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CN B (zh ) [https://www.google.com/ 面包] CN B (zh ) 表达重组人血白蛋白的酵母菌、其构建方法及应用和表达重组人血白蛋白的方法 Country Status 重组人血白蛋白及其表达载体的构建方法 Citations 肖生科 等: &quot;提高外源基因在巴斯德毕赤酵母中表达量的研究进展&quot;, 《生物技术通报》 陈孝康 等: &quot;表达乙肝病毒融合表面抗原基因SA-28的二倍体酵母工程菌的选育&quot;, 《生物工程学报》<br /><br /><br />5.根据权利要求1-4任一项所述的二倍体巴斯德毕赤酵母菌,其特征在于,所述二倍体巴斯德毕赤酵母菌是MFD1-MFD7菌 8.根据权利要求1-5任一项所述的二倍体巴斯德毕赤酵母菌在表达重组人血白蛋白中的应用 9.一种表达重组人血白蛋白的方法,包括培养权利要求1-5任一项所述的二倍体巴斯德毕赤酵母菌 10.根据权利要求9所述的方法,其特征在于,所述方法用于工业生产,在发酵培养二倍体巴斯德毕赤酵母菌的后期补 加蛋白胨 CN B (zh ) 表达重组人血白蛋白的酵母菌、其构建方法及应用和表达重组人血白蛋白的方法 Applications Claiming Priority<br />将发酵液划线于YPD平板分离单菌落,然后挑取获得的单菌落涂于MD平板上进行二倍体稳定性验证 结果显示,涂布后的MD平板在30&deg;C培养48h后,95%以上菌落均可以正常生长出单菌落,这说明大部分二倍体菌株在发酵结束时仍以二倍体形式稳定存在 实施例10:二倍体菌株表达的重组人血白蛋白的纯化及结构分析 采用本公司自有纯化工艺(专利申请号: .4和 .X)对实施例9中收获的发酵液进行纯化后,得到了高纯度的重组人血白蛋白,高压液相色谱法(HPLC)检测纯度可以达到99%以上(见附图8 ) 将纯化后的重组人血白蛋白进行质谱分子量检测(上海中科新生命生物科技有限公司),发现与天然人血白蛋白分子量一致<br />培养约20-25h时发酵罐中甘油耗完,此时开始以500ml/hr补料速度流加50%甘油(含12ml/L PTMl ),流加约4h后停止甘油补料,开始流加甲醇(含12ml/L PTMl)进行诱导表达 初始甲醇补料速度设置为60ml/hr,5小时后增加至180ml/hr,流加3小时后增加至270ml/hr,并以此速度补料至发酵结束 发酵至200h开始以200ml/hr速度补加90g/L的蛋白胨溶液,防止发酵后期因营养不足导致二倍体菌株单倍体化 整个发酵过程,甲醇补料期约为280h,发酵周期约为310h<br />目的蛋白N末端测序结果为:DAHKSEVAHRFKDLG(SEQ ID NO:8),C末端测序结果为KLVAASQAALGL (SEQ ID NO:9),两端序列与天然人血白蛋白完全相同 圆二色谱检测结果显示,纯化后的重组人血白蛋白与天然人血白蛋白二级结构一致<br />实施例9: 二倍体菌株发酵罐表达 将MFD2菌株接种到600ml YI3D培养基中,30 V 250rpm培养24h,至OD6tltl达到10-20 配制30L含有50g/L甘油的基本盐培养基,转移至发酵罐中高压蒸汽灭菌30min 灭菌结束后将发酵罐温度降低至30&deg;C,pH用28%氨水调节至6.0,设置转速300rpm,通气条件为0.2vvm 接种前在培养基中加入120ml PTMl微量元素,并调整溶解氧至100% 然后将600ml种子培养液接种至发酵罐中开始发酵,发酵过程中控制温度30&deg;C,pH6.0,通过搅拌及通气量控制溶解氧不低于20%<br />这说明本发明表达出的重组人血白蛋白的结构与天然人血白蛋白完全一致 1.一种二倍体巴斯德毕赤酵母菌,其特征在于,所述酵母菌含有两个染色体组,两个染色体组均包含人血白蛋白成熟肽编码序列,但分别包含不同的筛选标记,所述人血白蛋白成熟肽编码序列如SEQ ID N0.3所示 2.根据权利要求1所述的二倍体巴斯德毕赤酵母菌,其特征在于,所述筛选标记选自营养缺陷型筛选标记或抗生素筛选标记<br />

