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基本描述
| 规格或纯度 | 医药级 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 英文名称 | Vaccinia Capping Enzyme | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 单位定义 | 在37℃下一个小时内把:10 pmol (α-32P) GTP掺入一个80个核苷酸(80nt)转录产物所需要的酶量 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 储存温度 | -20°C储存 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 运输条件 | 超低温冰袋运输 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 产品介绍 |
产品基本信息 来源：携带牛痘病毒加帽酶基因的E.coli 反应条件：1x Capping Buffer (50 mM Tnis-HC1, pH 8.0; 5 mM KC1; 1 mMMgaly; 1 mM DTT). 37℃孵有 储存缓冲液：20mM Tnis-HC1 pH8.0;100mMNaC1; 1mM DTT; 0.1mMEDIA;0.1%TritonX-100;50% (wv) Glycerol 产品描述 体外转录获得的mRNA未经细胞内一系列修饰，不具备Cap 结构与PolyA 尾，容易降解，易激活免疫反应，不能与核糖体起始蛋白结合，无法启动蛋白翻译，因而在工业化mRNA生产中，需使用牛痘病毒加帽酶对IVT 的mRNA进行加帽修饰，使mRNA的5' 端获得Cap0 结构，进一步使用2'-O- 甲基转移酶将Cap0 转化为Cap1。酶法加帽引入的帽结构与真核生物体内天然帽结构完全一致，从根本上降低外源mRNA 免疫原性的同时，保护其不被降解，并提高翻译效率，增加细胞内蛋白产量。通过酶法加帽最大可获得100%的加帽效率，而通过化学合成帽类似物结构加帽，其加帽效率相对较低，并且帽类似物结构与天然帽结构有差异。 牛痘病毒加帽酶将 7-甲基鸟苷帽结构（m7Gppp，Cap0）加到 RNA 的 5′ 末端。在真核生物中，该结构与 mRNA 的稳定、转运和翻译密切相关。通过酶促反应为 RNA 加帽是一种简单有效的方法，能显著改善用于体外转录、转染和显微注射的RNA的稳定性和翻译能力。这种酶由两个亚基（D1 和 D12）组成 （图 1a），D1 亚基执行 RNA 三磷酸酶和鸟苷转移酶的功能，D12亚基执行鸟嘌呤甲基转移酶的功能，它们对于添加一个完整的 Cap0 结构 m7Gppp5′N 都是必须的（图 1b）。? 本制品可用于 T7 RNA Polymerase, GMP Grade（GMP-E121，Novoprotein）反应产生的 RNA 的加帽反应，加帽反应在一小时之内完成，效率接近 100%，并且保证正确的方向。? 重组牛痘病毒加帽酶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。
图1. 牛痘病毒加帽的结构和作用机理。a. 牛痘病毒加帽酶和 GTP（红色）、SAH（玫红）的共结晶结构 (PDB 4CKB) 。该酶由 D1 和 D12 两个亚基组成，兼具 RNA 三磷酸酶（蓝色）、鸟苷转移酶（橙色）和鸟嘌呤甲基转移酶（米黄色）的功能。本图引自 Ramanathan, A., Robb, G. B., & Chan, S. H. (2016). mRNA capping: biological functions and applications. Nucleic Acids Res 44(16), 7511–7526. b. mRNA 的帽结构是由一个7-甲基鸟苷通过一个 5′–5′三磷酸酯桥连接到 mRNA 链的 5′核苷上组成。Cap-0 结构由相邻的 RNA 链通过三种酶的依次反应形成。进一步形成 cap-1 结构需要 2-O 甲基转移酶的参与，该修饰可以降低RNA 在体内引起的细胞先天免疫反应。本图引自 Decroly, E., Ferron, F., Lescar, J. et al. (2012). Conventional and unconventional mechanisms for capping viral mRNA. Nat Rev Microbiol 10, 51–65。? 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 体内或体外翻译前 mRNA 的加帽和 mRNA 的 5’末端标记。? 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. 用于加帽反应的 RNA 在使用之前应进行纯化并溶解于无核酸酶水。溶液中不能含有 EDTA 和盐。 2. 在配置反应体系时，可以加入 0.5 μl 的 RNase 抑制剂（GMP-E125，Novoprotein），同时去掉等体积的 RNase-free Water。? 3. 在与加帽酶反应之前加热 RNA 可以消除转录产物 5′的二级结构。如果转录产物的 5′端结构复杂，可以把加热时间延长至10 分钟。 4. SAM 在 pH 7–8, 37°C 条件下不稳定，需要在反应开始之前新鲜配置。可以事先计算好 SAM 的用量，在反应开始前把分装的 32 mM 的储备液稀释成 2 mM 的工作液。为避免 SAM 降解，该工作液需要保存于冰上。 5. 如果已知转录产物的 5′端结构复杂，反应时间可延长至 60 分钟以提高加帽效率。 6. 在标记 5′端时，GTP 储备液应稀释到 mRNA 浓度的 1–3 倍。 Basic product?information Source: E. coli carrying vaccinia virus capping enzyme gene Reaction conditions: 1x Capping Buffer (50 mM Tnis-HC1, pH 8.0; 5 mM KC1; 1 mMMgaly; 1 mM DTT). Incubation at 37°C Storage buffer: 20mM Tnis-HC1 pH8.0; 100mMNaC1; 1mM DTT; 0.1mMEDIA; 0.1% TritonX-100; 50% (wv) Glycerol Product Description The mRNA obtained by in vitro transcription has not undergone a series of intracellular modifications, does not have the Cap structure and PolyA tail, is easily degraded, easily activates the immune response, cannot bind to the ribosome initiation protein, and cannot initiate protein translation. Therefore, in the industrialized mRNA production, It is necessary to use vaccinia virus capping enzyme to cap the IVT mRNA to obtain the Cap0 structure at the 5'end of the mRNA, and further use 2'-O-methyltransferase to convert Cap0 to Cap1. The cap structure introduced by enzymatic capping is completely consistent with the natural cap structure in eukaryotes, which fundamentally reduces the immunogenicity of exogenous mRNA while protecting it from degradation, improving translation efficiency, and increasing intracellular protein production. Enzymatic capping can achieve a maximum capping efficiency of 100%, while capping by chemically synthesized cap analog structures has relatively low capping efficiency, and the structure of cap analogs is different from the natural cap structure. The