CN113564185A - 一种碱性磷酸酶基因及其cho稳定细胞株和alp制备方法 - Google Patents
一种碱性磷酸酶基因及其cho稳定细胞株和alp制备方法 Download PDFInfo
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- CN113564185A CN113564185A CN202111043278.3A CN202111043278A CN113564185A CN 113564185 A CN113564185 A CN 113564185A CN 202111043278 A CN202111043278 A CN 202111043278A CN 113564185 A CN113564185 A CN 113564185A
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Abstract
一种碱性磷酸酶基因及其CHO稳定细胞株和ALP制备方法。本发明涉及一种编码碱性磷酸酶ALP的基因，核苷酸序列如SEQ ID No.1所示。通过对碱性磷酸酶核苷酸序列的优化，构建到适宜的表达载体中，进一步在哺乳细胞中稳定表达，其结构更接近天然的碱性磷酸酶蛋白，具有良好生物学活性和碱性磷酸酶活性，可以提高检测灵敏度。本发明还通过将SEQ ID NO：1所示基因构建到在pCDNA3基础上，导入GS基因改造后的表达载体上，以稳定方式转染进真核细胞基因组，经MSX抑制剂筛选、批培养、分批补料式培养，每步培养中结合高通量酶活筛选鉴定，最后得到表达高活性碱性磷酸酶CHO稳定细胞株，保藏于中国典型培养物保藏中心，其生物保藏编号为CCTCC NO：C2021225。
Description
技术领域
本发明属于生物技术领域，具体涉及一种碱性磷酸酶基因及其CHO稳定细胞株和ALP 制备方法。
背景技术
碱性磷酸酶(alkaline phosphatase)是一种专一性较广的磷酸酯水解酶，可以催化磷酸单脂的水解和磷酸基团的转移反应，在生物体内具有重要的生理功能。除此之外，碱性磷酸酶做为酶缀合物可应用于诊断领域，如基于抗原抗体反应的免疫诊断检测系统，比如化学发光平台或酶联免疫平台，或使DNA脱磷酸化。目前已知的天然ALP中，比活性最高的是源自牛小肠的碱性磷酸酶。
目前已知的高活性的ALP有两种获取方式，一是直接从小牛小肠中纯化并且是稳定的。该方法受限于原材料以及提取工艺流程，不能保证大量高品质供应。二是通过重组表达，尤其是酵母表达。但是酵母表达产物容易产生过度糖基化以及糖基化不均匀，不利于下游偶联应用，并且由于蛋白N末端切割不完全，导致有氨基酸残留，影响酶活性。尽管已有报道，运用哺乳动物细胞表达碱性磷酸酶，但是产量普遍偏低，不适合产业化。碱性磷酸酶抑制剂范围较广泛，如常见氨基酸如苯丙氨酸、亮氨酸、色氨酸、半胱氨酸等，或重金属如Be2+等，又如金属离子耦合剂EDTA等，导致表达的碱性磷酸酶活性偏低，需要优化培养基与培养方案。因此如何通过基因重组表达技术获取低糖基化，高活性，高产量的碱性磷酸酶是目前亟需解决的问题。
发明内容
有鉴于此，本发明的目的在于提供一种碱性磷酸酶基因，还提供相应的载体及其CHO稳定细胞株和ALP制备方法。
为达到上述目的，本发明提供如下技术方案：
1、一种编码碱性磷酸酶ALP的基因，将SEQ ID NO：2所示氨基酸序列的核苷酸序列进行密码子优化，优化后所述编码碱性磷酸酶ALP基因的核苷酸序列如SEQ ID No.1所示。
2、核苷酸序列如SEQ ID No.1所示的基因在构建表达碱性磷酸酶的载体中的应用。
合适的表达载体包括pCDNA3，pCDNA3.1 Hygro(+)等，也包括经过改造后的表达载体，如在pCDNA3基础上，导入GS基因(谷氨酰胺合成酶基因)或DHFR基因二氢叶酸还原酶基因)。
进一步，所述载体为pCDNA-GS。
3、核苷酸序列如SEQ ID No.1所示的基因在构建表达碱性磷酸酶的宿主细胞中的应用。
克隆核苷酸序列如SEQ ID No.1所示基因到改造后的表达载体上，以稳定方式转染进真核细胞基因组，经抑制剂筛选，其中GS系统用MSX(蛋氨酸亚氨基代砜)，DHFR基因用MTX(氨甲喋呤)，获取高表达细胞库。DHFR系统需对高表达细胞库进行加压筛选，逐步提到抑制剂浓度。
进一步，所述宿主细胞为CHO细胞，HEK293细胞，Expi293细胞，COS7细胞，NSO 细胞或BHK21细胞。
进一步，所述宿主细胞为CHO细胞。
