[发明专利]重组毒素蛋白的高水平表达

权利要求书

1.一种在假单胞菌宿主细胞中生产重组毒素蛋白的方法，所述方法包括：

将编码毒素蛋白的核苷酸序列连接到表达载体中；

用该表达载体转化假单胞菌宿主细胞；以及

在适合重组毒素蛋白表达的培养基中培养已转化的假单胞菌宿主细胞；

其中所述重组毒素蛋白选自CRM197、白喉毒素、霍乱全毒素、霍乱毒素B、百日咳毒素、破伤风毒素C片段、艰难梭菌毒素B和铜绿假单胞菌外毒素A，或者

其中所述重组毒素蛋白选自霍乱毒素B、霍乱全毒素、百日咳毒素、破伤风毒素C片段、艰难梭菌毒素B和铜绿假单胞菌外毒素A，或者

其中所述重组毒素蛋白选自霍乱毒素B、霍乱全毒素、百日咳毒素、破伤风毒素C片段和艰难梭菌毒素B。

2.权利要求1的方法，其中所述重组蛋白以0.2克/升至约12克/升的可溶性和/或活性毒素蛋白的产率生产。

3.权利要求2的方法，其中可溶性和/或活性毒素蛋白的产率为约0.2克/升至约12克/升，为约0.2g/L、约0.3g/L、约0.4g/L、约0.5g/L、约0.6g/L、约0.7g/L、约0.8g/L、约0.9g/L、约1g/L、约1.5g/L、约2g/L、约2.5g/L、约3g/L、约3.5g/L、约4g/L、约4.5g/L、约5g/L、约5.5g/L、约6g/L、约6.5g/L、约7g/L、约7.5g/L、约8g/L、约8.5g/L、约9g/L、约9.5g/L、约10g/L、约10.5g/L、约11g/L、约12g/L、约0.2g/L至约0.5g/L、约0.2g/L至约1g/L、约0.2至约2g/L、约0.3g/L至约0.6g/L、约0.3g/L至约1g/L、约0.3至约2g/L、约0.4至约0.7g/L、约0.4至约1g/L约0.4至约2g/L、约0.4至约3g/L、约0.5g/L至约1g/L、约0.5g/L至约2g/L、约0.5g/L至约3g/L、约0.5g/L至约4g/L、约0.5g/L至约5g/L、约0.5g/L至约6g/L、约0.5g/L至约7g/L、约0.5g/L至约8g/L、约0.5g/L至约9g/L、约0.5g/L至约10g/L、约0.5g/L至约11g/L、约0.5g/L至约12g/L、约1g/L至约2g/L、约1g/L至约3g/L、约1g/L至约4g/L、约1g/L至约5g/L、约1g/L至约6g/L、约1g/L至约7g/L、约1g/L至约8g/L、约1g/L至约9g/L、约1g/L至约10g/L、约1g/L至约11g/L、约1g/L至约12g/L、约2g/L至约3g/L、约2g/L至约4g/L、约2g/L至约5g/L、约2g/L至约6g/L、约2g/L至约7g/L、约2g/L至约8g/L、约2g/L至约9g/L、约2g/L至约10g/L、约2g/L至约11g/L、约2g/L至约12g/L、约3g/L至约4g/L、约3g/L至约5g/L、约3g/L至约6g/L、约3g/L至约7g/L、约3g/L至约8g/L、约3g/L至约9g/L、约3g/L至约10g/L、约3g/L至约11g/L、约3g/L至约12g/L、约4g/L至约5g/L、约4g/L至约6g/L、约4g/L至约7g/L、约4g/L至约8g/L、约4g/L至约9g/L、约4g/L至约10g/L、约4g/L至约11g/L、约4g/L至约12g/L、约5g/L至约6g/L、约5g/L至约7g/L、约5g/L至约8g/L、约5g/L至约9g/L、约5g/L至约10g/L、约5g/L至约11g/L、约5g/L至约12g/L、约6g/L至约7g/L、约6g/L至约8g/L、约6g/L至约9g/L、约6g/L至约10g/L、约6g/L至约11g/L、约6g/L至约12g/L、约7g/L至约8g/L、约7g/L至约9g/L、约7g/L至约10g/L、约7g/L至约11g/L、约7g/L至约12g/L、约8g/L至约9g/L、约8g/L至约10g/L、约8g/L至约11g/L、约8g/L至约12g/L、约9g/L至约10g/L、约9g/L至约11g/L、约9g/L至约12g/L、约10g/L至约11g/L、约10g/L至约12g/L或约11g/L至约12g/L。

4.权利要求1-3中任一项的方法，其中所述编码毒素蛋白的核苷酸序列与分泌信号编码序列融合，当所述分泌信号编码序列表达时，指引所述毒素蛋白向周质转移。