vaccinia virus capping enzyme adds the 7-methylguanosine cap structure (m7Gppp, Cap0) to the 5′ end of the RNA. In eukaryotes, this structure is closely related to the stability, transport and translation of mRNA. Enzymatic reaction to cap RNA is a simple and effective method, which can significantly improve the stability and translation ability of RNA used for in vitro transcription, transfection and microinjection. This enzyme is composed of two subunits (D1 and D12) (Figure 1a). The D1 subunit performs the functions of RNA triphosphatase and guanosine transferase, and the D12 subunit performs the functions of guanine methyltransferase. It is necessary to add a complete Cap0 structure m7Gppp5′N (Figure 1b). This product can be used for the capping reaction of RNA produced by T7 RNA Polymerase and GMP Grade (GMP-E121, Novoprotein) reactions. The capping reaction is completed within one hour, with an efficiency close to 100% and ensuring the correct direction. Recombinant vaccinia virus capping enzyme is expressed by large-scale fermentation in Escherichia coli. It is produced with medicinal-specific raw materials and strictly controlled host protein residues and nucleic acid residues. Product production and quality management procedures comply with GMP standards to ensure the production process and all raw materials Traceable.
Figure 1. The structure and mechanism of vaccinia virus capping. a. Co-crystal structure of vaccinia virus capping enzyme, GTP (red) and SAH (rose red) (PDB 4CKB). The enzyme consists of two subunits, D1 and D12, and has the functions of RNA triphosphatase (blue), guanosine transferase (orange) and guanine methyltransferase (beige). This figure is quoted from Ramanathan, A., Robb, GB, & Chan, SH (2016). mRNA capping: biological functions and applications. Nucleic Acids Res 44(16), 7511–7526. b. The cap structure of mRNA consists of a 7-Methylguanosine is composed of a 5'-5' triphosphate bridge connected to the 5'nucleoside of the mRNA chain. The Cap-0 structure is formed by the sequential reaction of three adjacent RNA strands. The further formation of cap-1 structure requires the participation of 2-O methyltransferase. This modification can reduce the cellular innate immune response caused by RNA in the body. This figure is quoted from Decroly, E., Ferron, F., Lescar, J. et al. (2012). Conventional and unconventional mechanisms for capping viral mRNA. Nat Rev Microbiol 10, 51–65. 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 Capping of mRNA before translation in vivo or in vitro and labeling of 5'end of mRNA. Applications
The intact mRNA expresses GFP protein in the cell, the capping enzyme is compared with the cap analog Precautions 1. The RNA used for the capping reaction should be purified and dissolved in nuclease-free water before use. The solution must not contain EDTA and salt. 2. When configuring the reaction system, 0.5 μl of RNase inhibitor (GMP-E125, Novoprotein) can be added, and an equal volume of RNase-free Water can be removed at the same time. 3. Heating the RNA before reacting with the capping enzyme can eliminate the 5′ secondary structure of the transcription product. If the structure of the 5′ end of the transcription product is complex, the heating time can be extended to 10 minutes. 4. SAM is unstable at pH 7-8, 37°C and needs to be freshly configured before the reaction starts. The amount of SAM can be calculated in advance, and the aliquoted 32 mM stock solution can be diluted to 2 mM working solution before the reaction starts. To avoid degradation of SAM, this working solution needs to be stored on ice. 5. If the 5′ end of the transcription product is known to have a complex structure, the reaction time can be extended to 60 minutes to increase the capping efficiency. 6. When labeling the 5′ end, the GTP stock solution should be diluted to 1–3 times the mRNA concentration.? |
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