4、一种高活性碱性磷酸酶CHO稳定细胞株的构建方法，具体包括以下步骤：
(1)合成核苷酸序列如SEQ ID NO.1所示的ALP基因，然后克隆到pCDNA-GS载体，得到重组表达载体pCDNA-GS-ALP；
(2)将重组表达载体pCDNA-GS-ALP电穿孔转染到CHOK1细胞中，进行亚克隆培养，并加入药物抑制剂筛选，测定酶活筛选鉴定表达高活性的克隆；
(3)步骤(2)挑出表达高活性的克隆再进行批培养、分批补料式培养，每步培养中通过高通量酶活筛选鉴定，最后得到表达高活性碱性磷酸酶CHO稳定细胞株。
进一步，所述的高活性碱性磷酸酶CHO稳定细胞株的构建方法，所述药物抑制剂为MSX。
5、一株稳定表达碱性磷酸酶CHO细胞株，保藏于中国典型培养物保藏中心，其生物保藏编号为CCTCC NO：C2021225。
6、一种碱性磷酸酶的制备方法，将表达高活性碱性磷酸酶CHO稳定细胞株按照细胞密度0.5*106-1*106个/ml接种至细胞器中，转速110rpm～130rpm，5％CO2培养，按照培养基厂家补料方案，第4,6,8,10,12天进行补料，每天检测葡萄糖含量，若含量低于4g，则补糖至4g/L，第14天，收取细胞上清即可。
进一步，碱性磷酸酶的制备方法还包括纯化步骤，纯化步骤具体为：
A.将收取的细胞上清加入55％硫酸铵进行沉淀，沉淀用20mM Tris,2mM MgCl2,0.08mM ZnCl2，1M硫酸铵pH8.0缓冲液A进行溶解；溶解后再次离心取上清过滤；
B.步骤A的滤液进行Phenyl HP疏水层析分离，用缓冲液A和缓冲液B进行线性洗脱，由100％A洗脱至100％B，所述缓冲液B为20mM Tris，2mM MgCl2,0.08mM ZnCl2，收集目标蛋白洗脱峰，透析样品至缓冲液B中。
C.阴离子交换柱：将透析后的蛋白液进行DEAE阴离子交换层析，用缓冲液B线性洗脱至缓冲液C线，所述缓冲液C为20mM Tris,2mM MgCl2,0.08mM ZnCl2，1M NaCl, pH8.0，对洗脱样品进行检测，收集并合并目的蛋白。
进一步，将收集的蛋白液透析至最终储存缓冲液3mol/l NaCl，0.1mmol/l ZnCl2，30 mmol/l TEA，5mmol/l MgCl2，pH7.5。
进一步，碱性磷酸酶的制备方法，所述表达高活性碱性磷酸酶CHO稳定细胞株为，待补充保藏信息。
本发明的有益效果在于：本发明通过对碱性磷酸酶核苷酸序列的优化，构建到适宜的表达载体中，进一步在哺乳细胞中得到稳定的表达，其结构更接近天然的碱性磷酸酶蛋白，具有良好的生物学活性和碱性磷酸酶活性，因此可以提高检测灵敏度。本发明还通过将SEQ ID NO：1所示基因构建到在pCDNA3基础上，导入GS基因改造后的表达载体上，以稳定方式转染进真核细胞基因组，经MSX抑制剂筛选、批培养、分批补料式培养，每步培养中结合高通量酶活筛选鉴定，最后得到表达高活性碱性磷酸酶CHO稳定细胞株，并将其保藏于中国典型培养物保藏中心，其生物保藏编号为CCTCC NO：C2021225。使碱性磷酸酶的表达量有大幅度的提高，可达到3～5g/liter，再经优化的蛋白纯化，得到更高高纯度和比活性碱性磷酸酶，适应工业化生产需求。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为密码子使用调整示意图，密码子调整后序列优化的最佳值是0.95。
图2为优化后SEQ ID NO：1所示核苷酸序列中密码子使用分布，显示相对密码子使用分布。
图3为优化后SEQ ID NO：1所示核苷酸序列中GC含量示意图。
图4为限制性内切酶酶切鉴定图。
图5为最终细胞培养上清液的SDS-PAGE蛋白电泳图，其中，M泳道为蛋白Marker，1是CHOK1-13E8克隆稳定细胞株培养基批培养表达上清，2是CHOK1-13E8克隆稳定细胞株补料培养表达上清液，3是HEK293T细胞瞬时表达培养基上清液，4是CHOK1细胞瞬时表达培养基上清液，5是Expi293细胞瞬时表达培养基上清液层析收集到的样品，6是BSA蛋白标准品(1ug)，7是BSA蛋白标准品(2ug)，蛋白marker分子量从上往下依次是 116KDa,67KDa,45KDa,35KDa,25KDa,18KDa,14KDa。目标碱性磷酸酶理论分子量约为 52.3KDa。
图6为取市售10种适合CHO细胞培养的无血清培养基以及相应的补料培养基，对CHOK1-13E8克隆稳定细胞株进行补料培养，并取细胞培养上清液进行SDS-PAGE蛋白电泳，其中，M泳道Marker，1～10分别对应不同培养基培养细胞上清液。
