CN106220726A - 重组人血白蛋白及其表达载体的构建方法 - Google Patents
重组人血白蛋白及其表达载体的构建方法 Download PDFInfo
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Abstract
本发明提供重组人血白蛋白，所述重组人血白蛋白的氨基酸序列为序列表SEQ ID No.2。本发明还提供一种重组人血白蛋白表达载体的构建方法，应用该方法制备得到的重组人血白蛋白表达载体能够分泌人血白蛋白。将该表达载体进行优化后，HSA表达量达150‑180mg/L。
Description
技术领域
本发明涉及重组人血白蛋白及其表达载体的构建方法。
背景技术
人血白蛋白（HSA）是人血浆中最丰富的蛋白质，占血浆总蛋白的40%-60%，在体内起着维持血浆胶体渗透压、运输营养等重要作用。人血白蛋白临床上主要用于失血创伤、烧伤引起的休克、脑水肿及损伤引起的颅压升高、肝硬化及肾病引起的水肿或腹水；在心肺分流术、烧伤的辅助治疗、血液透析的辅助治疗及成人呼吸窘迫综合征中也有广泛应用，是医院临床急救或重症手术的必备药品，在医院市场一直占据举足轻重的地位。人血白蛋白通常由血浆提取。由于浆站改制及血液制品安全监管力度的加强等诸多因素的影响，国内市场以人血白蛋白为代表的血液制品供应严重短缺，供需矛盾问题突出。采用基因工程方法生产重组人血白蛋白具有原料来源丰富、质量可控、可形成规模化生产等优点，同时可以彻底消除病毒污染危险，对于缓解国内人血白蛋白供不应求的局面、消除血源产品的安全风险具有十分重要的社会意义和经济意义，具有广阔的发展空间和市场前景。
发明内容
为了解决现有技术中存在的问题，本发明提供了重组人血白蛋白及其表达载体的构建方法，该表达载体能够分泌表达重组人血白蛋白。
本发明提供重组人血白蛋白，所述重组人血白蛋白的氨基酸序列为序列表SEQ IDNo.2。
本发明还提供一种重组人血白蛋白表达载体的构建方法，步骤如下：
（1）对人血白蛋白序列进行优化，上游插入Kpn I酶切位点，下游插入Xho I酶切位点，并在Kpn I酶切位点后加入Kozak序列，目的基因全长1845bp，核苷酸序列为序列表SEQ IDNo.1；
（2）进行PCR扩增，之后与pUC57载体连接，转化大肠杆菌DH5α感受态细胞，诱导表达后，得到质粒pUC57-HSA；
（3）用Kpn I+Xho I分别双酶切pUC57-HSA质粒和pcDNA3.1载体片段并连接，将连接产物转化BL21感受态细胞，诱导表达后提取质粒，得到pcDNA3.1-HSA；
（4）利用脂质体转染或电转染将pcDNA3.1-HSA质粒转染CHO-K1-S2细胞。
本发明还提供应用权利要求2的方法制备得到的重组人血白蛋白表达载体分泌人血白蛋白。
本发明还提供一种分泌表达人血白蛋白的方法，是将权利要求2制备得到的重组人血白蛋白表达载体接种至培养液中，在一定条件下进行培养。
作为优选，是将权利要求2制备得到的重组人血白蛋白表达载体用G418进行持续的筛选和细胞传代，提高细胞活力至85%后，进行单克隆，选择人血白蛋白表达量最佳的细胞株，将该细胞株接种至培养液中，在一定条件下进行培养。
作为优选，是将中国仓鼠卵巢细胞CHO-K1-S2-HSA CCTCC C2015171接种至培养液中，在一定条件下进行培养。
作为优选，所述培养液为：Hyclone 公司的SFM4CHO培养基。
作为优选，所述在一定条件下培养是于37±1℃、50mL/L CO2、120±30rmp条件下培养3-6天后，再于32±2℃、50mL/L CO2、120±20rmp继续培养。
作为更优选，是将CHO-K1-S2-HSA CCTCC C2015171以5×105cells/mL的细胞量接种，培养基为Hyclone 公司的SFM4CHO培养基，于37℃、50mL/L CO2、120rmp培养5天后，添加培养基，然后再于32℃、50mL/L CO2、120rmp继续培养6天。
作为优选，所述在一定条件下培养是将CHO-K1-S2-HSA CCTCC C2015171以5×105cells/mL～8×105cells/mL的细胞量接种，培养基为Hyclone 公司的SFM4CHO培养基，培养条件为溶氧量50%、pH7.2及转速100rpm，培养温度为37±1℃，采用流加的方式进行培养。
本发明的重组人血白蛋白表达载体能够分泌表达重组人血白蛋白，将该表达载体进行优化后，HSA表达量达150-180mg/L。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。在附图中：
