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mRNA Cap 2′-O-Methyltransferase
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| 规格或纯度 | 医药级 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 英文名称 | mRNA Cap 2′-O-Methyltransferase | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 单位定义 | 在37℃下一个小时内将10 pmol 80nt 的带帽RNA转录子甲基化所需酶量 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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mRNA帽结构2'-O-甲基转移酶基因的E.coli菌株。 反应条件：1× Capping Reaction Buffer (50 mM Tnis-HC1, pH 8.0; 5 mMKCl; 1 mMMgCl; 1 mM DTT),37C孵育 储存缓冲液：20mM Tnis-HCl pH8.0;100mMNaC1; 1mMDTT;0.1mM EDTA;0.1%TritonX-100; 50%(vv)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。 mRNA Cap 2′-O-Methyltransferas 可利用 S-腺苷甲硫氨酸（SAM）作为甲基供体， 在 RNA 5′末端紧邻帽结构（Cap 0）的第一个核苷酸的 2′-O 上添加甲基基团，形成带有 Cap 1 结构的 mRNA。Cap1 结构能增强 mRNA 的翻译效率，从而可提高转染后 mRNA 编码的蛋白表达水平。该酶只能以带 7-甲基鸟苷帽结构（m7GpppN）的 RNA 为底物，不会作用于 5′末端为pN、ppN、 pppN 或 GpppN 的 RNA。带 7-甲基鸟苷帽结构（m7GpppN）的 RNA 可通过 Vaccinia Capping System（Cat. No: GMP-M062, Novoprotein）来制备。? 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。?
图1. mRNA 帽结构 2′ -O-甲基转移酶作用机理。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 Cap0 结构的 mRNA 2′-O 甲基化（Cap1），以提高 mRNA 的翻译和表达效率。 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. 用于反应的 RNA 在使用之前应进行纯化并溶解于 RNase-free water。溶液中不能含有 EDTA 和盐； 2. 在配置反应体系时，建议使用 RNase 抑制剂以增强反应中 RNA 的稳定性。可以加入 0.5μl 的 RNase 抑制剂（Cat. No: GMP-E125, Novoprotein），同时去掉等体积的 RNase-free water； 3. 反应之前加热 RNA 可以去除 5′端的二级结构。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。可事先计算好 SAM 的用量，在反应开始前将 32 mM的储备液稀释成 4 mM 的工作液。为避免 SAM 降解，该工作液需要保存于冰上。 Basic product information Source: E. coli strain carrying the 2'-O-methyltransferase gene of the vaccinia virus mRNA cap structure. Reaction conditions: 1× Capping Reaction Buffer (50 mM Tnis-HC1, pH 8.0; 5 mMKCl; 1 mMMgCl; 1 mM DTT), incubated at 37C Storage buffer: 20mM Tnis-HCl pH8.0; 100mMNaC1; 1mMDTT; 0.1mM EDTA; 0.1% TritonX-100; 50% (vv) 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. mRNA Cap 2′-O-Methyltransferas can use S-adenosylmethionine (SAM) as a methyl donor, at the 2′- end of the RNA 5′ end next to the first nucleotide of the cap structure (Cap 0) Add a methyl group to O to form mRNA with Cap 1 structure. The Cap1 structure can enhance the translation efficiency of mRNA, thereby increasing the expression level of the protein encoded by the mRNA after transfection. This enzyme can only use RNA with a 7-methylguanosine cap structure (m7GpppN) as a substrate, and will not act on RNA with pN, ppN, pppN or GpppN at the 5′ end. RNA with 7-methylguanosine cap structure (m7GpppN) can be prepared by Vaccinia Capping System (Cat. No: GMP-M062, Novoprotein). 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.
Figure 1. mRNA cap structure 2′ -O-methyltransferase mechanism of action. The cap structure of mRNA consists of a 7-methylguanosine connected to the 5'nucleoside of the mRNA chain via a 5'-5' triphosphate bridge. 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, which 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 requirement
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 The mRNA of Cap0 structure is 2′-O methylated (Cap1) to improve the efficiency of mRNA translation and expression. Applications
The intact mRNA expresses GFP protein in the cell, the capping enzyme is compared with the cap analog Precautions 1. The RNA used in the reaction should be purified and dissolved in RNase-free water before use. The solution must not contain EDTA and salt; 2. When configuring the reaction system, it is recommended to use an RNase inhibitor to enhance the stability of RNA in the reaction. 0.5μl of RNase inhibitor (Cat. No: GMP-E125, Novoprotein) can be added, and the same volume of RNase-free water can be removed at the same time; 3. Heating RNA before the reaction can remove the secondary structure at the 5′ end. 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 32 mM stock solution can be diluted to 4 mM working solution before the reaction starts. To avoid degradation of SAM, this working solution needs to be stored on ice. |
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