产品描述
在mRNA大规模生产过程中，转录结束后需要对转录模板进行去除，DNase I 可以将单链或者双链 DNA 进行同等程度的随机分解，生成具有 5'-P 末端寡核苷酸。在Mg2+条件下，DNase I可以对双链DNA进行随意剪切。
DNase I（Deoxyribonuclease I，脱氧核糖核酸酶 I）最初分离自牛胰脏，分子量约为 39 kD，是一种脱氧核糖核酸内切酶，可将单链或双链 DNA 同等程度进行随机分解，生成具有 5'-P 末端寡核苷酸。DNase I 水解单链或双链 DNA，其活性非常依赖于 Ca2+水平，并能被 Mg2+或 Mn2+激活。
本制品为利用毕赤酵母大规模发酵表达的 GMP 级重组 DNase I，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
| 项目 |
标准 |
方法 |
| 外观 |
澄明液体 |
目视检查 |
| 可见异物 |
符合规定 |
中国药典2020版第四部第一法灯检法(通则0904) |
| pH值 |
7.0-8.0 |
中国药典2020版第四部pH值测定法(通则0631) |
| 活性 |
1.8KUml-2.2KU/ml |
004质粒DNA降解法 |
| 纯度 |
≥95% |
中国药典2020版第四部高效液相色谱法(通则0512) |
| RNA酶残留 |
293-RNA的降解不超过10% |
2U酶与293-RNA.37°C孵育1h |
| 细菌内毒素含量 |
≤10 EU/mg |
中国药典2020版第四部凝胶限度试验法(通则1143) |
| 外源性DNA残留 |
≤100 pg/mg |
荧光定量PCR法 |
| 宿主蛋白残留 |
≤50 ppm |
中国药典2020版第四部菌体蛋白残留量测定法(通则 3414) |
| 支原体检测 |
阴性 |
支原体检测试剂盒 |
| 重金属残留 |
≤10 ppm |
中国药典2020版第四部重金属检查法(通则0821) |
|
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.制备不含 DNA 的 RNA 样品。
2.RT-PCR 反应前 RNA 样品中，去除基因组 DNA 等可能的 DNA 污染。
3.体外 T7，T3，SP6 等 RNA 聚合酶催化的体外转录后去除 DNA 模板。
4.用于足迹法（Footprinting）分析 DNA-蛋白质相互作用。
5.与 DNA Polymerase I 一起用于切口平移。
6.二价锰离子存在条件下，使 DNA 片段化，产生 DNA 随机片段文库。
7.细胞凋亡 TUNEL 检测中部分剪切基因组 DNA 作为阳性对照。
保存体系
10 mM Tris-HCl (pH7.6); 2 mM CaCl2; 50% (v/v) Glycerol。
注意事项
1.最适 pH：7.0-8.0
2.活化剂：DNase I 需要二价阳离子以达到最大活性。
3.抑制剂：EDTA, EGTA, SDS。
4.特异性：降解 DNA 的双链特异性的核酸内切酶。
5.使用时将酶置于冰上操作，使用完毕后-20℃保存。
In the process of large-scale mRNA production, the transcription template needs to be removed after transcription. DNase I can randomly decompose single-stranded or double-stranded DNA to the same degree to generate oligonucleotides with 5'-P ends. Under Mg2+ conditions, DNase I can cut double-stranded DNA at will.
DNase I (Deoxyribonuclease I, deoxyribonuclease I) was originally isolated from bovine pancreas, with a molecular weight of about 39 kD. Has a 5'-P terminal oligonucleotide. DNase I hydrolyzes single-stranded or double-stranded DNA, its activity is very dependent on the level of Ca2+, and can be activated by Mg2+ or Mn2+.
This product is a GMP-grade recombinant DNase I expressed by large-scale fermentation of Pichia pastoris. It is produced with medicinal specifications raw materials and strictly controlled host protein residues, nucleic acid residues, etc., and conforms to GMP standard product production and quality management procedures to ensure the production process And all raw and auxiliary materials can be traced back.
Quality requirements
| Project |
Standard |
Method |
| Exterior |
Clear liquid |
Visual inspection |
| Visible foreign body |
Compliance |
Chinese Pharmacopoeia 2020 Edition Fourth Part 1 Lamp Inspection Method (General Rule 0904) |
| pH value |
7.0-8.0 |
Chinese Pharmacopoeia 2020 Edition Part IV pH Determination Method (General Principle 0631) |
| active |
1.8KUml-2.2KU/ml |
004 Plasmid DNA degradation method |
| Purity |
≥95% |
Chinese Pharmacopoeia 2020 Edition Part IV High Performance Liquid Chromatography (General Principle 0512) |
| RNase residue |
Degradation of 293-RNA does not exceed 10% |
2U enzyme and 293-RNA. Incubate at 37°C for 1h |
| Bacterial endotoxin content |
≤10 EU/mg |
Chinese Pharmacopoeia 2020 Edition Fourth Gel Limit Test Method (General Rule 1143) |
| Exogenous DNA residue |
≤100 pg/mg |
Fluorescence quantitative PCR |
| Host protein residue |
≤50 ppm |
Chinese Pharmacopoeia 2020 Edition Part IV Method for the Determination of Bacterial Protein Residues (General Rule 3414) |
| Mycoplasma detection |
Feminine |
Mycoplasma detection kit |
| Heavy metal residue |
≤10 ppm |
Chinese Pharmacopoeia 2020 Edition Fourth Heavy Metal Inspection Method (General Principle 0821) |
|
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. Prepare an RNA sample without DNA.
2. In the RNA sample before the RT-PCR reaction, remove possible DNA contamination such as genomic DNA.
3. In vitro T7, T3, SP6 and other RNA polymerases catalyze the removal of DNA template after in vitro transcription.
4. Used for Footprinting (Footprinting) to analyze DNA-protein interactions.
5. Used with DNA Polymerase I for nick translation.
6. In the presence of divalent manganese ions, the DNA is fragmented to generate a library of random DNA fragments.
7. In the apoptosis TUNEL test, part of the genomic DNA is sheared as a positive control.
Preservation system
10 mM Tris-HCl (pH7.6); 2 mM CaCl2; 50% (v/v) Glycerol.
Precautions
1. Optimal pH: 7.0-8.0
2. Activator: DNase I requires divalent cations to achieve maximum activity.
3. Inhibitors: EDTA, EGTA, SDS.
4. Specificity: double-strand specific endonuclease that degrades DNA.
5. Put the enzyme on ice during use, and store it at -20°C after use.
G2327352
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