Latest revision as of 06:34, 14 July 2020

CN B (zh ) 面包 CN B (zh ) 表达重组人血白蛋白的酵母菌、其构建方法及应用和表达重组人血白蛋白的方法 Country Status 重组人血白蛋白及其表达载体的构建方法 Citations 肖生科 等: "提高外源基因在巴斯德毕赤酵母中表达量的研究进展", 《生物技术通报》 陈孝康 等: "表达乙肝病毒融合表面抗原基因SA-28的二倍体酵母工程菌的选育", 《生物工程学报》


5.根据权利要求1-4任一项所述的二倍体巴斯德毕赤酵母菌,其特征在于,所述二倍体巴斯德毕赤酵母菌是MFD1-MFD7菌 8.根据权利要求1-5任一项所述的二倍体巴斯德毕赤酵母菌在表达重组人血白蛋白中的应用 9.一种表达重组人血白蛋白的方法,包括培养权利要求1-5任一项所述的二倍体巴斯德毕赤酵母菌 10.根据权利要求9所述的方法,其特征在于,所述方法用于工业生产,在发酵培养二倍体巴斯德毕赤酵母菌的后期补 加蛋白胨 CN B (zh ) 表达重组人血白蛋白的酵母菌、其构建方法及应用和表达重组人血白蛋白的方法 Applications Claiming Priority
将发酵液划线于YPD平板分离单菌落,然后挑取获得的单菌落涂于MD平板上进行二倍体稳定性验证 结果显示,涂布后的MD平板在30°C培养48h后,95%以上菌落均可以正常生长出单菌落,这说明大部分二倍体菌株在发酵结束时仍以二倍体形式稳定存在 实施例10:二倍体菌株表达的重组人血白蛋白的纯化及结构分析 采用本公司自有纯化工艺(专利申请号: .4和 .X)对实施例9中收获的发酵液进行纯化后,得到了高纯度的重组人血白蛋白,高压液相色谱法(HPLC)检测纯度可以达到99%以上(见附图8 ) 将纯化后的重组人血白蛋白进行质谱分子量检测(上海中科新生命生物科技有限公司),发现与天然人血白蛋白分子量一致
培养约20-25h时发酵罐中甘油耗完,此时开始以500ml/hr补料速度流加50%甘油(含12ml/L PTMl ),流加约4h后停止甘油补料,开始流加甲醇(含12ml/L PTMl)进行诱导表达 初始甲醇补料速度设置为60ml/hr,5小时后增加至180ml/hr,流加3小时后增加至270ml/hr,并以此速度补料至发酵结束 发酵至200h开始以200ml/hr速度补加90g/L的蛋白胨溶液,防止发酵后期因营养不足导致二倍体菌株单倍体化 整个发酵过程,甲醇补料期约为280h,发酵周期约为310h
目的蛋白N末端测序结果为:DAHKSEVAHRFKDLG(SEQ ID NO:8),C末端测序结果为KLVAASQAALGL (SEQ ID NO:9),两端序列与天然人血白蛋白完全相同 圆二色谱检测结果显示,纯化后的重组人血白蛋白与天然人血白蛋白二级结构一致
实施例9: 二倍体菌株发酵罐表达 将MFD2菌株接种到600ml YI3D培养基中,30 V 250rpm培养24h,至OD6tltl达到10-20 配制30L含有50g/L甘油的基本盐培养基,转移至发酵罐中高压蒸汽灭菌30min 灭菌结束后将发酵罐温度降低至30°C,pH用28%氨水调节至6.0,设置转速300rpm,通气条件为0.2vvm 接种前在培养基中加入120ml PTMl微量元素,并调整溶解氧至100% 然后将600ml种子培养液接种至发酵罐中开始发酵,发酵过程中控制温度30°C,pH6.0,通过搅拌及通气量控制溶解氧不低于20%
这说明本发明表达出的重组人血白蛋白的结构与天然人血白蛋白完全一致 1.一种二倍体巴斯德毕赤酵母菌,其特征在于,所述酵母菌含有两个染色体组,两个染色体组均包含人血白蛋白成熟肽编码序列,但分别包含不同的筛选标记,所述人血白蛋白成熟肽编码序列如SEQ ID N0.3所示 2.根据权利要求1所述的二倍体巴斯德毕赤酵母菌,其特征在于,所述筛选标记选自营养缺陷型筛选标记或抗生素筛选标记