM DTT; 1mM EDTA; 0.1% TritonX-100; 50% (v/v) Glycerol, pH 7.5. Applications
The intact mRNA expresses GFP protein in the cell, the capping enzyme is compared with the cap analog Precautions 1. Thermal inactivation conditions: 65°C, 20 min. 2. The enzyme can only use RNA as a substrate. 3. The enzyme adds AMP to the 3′ end of RNA with high selectivity, and does not add the same length of Poly (A) to all RNA molecules. 4. The enzyme uses M-MuLV reverse transcriptase reaction buffer, which can also be used for reaction. 5. The enzyme requires divalent cations such as Mg2+ to be active. 6. The length of RNA plus A tail is affected by the amount of enzyme, ATP, reaction time and other factors. The amount of A required for different experiments will be different. The length of A can be adjusted by reducing the reaction time. The enzyme is at 37°C. About 30 A bases can be added in 30 minutes of reaction, and about 100 A bases can be added in 1 hour. 7. EDTA inhibits the enzyme activity. If the reaction is stopped, it can be purified directly by adding EDTA to a final concentration of 10mM. |
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