图7为碱性磷酸酶表达上清用疏水层析柱纯化图。
图8为最终纯化后碱性磷酸酶的SDS-PAGE蛋白电泳图，其中，1泳道是蛋白Marker，2是层析收集到的样品，蛋白marker分子量同图5。
图9为最终纯化后碱性磷酸酶SEC-HPLC图。
图10为最终纯化后碱性磷酸酶比活性对比数据。
保藏说明：
培养物名称：中国仓鼠卵巢细胞CHO-K1/AP-13E8
保藏机构：中国典型培养物保藏中心
保藏机构简称：CCTCC
地址：中国武汉武汉大学
保藏日期：2021年8月10日
保藏中心登记入册编号：CCTCC NO：C2021225。
具体实施方式
下面将结合附图，对本发明的优选实施例进行详细的描述。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。
实施例1
密码子优化，基因合成与构建表达载体
按照哺乳动物细胞密码子偏好性对SEQ ID NO：2所示的碱性磷酸酶蛋白序列(在碱性磷酸酶蛋白N端加入信号肽)进行密码子优化，优化参数包括密码子偏好性，GC含量等，优化后碱性磷酸酶的核苷酸序列如SEQ ID NO：1所示。图1为密码子使用调整示意图，密码子调整后序列优化的最佳值是0.95，图2为优化后SEQ ID NO：1所示核苷酸序列中密码子使用分布，显示相对密码子使用分布，图3优化后SEQ ID NO：1所示核苷酸序列中GC 含量示意图。将优化好的基因序列，采用重叠PCR技术合成碱性磷酸酶基因，并在基因的5’端添加kozak序列与限制性内切酶酶切位点，在3’端添加终止密码子与酶切位点与保护碱基(EcoRI/BamHI)。合成后可将其构建在表达碱性磷酸酶的载体中，合适的表达载体包括pCDNA3，pCDNA3.1 Hygro(+)等，也包括经过改造后的表达载体，如在pCDNA3基础上，导入GS基因(谷氨酰胺合成酶基因)或DHFR基因二氢叶酸还原酶基因)。
本实施例合成碱性磷酸酶基因完成后，酶切该基因，连接到pCDNA-GS载体上。使用商业化质粒抽提试剂盒抽提质粒。通过限制性内切酶分析和测序分析以此方式插入的基因序列是否无误，限制性内切酶酶切鉴定结果如图4所示。该表达载体被称为pCDNA-GS-ALP。宿主细胞可采用如CHOK1细胞(中华仓鼠卵巢细胞)，HEK293T细胞，Expi293细胞，COS7 细胞，NSO细胞或BHK21细胞等。
实施例2
HEK293T，Expi293，CHOK1细胞瞬时表达
转染前一天将细胞传至0.5×106～0.7*106个/ml，转染当天调整宿主细胞HEK293T， Expi293，CHOK1悬浮细胞密度至1×106～1.2*106个/ml，保证活力95％以上，细胞状态良好。按照1ml细胞，加入1ug质粒，6ug PEI转染试剂进行转染。6天后取细胞上清进行SDS-PAGE 检测表达情况，电泳结果如图5中泳道3、4、5所示，3是HEK293T细胞瞬时表达培养基上清液，4是CHOK1细胞瞬时表达培养基上清液，5是Expi293细胞瞬时表达培养基上清液层析收集到的样品。瞬时表达上清的表达水平低，表达产物纯度低，给后续纯化带来困难。继续研究构建CHOK1稳定细胞株，提高表达水平。
实施例3
CHOK1细胞培养，电穿孔，铺96孔板与压力筛选
将宿主细胞CHOK1培养至对数生长期，保证活力95％以上。电穿孔转染：细胞计数，取1×107个细胞，800rpm/5min离心，去上清，离心后细胞用2ml PBS重悬洗涤一遍。用700ul CD-CHO培养基(不含谷氨酰胺)重悬细胞，加入线性化质粒(0.526mg/ml)20ug，吹打混合均匀，转移至0.4cm电转杯中。设置电转仪，进行电击，电转指数波参数：250V，960uF，电阻无穷大，加入300ul CD-CHO培养基(不含谷氨酰胺)，静置5min。用CD-CHO培养基 (不含谷氨酰胺)稀释100倍，使得细胞密度为0.1×106个/ml。按每孔50ul/5000个细胞进行96孔板铺板，放置CO2培养箱中培养。转染24h后每孔补加150ul CD-CHO培养基(不含谷氨酰胺)，66.6uM MSX(蛋氨酸亚氨基代砜，methionine+sulfoximine)，放置CO2培养箱中培养。转染21～28天，取长出克隆的细胞上清进行ALP表达量与表达活性测定，用高通量方法筛选高表达碱性磷酸酶克隆，选定若干表达量高的克隆转移到24孔板中，长2～3天后转移至6孔板，细胞密度达到80％后冻存细胞。
