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主页疫苗生产疫苗和药物开发用mRNA合成

疫苗和药物开发用mRNA合成

合成 mRNA 有可能重现成熟 mRNA 功能，有望应用于一系列生物制药应用。这类治疗应用包括体内递送 mRNA 进行蛋白质替代、干细胞诱导或癌症免疫治疗。最近的重要制药应用包括针对传染病的新疫苗途径——尤其是针对 SARS-CoV-2 的 mRNA 疫苗。

索取更多信息

如需完整的 mRNA 疫苗产品和解决方案，请访问我们的疫苗开发和生产页面。

mRNA 疫苗如何诱导免疫

使 RNA (mRNA) 疫苗基于 mRNA 编码蛋白质的中心法则。这种直接的疫苗途径以编码全部或部分抗原蛋白的遗传元件作为可翻译分子，诱导免疫反应。 编码靶标抗原的 mRNA 被抗原递送细胞摄入并翻译成靶标病原体蛋白，进而诱导免疫反应。此方法可精密模仿病毒感染细胞的自然过程。

mRNA 作为疫苗途径的优势

通常，mRNA疫苗通过酶促过程在体外合成生产。相对于更传统的途径，mRNA 疫苗开发和制造过程相对简单，且可在短时间内完成。这一性质使其适合加速开发和放大生产，迄今经证对于紧急公共卫生事件至关重要。创新的 mRNA 疫苗设计和制剂可增强热稳定性，使疫苗无需冷链配送，从而降低生产成本并提高全球覆盖范围。 

mRNA 合成的质粒 DNA(pDNA) 制备

对于 mRNA 疫苗设计，通常通过体外转录质粒 DNA (pDNA) 模板制备功能性合成 mRNA。质粒载体通常包含以下元件：T7、SP6或T3 RNA聚合酶（均源自噬菌体）特异性识别的上游启动子； 5' UTR、cDNA、3' UTR、下游poly A尾； 以及poly A尾下游的独特切割位点。

体外转录 (IVT) 质粒制备步骤。

图 1.体外转录（IVT）质粒制备。基因合成后，使用质粒载体 (pDNA) 克隆目标 DNA。pDNA 在细菌培养物中扩增，然后通过核酸纯化方法（例如离心柱搭配硅胶模膜）进行纯化。

mRNA 体外 转录和加帽

噬菌体 RNA 聚合酶常用于转录线性化质粒 DNA。首先用指定的独特限制位点切割酶将 pDNA 线性化。酶解后，可通过苯酚-氯仿方案或GenErude PCR纯化试剂盒等方法纯化线性化的 pDNA。如需进行大规模纯化，通常建议使用切向流过滤(TFF)，因为此过滤技术支持轻松放大。线性化后，在含有重组RNA聚合酶（T7、T3 或 SP6）和三磷酸核苷以及帽类似物（如 CleanCap®试剂或 ARCA（抗反向帽类似物））的混合溶液中进行体外转录和加帽。N1-甲基伪尿苷-5'-三磷酸（N1-甲基伪尿苷、1-甲基伪尿苷）等修饰核苷可代替GTP抑制先天免疫系统，用于现有mRNA疫苗。  由于与GTP竞争，ARCA对mRNA的平均加帽效率为70~80%——与标准mRNA合成相比，mRNA产量减少了约四分之一。  而CleanCap®试剂表现出 94% 的加帽效率，且不影响 mRNA 产量。也可在无帽类似物的情况下转录加帽，此时需要转用痘苗病毒编码的加帽复合物（加帽酶、2'-O-甲基转移酶、GTP 和 S-腺苷甲硫氨酸 (SAM)）。加帽效率因目标 mRNA 的二级结构而异。最后，当模板pDNA的poly A尾长度不足（最多150个碱基）时，可使用poly A酶进行延伸。