图1为重组质粒pcDNA3.1-HSA的酶切结果；
图2为重组质粒pcDNA3.1-HSA转染CHO-K1-SFS细胞后的Western blot检测结果，其中，M为Marker，1为转染后24h上清，2为转染后48h上清；
图3为HSA标准曲线；
图4 为CHO-K1-S2-HSA细胞的冻存与复苏分析，其中1为CHO-K1-S2-HSA复苏后第3代；2为CHO-K1-S2-HSA复苏后第4代；3为CHO-K1-S2-HSA复苏后第5代；4为CHO-K1-S2-HSA冻存前；5为HSA标准品（Sigma）100μg/mL，M为Marker；
图5 为CHO-K1-S2-HSA细胞在传代过程中白蛋白含量检测结果；其中1为CHO-K1-S2-HSA 第14代；2为CHO-K1-S2-HSA 第13代；3为CHO-K1-S2-HSA 第12代；4为CHO-K1-S2-HSA第10代；5为CHO-K1-S2-HSA 第5代；6为CHO-K1-S2-HSA 第0代；7为HSA标准品（Sigma）10μg/ml变性样品；8为HSA标准品（Sigma）10μg/ml非变性样品；M为Marker；
图6为CHO-K1-S2-HSA细胞加与不加G418时，上清中的白蛋白含量分析；其中M为Marker；1为CHO-K1-S2-HSA 加G418第1代；2为CHO-K1-S2-HSA 无G418第1代；3为CHO-K1-S2-HSA 加G418第2代；4为CHO-K1-S2-HSA 无G418第2代；5为CHO-K1-S2-HSA 加G418第3代；6为CHO-K1-S2-HSA 无G418第3代；7为CHO-K1-S2-HSA 加G418第5代；8为CHO-K1-S2-HSA 无G418第5代；
图7为CHO-K1-S2-HSA细胞上清冻干后的白蛋白检测，其中M为Marker；1为冻干1mg/mL；2为冻干10mg/mL；3为冻干100mg/mL；4为冻干500mg/mL；
图8为CHO-K1-S2-HSA细胞的生长曲线。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为市售。
实施例1
一、人血白蛋白哺乳动物真核表达载体的构建
1、人血白蛋白(HSA)基因的获得
在GENBANK、GOOGLE学术等查询有关HSA相关信息及文献资料，HSA 基因登录号：NM_000477.5。
前原白蛋白共609个氨基酸，登录号为NP_000468.1，其中前24个编码信号肽（引导新合成的蛋白质向分泌通路转移的短肽链（长度5-30个氨基酸）。
2、重组表达载体pcDNA3.1-HSA的构建
根据哺乳动物细胞密码子偏好性，主要针对CHO细胞进行密码子优化，上游插入Kpn I酶切位点，下游插入Xho I酶切位点，并在Kpn I酶切位点后加入Kozak序列，目的基因全长1845bp，由上海生工生物技术有限公司合成。合成的具体核苷酸序列如下：
GGTACC GCCACCATGAAGTGGGTCACCTTTATCTCTCTCCTGTTCTTGTTCAGCTCTGCATACTCCCGTGGTGTGTTCCGCAGGGACGCTCATAAATCTGAGGTGGCTCACAGGTTCAAGGACCTGGGCGAAGAGAACTTTAAAGCTCTCGTACTCATCGCATTCGCTCAGTACTTGCAGCAGTGTCCCTTTGAGGACCACGTGAAACTGGTGAACGAGGTAACCGAATTTGCCAAAACATGCGTGGCCGATGAGAGTGCAGAGAATTGTGATAAGTCCTTGCATACCCTCTTCGGTGACAAGCTGTGTACAGTCGCCACTCTGAGGGAAACATATGGGGAGATGGCAGATTGCTGCGCCAAACAGGAACCTGAACGAAACGAGTGTTTTCTGCAGCACAAGGACGATAATCCAAATTTGCCCCGTCTGGTTAGACCCGAGGTGGACGTGATGTGCACCGCATTTCATGACAACGAGGAAACCTTCCTGAAGAAATACCTCTACGAAATCGCCCGACGTCACCCCTATTTTTACGCTCCCGAGCTTCTGTTCTTTGCCAAGAGATACAAAGCAGCTTTTACCGAATGCTGTCAGGCCGCCGATAAAGCCGCCTGTTTGTTGCCAAAGCTTGACGAACTGAGAGATGAGGGCAAGGCATCCTCCGCAAAACAACGGCTGAAATGTGCCAGCTTGCAAAAGTTCGGGGAACGCGCCTTCAAAGCATGGGCAGTGGCAAGGCTCAGTCAACGGTTCCCCAAAGCCGAGTTTGCAGAGGTATCCAAACTTGTCACTGATCTTACTAAGGTTCATACTGAGTGTTGTCACGGAGATCTTCTTGAGTGCGCTGACGACCGGGCCGATCTCGCCAAGTATATCTGCGAGAACCAGGACTCAATTAGCAGTAAGCTGAAGGAGTGTTGCGAAAAGCCACTGCTGGAAAAATCCCACTGTATAGCCGAGGTAGAAAATGATGAGATGCCAGCCGACCTTCCTAGCCTCGCTGCTGATTTCGTGGAATCAAAAGATGTCTGCAAGAACTATGCCGAAGCCAAGGACGTGTTTCTCGGTATGTTTCTGTACGAATACGCTCGCCGACACCCAGATTATAGTGTCGTCCTGCTCCTGCGCCTCGCTAAGACATACGAGACTACATTGGAAAAGTGTTGTGCCGCCGCAGACCCCCATGAGTGCTATGCTAAGGTCTTTGATGAGTTCAAGCCTCTGGTTGAGGAGCCTCAGAACCTCATTAAGCAGAATTGCGAACTGTTCGAGCAGCTGGGCGAGTATAAGTTCCAGAACGCATTGCTTGTTCGCTATACTAAGAAAGTTCCTCAGGTGTCTACTCCTACACTTGTGGAGGTGTCCCGGAATCTGGGCAAGGTAGGAAGCAAATGCTGCAAACACCCCGAGGCTAAACGGATGCCATGTGCAGAGGATTATCTTTCAGTGGTTCTGAATCAACTGTGCGTGCTGCATGAAAAAACCCCAGTGTCAGACAGAGTTACCAAGTGCTGCACCGAGAGCCTGGTCAATAGGCGACCTTGCTTCAGCGCTCTGGAAGTGGATGAGACTTACGTGCCTAAGGAATTCAACGCTGAGACATTCACCTTTCATGCCGACATTTGCACATTGTCTGAGAAAGAGAGACAGATCAAGAAACAGACCGCTCTGGTGGAACTCGTGAAGCACAAACCCAAGGCTACAAAGGAGCAGTTGAAGGCTGTTATGGACGACTTTGCCGCTTTTGTCGAAAAGTGCTGTAAGGCTGATGACAAGGAGACATGTTTCGCCGAAGAAGGAAAGAAGCTGGTCGCAGCCTCTCAAGCTGCCCTCGGGCTGCTCGA G。
其中下划线分别为Kpn I和Xho I酶切位点，斜体为Kozak序列。
设计一套引物：
上游F1：GGTACCGCCACCATGAAGTGGGTCACC；
下游R1：CTCGAGCAGCCCGAGGGCAGCTTG。
以合成的基因为模板，用F1/R1为引物扩增（1845bp），PCR反应体系为dNTPs(10mM)18.5μL，10×PCR 缓冲液 2.5μL，F1(25mM) 0.5μL，R1(25mM) 0.5μL，Taq DNA聚合酶0.5μL，模板基因 2μL，加ddH2O 补至25μL。反应条件94℃预变性5min；94℃变性30s，61℃退火45s，72℃延伸60s，共循环35次；72℃延伸10min，4℃保存。
将PCR产物与pUC57载体于16℃连接2h，转化大肠杆菌DH5α感受态细胞，并涂布氨苄青霉素抗性LB平板。37℃恒温培养12-15h后，挑取单一菌落接种于含50mg/L 的Amp+的LB液体培养基中，37℃ 200rpm振荡培养8h后提取质粒，由上海生工生物技术有限公司进行测序。测序正确的质粒命名为pUC57-HSA。
用Kpn I+Xho I分别双酶切pUC57-HSA质粒和pcDNA3.1载体片段，凝胶纯化回收后用T4 DNA连接酶于16℃连接过夜，次日将连接产物转化BL21感受态细胞，并涂布含Amp+的LB平板，37℃培养约15h后，随机挑取5个单克隆菌斑加入含50mg/L 的Amp+的LB液体培养基中，37℃ 200rpm振荡培养8h后提取质粒，用Kpn I+Xho I进行酶切鉴定。酶切鉴定结果参见图1，与预期相符（1845bp，5428bp）。双酶切鉴定为阳性的质粒由上海生工生物技术有限公司进行测序。核苷酸序列参见序列表SEQ ID No.1。
测序：由TAKARA测序，并与Genbank中的标准人血清白蛋白序列（NP_000468.1）blast比对，氨基酸序列同源性为100%。氨基酸序列参见序列表SEQ ID No.2。