实施例4
高通量方法筛选高表达碱性磷酸酶克隆
待96孔中克隆大小长至30％以上覆盖面积时，从96孔吸取克隆上清，用Assaybuffer (1mol/L二乙醇胺，0.5mmol/L MgCl2,pH9.8)将蛋白样品按照1/10比例进行稀释(5ul 96 孔上清加到45ul Assay Buffer中)，按照每150ul Assay buffer加2.5ul pNPP(0.67M)，根据实际需要测定的样品数，配制底物混合液并置于金属浴中37℃预热。取5ul酶加入至PCR管中，置于金属浴，并用排枪加入75ul底物混合液，开始计时，3分钟后，每孔加入75ul终止液终止反应。测定405nm的吸光值，计算其对应上清ALP蛋白活性，表1为高通量筛选高表达碱性磷酸酶克隆实验中少量数据样本展示。选定若干表达量高的克隆转移到24孔板中，长2～3天后转移至6孔板，当细胞密度达到80％后冻存细胞。
表1高表达克隆高通量筛选，部分克隆96孔板上清活性数据
实施例5
批培养，Fed-batch与培养基筛选
实施例4中高表达克隆，扩大陪养至24孔，6孔，直至转移到悬浮培养，培养基为CDFortiCHOTMMedium(ThermoFisher，A1148301)，冻存细胞。按照细胞密度0.5*106个/ml接种20ml细胞至三角细胞培养瓶中，转速120rpm，5％CO2摇床培养箱中培养。待细胞存活率降至70％以下时，收获细胞培养上清进行酶活分析，表2为高表达克隆批培养筛选，部分克隆摇瓶批培养活性数据。
表2高表达克隆批培养筛选，部分克隆摇瓶批培养活性数据
具有高活力细胞进入Fed-batch筛选，按照细胞密度0.5*106个/ml接种20ml细胞至三角细胞培养瓶中，转速120rpm，5％CO2摇床培养箱中培养，第四天开始测糖含量，如果糖含量低于4g/L，补糖至4g/L，第5天开始调节摇床温度至33℃，第5,7,9,11天按照8％的体积比进行补料CD EfficientFeedTMC AGTTMNutrient Supplement(ThermoFisher，A1327504)。14 天后收取细胞上清，进行表达水平鉴定以及酶活力分析。图5为细胞培养上清液的SDS-PAGE 蛋白电泳图，其中，M泳道Marker，1是CHOK1-13E8克隆稳定细胞株培养基批培养表达上清，2是CHOK1-13E8克隆稳定细胞株补料培养表达上清液，3是HEK293T细胞瞬时表达培养基上清液，4是CHOK1细胞瞬时表达培养基上清液，5是Expi293细胞瞬时表达培养基上清液层析收集到的样品，6是BSA蛋白标准品(1ug)，7是BSA蛋白标准品(2ug)，蛋白marker分子量从上往下依次是116KDa,67KDa,45KDa,35KDa,25KDa,18KDa,14KDa。目标碱性磷酸酶理论分子量约为52.3KDa。
表3高表达克隆补料培养筛选，部分克隆摇瓶补料14天培养活性数据
| 克隆号 | 序号 | OD405 | 去背景 | 稀释倍数 | 对应稀释后酶活 |
| 5D8 | 1 | 0.633 | 0.511 | 2000 | 213.42 |
| 9C9 | 9 | 0.731 | 0.609 | 2000 | 254.25 |
| 10F4 | 11 | 0.543 | 0.421 | 2000 | 175.92 |
| 13E8 | 12 | 0.805 | 0.683 | 2000 | 285.08 |
| 18E3 | 14 | 0.767 | 0.645 | 2000 | 269.25 |
| 19E11 | 15 | 0.435 | 0.313 | 2000 | 130.92 |
| 20C3 | 16 | 0.635 | 0.513 | 2000 | 214.25 |
| 20F7 | 17 | 0.735 | 0.613 | 2000 | 255.92 |
综合上述筛选数据分析，13E8克隆在筛选过程中始终保持活性数值高，尤其在筛选第三阶段补料培养筛选步骤酶活性高，因而取最优高表达克隆13E8建立工作库，并保藏于中国典型培养物保藏中心，其生物保藏编号为CCTCC NO：C2021225。培养基筛选，取市售10种适合CHO细胞培养的无血清培养基以及相应的补料培养基，分别编号为A：CDFortiCHOTM Medium(ThermoFisher)；B：CD CHO Medium(ThermoFisher)；C：CD OPT CHOMedium (ThermoFisher)；D：CD CHO 011(健顺生物)；E：CD CHO 012(健顺生物)；F：CD CHO014(健顺生物)；G：CD CHO 015(健顺生物)；H：CD CHO 017(健顺生物)；I：CD CHO 018(健顺生物)；J：CHO细胞Growth A(FUJIFILM Irvine Scientific)；按照厂家提供的补料方案进行培养，并添加2mM MgCl2,0.1mM ZnCl2，至细胞存活率低于70％收取细胞上清，进行表达水平以及酶活力分析。
