| 吴倩萍,顾娟,刘海萍,等.冻融法控制制药用转基因蓝藻在环境中释放[J].中国海洋药物,2018,37(2):25-31. |
| 冻融法控制制药用转基因蓝藻在环境中释放 |
| Controlling environmental release of transgenic cyanobacteria used to prepare recombinant drug by freezing-thawing way |
| 投稿时间：2017-10-16 修订日期：2017-12-18 |
| DOI： |
| 中文关键词: 重组蛋白药物 转基因蓝藻 冻融法 环境释放 转基因蓝藻安全性 |
| English Keywords:Recombinant protein drug transgenic cyanobacteria the freezing-thawing environmental release safety of transgenic cyanobacteria |
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| 中文摘要: |
| 摘要:目的 用转基因生物制备重组药物中的1个关键问题就是防止其环境释放。为控制制备抗对虾白斑综合征病毒用的转vp28基因鱼腥藻7120蓝藻细胞在应用中的生长和繁殖能力，本研究探讨用冻融法在不破坏重组VP28蛋白活性的同时杀灭藻细胞。方法 通过把蓝藻细胞在2种低温环境(-20、-80 ℃)和室温中处理，选取最适温度以多次冻融法破坏藻细胞。用液体和固体培养观察其后续生长，用光学和扫描电镜检测细胞的损伤。结果 -20和-80 ℃与室温处理连用，都可使处理的藻细胞失去生长能力。为操作简便，选用冷冻温度为-20 ℃、反复冻融3次（24 h/次），使蓝藻细胞100%失去繁殖能力，藻细胞在冻融处理后丝状体断裂，细胞遭破坏，固体培养发现无藻细胞生长，蛋白印迹法检测VP28蛋白无丢失。结论 冻融法可控制转基因鱼腥藻在环境中的释放。该方法成本低廉、操作简单，且无次生污染，既保障该药物在应用中的安全性，也可为其他转基因蓝藻在环境中的安全释放提供借鉴。 |
| English Summary: |
| Abstract: Objective The key point of preparing recombinant drug from transgenic organisms is prevention of their release into environment . To control the growth and reproduction of transgenic Anabaena sp. PCC 7120 harbering vp28 gene , freezing-thawing was developed to kill the cyanobacteria cells and keep the activity of recombinant VP28 protein which against the white spot syndrome virus (WSSV) . Methods The cyanobacteria cells were treated with -20 or -80 ℃ , and then with room temperature . Comparing deferent repeats of the treatment and testing the growth of treated cells in liquid and solid media , the optimum model has been selected . Meanwhile , the morphology and structure of treated cells , were observed with optical and scanning electron microscopy . Results Both treatments with -20 or -80 ℃ plus room temperature , can destroy treated cells growth , and for easy operation -20 ℃ was selected . After freezing in -20 ℃ for 24 h and thawing in room temperature , then repeated tree times , the growth of cyanobacterial cells was lost completely and the cell could not grow on the solid medium . Western blotting showed no loss of VP28 protein . Conclusion Our freezing-thawing way can control the release of transgenic cyanobacteria into environment effectively , which also has advantages of low cost , simple procedure and no secondary pollution . It can also provide reference for the safe release of other transgenic cyanobacteria in the environment . |
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