将测序鉴定正确的pcDNA3.1-HSA质粒转化大肠杆菌后用OMEGA公司的去内毒素质粒小提试剂盒重新提取，取10μL重组质粒10倍稀释后用分光光度计测定OD260/280，以1.8-2.0为宜，其余质粒-20℃分装保存。
3、批培养CHO-K1-S2-HSA C0细胞的制备
3.1 CHO-K1-SFS细胞的复苏及传代
复苏无血清全悬浮CHO-K1-SFS细胞(本实验室驯化的无血清全悬浮CHO-K1-S2细胞，见《中国生物制品学杂志》2014年2月第27期第2期《无血清悬浮培养CHO-K1-SFS细胞系的建立及其生物学特性》)，传代3-5代。
细胞复苏：从液氮罐中取出1支冻存的无血清全悬浮CHO-K1-SFS细胞，立即放入37℃的温水中快速晃动，直至完全溶解。然后900rpm 离心10min，弃去上清，细胞中加入20mL的无血清SFM4CHO培养基（Hyclone 公司的SFM4CHO培养基），于37℃、50mL/LCO2 120rpm培养。24h后补加20mL的SFM4CHO培养基，继续37℃、50mL/LCO2 120rpm培养。
细胞传代：48h后取1mL细胞悬液稀释后进行台盼蓝计数和计算细胞活力。取出部分细胞加入新鲜的SFM4CHO培养基，使得最终的细胞密度为5×105cells/mL，37℃、50mL/LCO2 120rpm继续培养。48h后同上进行传代，如此直至细胞活力≥95%后方能进行转染。800rpm 离心15min收集此时的细胞上清，0.22μm的滤膜过滤，命名为旧SFM培养基，-20℃分装保存，用于细胞冻存液的配制。
3.2 转染
待细胞活力≥95%时，利用脂质体转染或电转染将pcDNA3.1-HSA质粒转染CHO-K1-SFS细胞，24h、48h后分别收集细胞上清，利用Western blot检测有白蛋白表达。检测结果参见图2。
（1）脂质体转染：使用Invitrogen公司的Lipofectamine2000进行转染，按照说明书进行操作。具体如下：
取出1mL悬浮细胞进行细胞计数，确定细胞密度和活力，细胞活力≥95%方可进行电转。取总数为1-2×106cells/组细胞转移至离心管中，室温下800rpm离心10min，弃去上清。细胞中加入2mL的opti-MEM重悬细胞，800rpm离心10min，弃去上清。如此重复清洗细胞2次，然后加入2mL的opti-MEM轻轻混匀后加入6孔板中。
转染复合物：①将4μg pcDNA3.1-HSA稀释于250μL opti-MEM 中；②将10μLLipofectamine2000稀释于250μL opti-MEM中，轻轻混匀，室温孵育5min。③将步骤①的混合液逐滴加入步骤②的混合液中，并轻轻混匀，室温孵育20min。
20min后，将转染复合物加入至6孔板中的细胞中，轻轻摇匀。37℃、50mL/L CO2静置培养。5h轻轻弃去上清，加入2mL 无血清SFM4CHO培养基，37℃ 50mL/L CO2静置培养。
（2）电转染：转染前一天，将细胞传代，保证实验当天细胞生长至对数生长期，悬浮细胞密度约在1-2×106cells/mL。电转：使用LONZA公司的4D-NucleofectorTM电转仪。
实验前准备：将试剂盒取出放置于室温中约30min；加培养基（不含抗生素）到6孔板中，每孔1.5mL，放于37℃、50mL/LCO2培养箱预热。取出1mL悬浮细胞进行细胞计数，确定细胞密度和活力，细胞活力≥95%方可进行电转。取总数为1-2×106cells/组细胞转移至离心管中，室温下800rpm离心10min，弃去上清。
配置电转缓冲液：82μL NuclefectorTM Solution+18μL Supplement，轻轻吹打混匀。用电转缓冲液重悬细胞，使其形成单细胞悬液（注：电转缓冲液为现用现配，电转缓冲液重悬细胞后应尽快电转，同时切忌气泡）。将10μg重组质粒pcDNA3.1-HSA加至上述细胞悬液中，轻轻混匀。迅速转移至电转杯中，选择程序DT133进行细胞的电转。电转完成后取出预热的培养基，用pipettes吸取约500μL加入电转杯中混合细胞2-3次，然后再用pipettes将电转杯中的所有悬液吸出加入对应的6孔板中，如此重复2-3次，至37℃、50mL/L CO2培养箱中静止培养。
Western blot检测：配制12%SDS-PAGE分离胶，分别收集转染前和转染后的细胞上清进行SDS-PAGE电泳。然后将蛋白条带在低温、恒压60V转印至PVDF膜，2h后取出膜用5%的脱脂奶粉室温摇床封闭2h，加入1:150的自制的兔抗pHSA IgG-HRP，室温摇床孵育2h，经PBST充分洗涤后进行DAB显色。
3.3 加药筛选