表4高表达克隆培养基筛选，13E8克隆摇瓶补料14天培养活性数据
图6为取市售10种适合CHO细胞培养的无血清培养基以及相应的补料培养基，对CHOK1-13E8克隆稳定细胞株进行补料培养，并取细胞培养上清液进行SDS-PAGE蛋白电泳，其中，M泳道Marker，1～10分别对应以下培养基培养细胞上清液，1是CD FortiCHOTMMedium(ThermoFisher)；2是CD CHO Medium(ThermoFisher)；3是CD OPT CHO Medium(ThermoFisher)；4是CD CHO 011(健顺生物)；5是CD CHO 012(健顺生物)；6是 CD CHO 014(健顺生物)；7是CD CHO 015(健顺生物)；8是CD CHO 017(健顺生物)； 9是CD CHO 018(健顺生物)；10是CHO细胞Growth A(FUJIFILM Irvine Scientific)；， 11是BSA蛋白标准品(1ug)，12是BSA蛋白标准品(2ug)，蛋白marker分子量从上往下依次是116KDa,67KDa,45KDa,35KDa,25KDa,18KDa,14KDa。目标碱性磷酸酶理论分子量约为52.3KDa。
综合上述筛选数据分析，13E8克隆在编号J培养基CHO细胞Growth A(FUJIFILMIrvine Scientific)酶活性数值最高(表4)，表达水平高(图6)，约为5g/L，最终确定J培养基为放大生产用培养基。
实施例6
放大生产与纯化
将克隆13E8按照细胞密度0.5*106个/ml接种1L细胞至三角细胞培养瓶中，培养基为 CHO细胞Growth A(FUJIFILM Irvine Scientific)，转速120rpm，5％CO2摇床培养箱中培养，按照培养基厂家补料方案，第4,6,8,10,12天进行补料，每天检测葡萄糖含量，若含量低于4g，则补糖至4g/L。第14天，收取细胞上清大约1L体积，进行纯化。
纯化步骤如下：
A.55％硫酸铵沉淀
待纯化的细胞表达上清碱性磷酸酶表达水平为5g/L，取其中50ml进行纯化。大约向收取的细胞上清中加入称取的硫酸铵粉末，称取量按照326g/1L细胞上清的比例缓慢添加。放置4度搅拌40min，离心8000rpm/15min，沉淀用20mM Tris,2mM MgCl2,0.08mM ZnCl2,1M 硫酸铵pH8.0(缓冲液A)进行溶解，再次离心11000rpm/15min，取出上清后，过滤。
B.疏水层析
用Phenyl HP疏水层析柱进行分离，先用缓冲液A平衡助料至基线，再用缓冲液A和缓冲液B(20mM Tris,2mM MgCl2,0.08mM ZnCl2)进行线性洗脱25个柱体积，流速按照2ml/min，由100％A洗脱至100％B，收集第一个蛋白洗脱峰1(如图7所示)，并透析蛋白样品至缓冲液B中。
C.阴离子交换柱
将透析后的蛋白样品液进行DEAE阴离子交换层析，用缓冲液B进行洗杂后，用含NaCl 浓度为0.5M的缓冲液C(20mM Tris,2mM MgCl2,0.08mM ZnCl2，0.5M NaCl,pH8.0)进行洗脱，收集并合并目的蛋白，将收集的蛋白液透析至最终储存缓冲液3mol/l NaCl，0.1mmol/l ZnCl2，30mmol/l TEA，5mmol/l MgCl2，pH7.5。对最终蛋白进行SDS-PAGE电泳分析(图 8)，确定高活性碱性磷酸酶产率为1g/L以上，SEC-HPLC分析纯度，为96.5％(图9)，比活性检测结果如图10，可高达7750U/mg，参照品为罗氏碱性磷酸酶(货号03535452103)，可见本发明碱性磷酸酶活性高于市售罗氏产品。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
序列表
<110> 金标优尼科（无锡）生物科技有限公司