mRNA合成步骤

图 2.mRNA合成通过pDNA线性化、无细胞方法mRNA的体外转录，以及利用帽类似物或病毒编码加帽复合物进行的mRNA加帽实现。

体外转录 (IVT) mRNA 合成试剂
产品编号产品名称说明价格
TMS-010阿尔新蓝染色液
TMS-008Alizarin-Red Staining SolutionAlizarin red is a commonly used stain to identify calcium containing osteocytes in differentiated culture of both human and rodent mesenchymal stem cells (MSCs).
CBL154Anti-H-CAM Antibody, clone F10-44-2clone F10-44-2, Chemicon®, from mouse
CBL154Anti-H-CAM Antibody, clone F10-44-2clone F10-44-2, Chemicon®, from mouse
CBL415Anti-Thy-1 Antibody, clone F15-42-1clone F15-42-1, Chemicon®, from mouse
CLS431751Corning®细胞过滤网pore size 70 μm, white, sterile, pkg of (individually wrapped), pack of 50 ea, Corning 431751
D2650二甲基亚砜Hybri-Max™, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
ES-020-BFetal Bovine SerumUS Origin, Mesenchymal Stem Cell Qualified FBS, sterile-filtered, suitable for stem cell culture
F0291hBFGFFGF-Basic, recombinant, expressed in E. coli, suitable for cell culture
SCC038Human Adipose Mesenchymal Stem CellsHuman Adipose Mesenchymal Stem Cells (MSCs) are isolated from human adipose tissue and are cryopreserved at P2 to ensure optimal phenotype and the highest viability and plating efficiency.
SCC034人间充质干细胞（骨髓）Human mesenchymal stem cells derived from bone marrow that have a capacity for expansion in vitro while maintaining their potential for differentiation to multiple lineages including adipocytes, osteoblasts and chondrocytes.
SCM045人间充质-XF扩增培养基Xeno-free mesenchymal stem cell media specially formulated for the rapid expansion of human mesenchymal stem cells isolated from a variety of sources, including adipose and bone marrow tissues.
FC010人血浆纤连蛋白纯化蛋白from human plasma, liquid, 1 mg/mL (100 MG pack size is lyophilized), purified protein, suitable for cell culture
SCM015间充质干细胞扩展培养基（1x）The Mesenchymal Stem Cell Expansion Medium (1x) has been optimized & validated for Stem cell culture. This medium is available in a 500mL format.
SCM016Mesenchymal Stem Cell Freezing Medium (1x)The Mesenchymal Stem Cell Freezing Medium (1x) has been optimized & validated for Stem cell culture. This medium is available in a 50ml format.
O1391油红O 溶液0.5% in isopropanol
SCM121OsteoMAX-XF分化培养基Defined Xeno-Free osteogenesis differentiation medium that is specially formulated to readily differentiate human mesenchymal stem cells (MSCs) into mature osteocytes as assessed by alizarin red & alkaline phosphatase staining.
SCM151PLTGold® Human Platelet LysateHeparin-free Human Platelet Lysate (hPL) for Mesenchymal Stem Cell Culture. 100ml.
SCM152PLTGold® Human Platelet LysateHeparin-free Human Platelet Lysate (hPL) for Mesenchymal Stem Cell Culture. 500ml.
SCM141PLTMax人血小板裂解物PLTMax Human Platelet Lysate is a growth factor rich supplement that is a superior alternative to fetal bovine serum (FBS) for human mesenchymal stem cell (MSC) culture. 100ml.