转染完成后将细胞置于6孔板中静止培养，24h后加400μg/mL的G418进行持续的筛选和细胞传代，此过程中细胞一直于37℃、50mL/L CO2培养箱中静止培养。待细胞数目达到约8×106时，将静止培养改为悬浮培养（37℃ 50mL/L CO2培养箱中120rpm），细胞传代3~5代后，细胞活力由最初的50%可提高到85%，最终得到批培养的CHO-K1-S2-HSA C0细胞。
（1）静止培养的细胞传代：转染24h后观察细胞形态又圆又亮。将6孔板中的1孔细胞传至2孔，2mL/孔，加入终浓度为400μg/mL的G418 SFM4CHO筛选培养基进行培养。48h后每孔中补加2mL培养基。再培养48h将6孔板中的1孔细胞传代至1个T25的细胞瓶中，10mL/瓶。继续培养72h后将1个T25细胞瓶传代至1个T100的细胞瓶中，30mL/瓶。传代过程中维持G418的终浓度为400μg/mL，细胞均置于37℃、50mL/LCO2培养箱中静置培养。
（2）由静止培养改为悬浮培养：取少量细胞进行计数，待细胞总数达到8×106时，收集T100细胞瓶中的细胞，900rpm 离心10min，将上清保存用于Western blot检测，细胞中加入10mL新鲜的含400μg/mL G418的无血清SFM4CHO培养基，于37℃、50mL/LCO2 120rpm培养，此时细胞密度约为8×105cells/mL，活力约为50-70%。48h进行细胞计数和活力测定，900rpm 离心10min，将上清保存用于Western blot检测，细胞中加入适当的新鲜SFM4CHO培养基，维持细胞密度在8×105cells/mL左右。48h后同上进行传代，传代过程中维持G418的终浓度为400μg/mL，细胞均置于37℃、50mL/L CO2培养箱中120rpm培养，直至细胞活力提高到85%以上。
4、第1次单克隆细胞CHO-K1-S2-HSA C1的获得
将批培养的CHO-K1-S2-HSA C0细胞计数后进行稀释至每孔5.5/11/22/44/88/176cells单克隆培养（期间G418浓度维持在400μg/ml），15天后观察有约200个单一克隆细胞及50个两克隆的细胞，分别取克隆细胞上清进行Western blot检测。选择其中Western blot检测条带最深的1株细胞进行放大培养（96孔板、24孔板、6孔板、细胞瓶、摇瓶），命名为CHO-K1-S2-HSA C1，并将放大培养后的细胞进行冻存。
细胞冻存方法为：取1mL细胞进行计数。收集细胞，900rpm 离心10min，收集上清用于Western blot检测，细胞中加入适量的细胞冻存液（93%旧SFM培养基+7%DMSO），混合均匀后1.5mL/支分装，每支冻存管中的细胞数目不少于2×107cells。先于4℃ 放置2h，然后-70℃过夜，之后取出冻存管，移入液氮罐中进行长期保存。
将CHO-K1-S2-HSA C1细胞以5×105cells/mL的细胞量接种，平行接种3瓶。接种细胞后，在培养第三天时，取上述细胞一瓶，进行计数和1000rpm离心10min收集细胞上清。在接种第6天时，如上进行细胞计数和上清收集。然后将培养箱温度下调至32℃，继续培养3天（共9天），进行细胞计数和上清收集。 用Abcam公司的ELISA检测试剂盒检测（ab108788Albumin Human ELISA Kit）CHO-K1-S2-HSA C1细胞上清中的HSA含量。
将试剂盒中的标准品进行倍比稀释后，绘制标准曲线（见图3）。根据标准曲线，计算细胞上清中的HSA浓度（见表1）。
表1 不同培养时间下CHO-K1-S2-HSA C1细胞中的HSA含量
5、第2次单克隆细胞CHO-K1-S2-HSA的获得、鉴定及细胞建库
5.1 CHO-K1-S2-HSA C0第2次单克隆筛选
将第1次单克隆获得的CHO-K1-S2-HSA C1这株细胞进行再次单克隆，得到1株白蛋白表达较强的细胞，将该细胞株命名为CHO-K1-S2-HSA。
5.2 CHO-K1-S2-HSA细胞的ELISA检测
将CHO-K1-S2-HSA细胞以5×105cells/mL的细胞量接种，平行接种3瓶。接种细胞后，在培养第三天时，取上述细胞一瓶，进行计数和1000rpm离心10min收集细胞上清。在接种第6天时，如上进行细胞计数和上清收集。然后将培养箱温度下调至32℃，继续培养3天（共9天），进行细胞计数和上清收集。 用Abcam公司的ELISA检测试剂盒检测（ab108788 AlbuminHuman ELISA Kit）CHO-K1-S2-HSA细胞上清中的HSA浓度，方法同CHO-K1-S2-HSA C1（见表2）。