<120> 一种碱性磷酸酶基因及其CHO稳定细胞株和ALP制备方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1461
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ctgatccccg ccgaagagga gaaccccgcc ttctggaaca gacaggccgc ccaggccctg 60
gacgtggcta agaagctgca gcccatccag accgctgcca aaaacgtgat cctgttcctg 120
ggcgacggaa tgggcgtgcc caccgtgacc gctaccagaa ttttgaaggg acagatgaac 180
ggaaagctgg gacccgagac ccccctggcc atggatcagt tcccttacgt ggctctgtct 240
aagacataca acgtggacag acaggtgccc gacagcgccg gcaccgctac agcttacctg 300
tgcggcgtga aaggaaacta cagaaccatc ggggtgagcg ccgccgctag atacaaccag 360
tgcaacacca ccaggggcaa cgaagtgacc agcgtgatca acagagccaa aaaggccggc 420
aaggccgtgg gggtggtgac cacaacaaga gtgcagcacg cttctcccgc cggcgcctac 480
gctcacacag tgaatagaaa ctggtacagc gacgccgacc tgcccgccga cgctcagaaa 540
aacggatgtc aggatatcgc cgcccagctg gtgtataata tggacatcga cgtgatcctg 600
ggcggaggcc ggatgtacat gttccccgaa ggaacccccg accccgagta tcccgacgac 660
gccagcgtga acggcgtgag aaaggacaag cagaacctgg tgcaggagtg gcaggccaag 720
caccagggcg cccagtacgt gtggaacaga accgccctgc tgcaggccgc tgacgacagt 780
agcgtgaccc acctgatggg cctgttcgag cccgccgata tgaagtacaa cgtgcagcag 840
gaccatacca aagaccccac cctggccgaa atgaccgaag ccgccctgca ggtgctgagc 900
agaaaccccc gcggctttta cctgtttgtg gaaggaggca gaatcgacca tggccaccac 960
gacggcaagg cctacatggc tctgaccgag gccatcatgt tcgacaacgc tatcgccaag 1020
gccaacgaac tgaccagcga actggacacc ctgatcctgg tgaccgccga ccacagccac 1080
gtgttctcct tcggcggata cacactgaga ggcaccagca tcttcggcct ggcccccgga 1140
aaagccctgg acagcaagtc ctatacctcc atcctgtatg gcaacggccc cggctacgct 1200
ctgggcggag gatctagacc agacgtgaac ggcagcacca gcgaagaacc cagctacaga 1260
cagcaggccg ccgtgcccct ggcctctgaa acccacggcg gagaagacgt ggccgtgttc 1320
gctaggggac cccaggccca cctggtgcat ggagtgcagg aggaaacctt cgtggcccac 1380
atcatggcct ttgccggctg tgtggagccc tacacagact gtaacctgcc cgcccctgcc 1440
acagctacca gcattcccga c 1461
<210> 2
<211> 487
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Leu Ile Pro Ala Glu Glu Glu Asn Pro Ala Phe Trp Asn Arg Gln Ala