mRNA纯化和分析

mRNA 纯化第一步是使用脱氧核糖核酸酶或 DNase 去除线性化的 pDNA 模板。然后用氯化锂(LiCl)沉淀 mRNA，并用 75% 乙醇洗涤。所得 mRNA 沉淀可用水或重悬缓冲液复溶。但在放大生产过程中应避免使用这类沉淀方法，因为这种方法有些难以放大，且 GMP 生产规范要求避免使用有害溶剂。建议使用GenElute™ mRNA Miniprep Kit纯化试剂盒进行小规模纯化。如需扩大规模，可以使用Pellicon®超滤盒进行切向流过滤 (TFF)，此方式支持线性放大。

mRNA合成过程中的dsRNA去除

双链 RNA (dsRNA)是一种转录副产物，是 mRNA 合成的主要伴随产物。dsRNA 可刺激先天免疫反应，减少递送的 mRNA 翻译。因此，利用合成 mRNA 开发疫苗时，应去除 dsRNA。可利用基于纤维素纤维的纤维素滤纯化方法去除各种规模的 dsRNA。

合成mRNA纯化和分析试剂
产品编号产品名称说明价格
TMS-010阿尔新蓝染色液
TMS-008Alizarin-Red Staining SolutionAlizarin red is a commonly used stain to identify calcium containing osteocytes in differentiated culture of both human and rodent mesenchymal stem cells (MSCs).
CBL154Anti-H-CAM Antibody, clone F10-44-2clone F10-44-2, Chemicon®, from mouse
CBL154Anti-H-CAM Antibody, clone F10-44-2clone F10-44-2, Chemicon®, from mouse
CBL415Anti-Thy-1 Antibody, clone F15-42-1clone F15-42-1, Chemicon®, from mouse
CLS431751Corning®细胞过滤网pore size 70 μm, white, sterile, pkg of (individually wrapped), pack of 50 ea, Corning 431751
D2650二甲基亚砜Hybri-Max™, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
ES-020-BFetal Bovine SerumUS Origin, Mesenchymal Stem Cell Qualified FBS, sterile-filtered, suitable for stem cell culture
F0291hBFGFFGF-Basic, recombinant, expressed in E. coli, suitable for cell culture
SCC038Human Adipose Mesenchymal Stem CellsHuman Adipose Mesenchymal Stem Cells (MSCs) are isolated from human adipose tissue and are cryopreserved at P2 to ensure optimal phenotype and the highest viability and plating efficiency.
SCC034人间充质干细胞（骨髓）Human mesenchymal stem cells derived from bone marrow that have a capacity for expansion in vitro while maintaining their potential for differentiation to multiple lineages including adipocytes, osteoblasts and chondrocytes.
SCM045人间充质-XF扩增培养基Xeno-free mesenchymal stem cell media specially formulated for the rapid expansion of human mesenchymal stem cells isolated from a variety of sources, including adipose and bone marrow tissues.
FC010人血浆纤连蛋白纯化蛋白from human plasma, liquid, 1 mg/mL (100 MG pack size is lyophilized), purified protein, suitable for cell culture
SCM015间充质干细胞扩展培养基（1x）The Mesenchymal Stem Cell Expansion Medium (1x) has been optimized & validated for Stem cell culture. This medium is available in a 500mL format.
SCM016Mesenchymal Stem Cell Freezing Medium (1x)The Mesenchymal Stem Cell Freezing Medium (1x) has been optimized & validated for Stem cell culture. This medium is available in a 50ml format.
O1391油红O 溶液0.5% in isopropanol
SCM121OsteoMAX-XF分化培养基Defined Xeno-Free osteogenesis differentiation medium that is specially formulated to readily differentiate human mesenchymal stem cells (MSCs) into mature osteocytes as assessed by alizarin red & alkaline phosphatase staining.
SCM151PLTGold® Human Platelet LysateHeparin-free Human Platelet Lysate (hPL) for Mesenchymal Stem Cell Culture. 100ml.
SCM152PLTGold® Human Platelet LysateHeparin-free Human Platelet Lysate (hPL) for Mesenchymal Stem Cell Culture. 500ml.
SCM141PLTMax人血小板裂解物PLTMax Human Platelet Lysate is a growth factor rich supplement that is a superior alternative to fetal bovine serum (FBS) for human mesenchymal stem cell (MSC) culture. 100ml.
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