表2不同培养时间CHO-K1-S2-HSA细胞中的HSA含量
检测结果发现，CHO-K1-S2-HSA C1及CHO-K1-S2-HSA细胞中HSA的表达量均随着培养时间的延长而增加，且每组中CHO-K1-S2-HSA的表达量均较CHO-K1-S2-HSA C1的表达量高，最高可达为28.32mg/L。故仅进行CHO-K1-S2-HSA细胞培养条件的进一步优化。
将CHO-K1-S2-HSA细胞放大培养至50ml摇瓶中，传代后建立主细胞库。并将该株细胞中国仓鼠卵巢细胞CHO-K1-S2-HSA进行保藏，保藏单位为：中国典型培养物保藏中心(CCTCC)，保藏地址：武汉市武汉大学，保藏编号：CCTCC C2015171，保藏日期：2015年11月5日。
取上述冻存的1支CCTCC C2015171细胞进行复苏，1200rpm离心10min，弃上清，细胞中加入30mL含400μg/mLG418的SFM4CHO培养基中，37℃ CO2培养箱中120rpm振荡培养。48h后补加20mL上述培养基。继续培养24h后将此瓶细胞传至3瓶，每瓶30mL。再培养48h后每瓶中补加20mL上述培养基。再培养48h后以1:4进行传代，共计12瓶，50mL/瓶。培养约72h后收集细胞，计数后进行冻存。建立工作细胞库。
6 CHO-K1-S2-HSA细胞的鉴定
6.1 冻存及复苏分析
将冻存的CHO-K1-S2-HSA复苏后进行Western blot检测，结果仍有白蛋白表达（见图4）。
6.2 传代分析
将CHO-K1-S2-HSA这株细胞放大至50ml摇瓶中，此时标记为CHO-K1-S2-HSA第0代。将CHO-K1-S2-HSA第0代传代14代，至10代左右白蛋白含量略有下降（见图5）。
6.3 加与不加G418分析
将CHO-K1-S2-HSA第0代分别传代5代，进行加与不加G418上清白蛋白含量分析比较，结果无明显差异（见图6）。
6.4 冻干样品中的白蛋白检测
将收集的2.5L CHO-K1-S2-HSA细胞上清进行冻干，共冻干约31.8g，分别用SFM4CHO培养基溶解为1mg/mL、10mg/mL、100mg/mL及500mg/mL，进行Western blot检测，结果见图7。注：因冻干时间太短，有部分样品未完全冻干。
6.5 生长曲线的绘制
复苏1支CHO-K1-S2-HSA细胞，按5.0×105cells/mL的密度传代，共接种三瓶细胞，培养9天（37℃、50mL/L CO2、120rmp），每天取500mL细胞培养液10倍稀释计数，三瓶细胞计数的平均值为当天的细胞密度，图8为CHO-K1-S2-HSA细胞的生长曲线。
7 优化、稳定白蛋白表达的工艺流程及放大
7.1 CHO-K1-S2-HSA细胞培养条件的优化
将复苏后的CHO-K1-S2-HSA细胞以5×105cells/mL的细胞量接种至50ml摇瓶，做4个平行，培养基为Hyclone 公司的SFM4CHO培养基。第一组细胞37℃、50mL/L CO2、120rmp培养3天，从第3天开始降温至32℃，32℃、50mL/L CO2、120rmp继续培养7天；第二组为37℃、50mL/L CO2、120rmp培养4天，32℃、50mL/L CO2、120rmp继续培养6天；第三组为37℃、50mL/L CO2、120rmp培养5天，32℃、50mL/L CO2、120rmp继续培养6天；第四组为37℃、50mL/L CO2、120rmp培养6天，32℃、50mL/L CO2、120rmp继续培养4天。最后一天时进行细胞计数、收集上清进行ELISA检测，结果见表3。
表3 CHO-K1-S2-HSA细胞优化培养条件
虽然调整了降温时间，但总的培养时间未能延长，一般在培养第十天后开始出现大量死亡，并且培养基开始粘稠浑浊，在第十一天或第十二天时，细胞基本全部死亡，限制了HSA的表达。CHO-K1-S2-HSA细胞在第5天降温后表达量提高较多，可尝试在第5天降温后补加新鲜培养基，这样HSA的表达量还会有提升的空间。
7.2 放大培养
将CHO-K1-S2-HSA细胞从50mL摇瓶进一步扩大培养至5L生物反应器中，人工监测培养液中葡萄糖、谷氨酰胺及主要代谢物乳酸及氨的浓度变化，接种量为：5×105cells/mL～8×105cells/mL，培养条件为溶氧量50%、pH7.2及转速100rpm，培养温度为37±1℃，培养基为Hyclone 公司的SFM4CHO培养基，采用流加的方式进行培养，利用Abcam公司的ELISA试剂盒进行检测，最终实现了HSA的表达量为150-180mg/L。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 重组人血白蛋白及其表达载体的构建方法