1 5 10 15
Ala Gln Ala Leu Asp Val Ala Lys Lys Leu Gln Pro Ile Gln Thr Ala
20 25 30
Ala Lys Asn Val Ile Leu Phe Leu Gly Asp Gly Met Gly Val Pro Thr
35 40 45
Val Thr Ala Thr Arg Ile Leu Lys Gly Gln Met Asn Gly Lys Leu Gly
50 55 60
Pro Glu Thr Pro Leu Ala Met Asp Gln Phe Pro Tyr Val Ala Leu Ser
65 70 75 80
Lys Thr Tyr Asn Val Asp Arg Gln Val Pro Asp Ser Ala Gly Thr Ala
85 90 95
Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Tyr Arg Thr Ile Gly Val
100 105 110
Ser Ala Ala Ala Arg Tyr Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu
115 120 125
Val Thr Ser Val Ile Asn Arg Ala Lys Lys Ala Gly Lys Ala Val Gly
130 135 140
Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Ala Tyr
145 150 155 160
Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Leu Pro Ala
165 170 175
Asp Ala Gln Lys Asn Gly Cys Gln Asp Ile Ala Ala Gln Leu Val Tyr
180 185 190
Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Met Tyr Met Phe
195 200 205
Pro Glu Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Ala Ser Val Asn
210 215 220
Gly Val Arg Lys Asp Lys Gln Asn Leu Val Gln Glu Trp Gln Ala Lys
225 230 235 240
His Gln Gly Ala Gln Tyr Val Trp Asn Arg Thr Ala Leu Leu Gln Ala
245 250 255
Ala Asp Asp Ser Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Ala
260 265 270
Asp Met Lys Tyr Asn Val Gln Gln Asp His Thr Lys Asp Pro Thr Leu
275 280 285
Ala Glu Met Thr Glu Ala Ala Leu Gln Val Leu Ser Arg Asn Pro Arg
290 295 300
Gly Phe Tyr Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His His
305 310 315 320
Asp Gly Lys Ala Tyr Met Ala Leu Thr Glu Ala Ile Met Phe Asp Asn
325 330 335
Ala Ile Ala Lys Ala Asn Glu Leu Thr Ser Glu Leu Asp Thr Leu Ile
340 345 350
Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Thr
355 360 365
Leu Arg Gly Thr Ser Ile Phe Gly Leu Ala Pro Gly Lys Ala Leu Asp
370 375 380
Ser Lys Ser Tyr Thr Ser Ile Leu Tyr Gly Asn Gly Pro Gly Tyr Ala
385 390 395 400
