【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(24h/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。更多还原

【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 had 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,could 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 three 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 could control the release of transgenic cyanobacteria into environment effectively,which also has advantages of low cost,simple procedure and no secondary pollution.It could also provide reference for the safe release of other transgenic cyanobacteria in the environment.更多还原