Leu Gly Gly Gly Ser Arg Pro Asp Val Asn Gly Ser Thr Ser Glu Glu
405 410 415
Pro Ser Tyr Arg Gln Gln Ala Ala Val Pro Leu Ala Ser Glu Thr His
420 425 430
Gly Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His Leu
435 440 445
Val His Gly Val Gln Glu Glu Thr Phe Val Ala His Ile Met Ala Phe
450 455 460
Ala Gly Cys Val Glu Pro Tyr Thr Asp Cys Asn Leu Pro Ala Pro Ala
465 470 475 480
Thr Ala Thr Ser Ile Pro Asp
485
Claims (10)
1.一种编码碱性磷酸酶ALP的基因，其特征在于，所述基因的核苷酸序列如SEQ IDNo.1所示。
2.权利要求1所述的基因在构建表达碱性磷酸酶的载体中的应用。
3.权利要求1所述的基因在构建表达碱性磷酸酶的宿主细胞中的应用。
4.根据权利要求3所述的应用，其特征在于，所述宿主细胞为CHO细胞，HEK293细胞，Expi293细胞，COS7细胞，NSO细胞或BHK21细胞。
5.一种高活性碱性磷酸酶CHO稳定细胞株的构建方法，其特征在于，包括以下步骤：
(1)合成核苷酸序列如SEQ ID NO.1所示的ALP基因，然后构建到pCDNA-GS载体，得到重组表达载体pCDNA-GS-ALP；
(2)将重组表达载体pCDNA-GS-ALP电穿孔转染到CHOK1细胞中，进行亚克隆培养，并加入药物抑制剂筛选，测定酶活筛选鉴定表达高活性的克隆；
(3)步骤(2)挑出表达高活性的克隆再进行批培养、分批补料式培养，每步培养中通过高通量酶活筛选鉴定，最后得到表达高活性碱性磷酸酶CHO稳定细胞株。
6.根据权利要求6所述的高活性碱性磷酸酶CHO稳定细胞株的构建方法，其特征在于，所述药物抑制剂为MSX。
7.一株稳定表达碱性磷酸酶CHO细胞株，保藏于中国典型培养物保藏中心，其生物保藏编号为CCTCC NO：C2021225。
8.一种碱性磷酸酶的制备方法，其特征在于，将表达高活性碱性磷酸酶CHO稳定细胞株按照细胞密度0.5*106-1*106个/ml接种至细胞器中，转速110rpm～130rpm，5％CO2培养，按照培养基厂家补料方案，第4,6,8,10,12天进行补料，每天检测葡萄糖含量，若含量低于4g，则补糖至4g/L，第14天，收取细胞上清即可。
9.根据权利要求8所述的碱性磷酸酶的制备方法，其特征在于，所述制备方法还包括纯化步骤，纯化步骤具体为：
A.将收取的细胞上清加入55％硫酸铵进行沉淀，沉淀用20mM Tris,2mM MgCl2,0.08mMZnCl2，1M硫酸铵pH8.0缓冲液A进行溶解；溶解后再次离心取上清过滤；
B.步骤A的滤液进行Phenyl HP疏水层析分离，用缓冲液A和缓冲液B进行线性洗脱，由100％A洗脱至100％B，所述缓冲液B为20mM Tris，2mM MgCl2,0.08mM ZnCl2，收集目标蛋白洗脱峰，透析样品至缓冲液B中。
C.阴离子交换柱：将透析后的蛋白液进行DEAE阴离子交换层析，用缓冲液B线性洗脱至缓冲液C线，所述缓冲液C为20mM Tris,2mM MgCl2,0.08mM ZnCl2，1M NaCl,pH8.0，对洗脱样品进行检测，收集并合并目的蛋白，将收集的蛋白液透析至最终储存缓冲液3mol/lNaCl，0.1mmol/l ZnCl2，30mmol/l TEA，5mmol/l MgCl2，pH7.5。
10.根据权利要求8所述的碱性磷酸酶的制备方法，其特征在于，所述表达高活性碱性磷酸酶CHO稳定细胞株为保藏于中国典型培养物保藏中心，其生物保藏编号为CCTCC NO：C2021225的细胞株。
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| CN111662917A (zh) * | 2020-06-11 | 2020-09-15 | 济南国科医工科技发展有限公司 | 一种高比活力碱性磷酸酶工程菌、工程菌构建及碱性磷酸酶纯化方法 |
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