<120> 西北民族大学
<170> PatentIn version 3.5
<210> 1
<211> 1845
<212> DNA
<213> 人工序列
<400> 1
ggtaccgcca ccatgaagtg ggtcaccttt atctctctcc tgttcttgtt cagctctgca 60
tactcccgtg gtgtgttccg cagggacgct cataaatctg aggtggctca caggttcaag 120
gacctgggcg aagagaactt taaagctctc gtactcatcg cattcgctca gtacttgcag 180
cagtgtccct ttgaggacca cgtgaaactg gtgaacgagg taaccgaatt tgccaaaaca 240
tgcgtggccg atgagagtgc agagaattgt gataagtcct tgcataccct cttcggtgac 300
aagctgtgta cagtcgccac tctgagggaa acatatgggg agatggcaga ttgctgcgcc 360
aaacaggaac ctgaacgaaa cgagtgtttt ctgcagcaca aggacgataa tccaaatttg 420
ccccgtctgg ttagacccga ggtggacgtg atgtgcaccg catttcatga caacgaggaa 480
accttcctga agaaatacct ctacgaaatc gcccgacgtc acccctattt ttacgctccc 540
gagcttctgt tctttgccaa gagatacaaa gcagctttta ccgaatgctg tcaggccgcc 600
gataaagccg cctgtttgtt gccaaagctt gacgaactga gagatgaggg caaggcatcc 660
tccgcaaaac aacggctgaa atgtgccagc ttgcaaaagt tcggggaacg cgccttcaaa 720
gcatgggcag tggcaaggct cagtcaacgg ttccccaaag ccgagtttgc agaggtatcc 780
aaacttgtca ctgatcttac taaggttcat actgagtgtt gtcacggaga tcttcttgag 840
tgcgctgacg accgggccga tctcgccaag tatatctgcg agaaccagga ctcaattagc 900
agtaagctga aggagtgttg cgaaaagcca ctgctggaaa aatcccactg tatagccgag 960
gtagaaaatg atgagatgcc agccgacctt cctagcctcg ctgctgattt cgtggaatca 1020
aaagatgtct gcaagaacta tgccgaagcc aaggacgtgt ttctcggtat gtttctgtac 1080
gaatacgctc gccgacaccc agattatagt gtcgtcctgc tcctgcgcct cgctaagaca 1140
tacgagacta cattggaaaa gtgttgtgcc gccgcagacc cccatgagtg ctatgctaag 1200
gtctttgatg agttcaagcc tctggttgag gagcctcaga acctcattaa gcagaattgc 1260
gaactgttcg agcagctggg cgagtataag ttccagaacg cattgcttgt tcgctatact 1320
aagaaagttc ctcaggtgtc tactcctaca cttgtggagg tgtcccggaa tctgggcaag 1380
gtaggaagca aatgctgcaa acaccccgag gctaaacgga tgccatgtgc agaggattat 1440
ctttcagtgg ttctgaatca actgtgcgtg ctgcatgaaa aaaccccagt gtcagacaga 1500
gttaccaagt gctgcaccga gagcctggtc aataggcgac cttgcttcag cgctctggaa 1560
gtggatgaga cttacgtgcc taaggaattc aacgctgaga cattcacctt tcatgccgac 1620
atttgcacat tgtctgagaa agagagacag atcaagaaac agaccgctct ggtggaactc 1680
gtgaagcaca aacccaaggc tacaaaggag cagttgaagg ctgttatgga cgactttgcc 1740
gcttttgtcg aaaagtgctg taaggctgat gacaaggaga catgtttcgc cgaagaagga 1800
aagaagctgg tcgcagcctc tcaagctgcc ctcgggctgc tcgag 1845
<210> 2
<211> 609
<212> PRT
<213> 人工蛋白
<400> 2
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala
20 25 30
His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu
35 40 45
Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val
50 55 60
Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp
65 70 75 80
Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp
85 90 95
Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala
100 105 110
Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln
115 120 125
His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val
130 135 140
Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys
145 150 155 160
Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro
165 170 175
Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys
180 185 190
Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu
195 200 205
Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys
210 215 220
Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val
225 230 235 240
Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser
245 250 255
Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly
260 265 270
Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile
275 280 285
Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu
290 295 300
Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp
305 310 315 320
Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser
325 330 335
Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly
340 345 350
Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val
355 360 365
Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys
370 375 380
Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu
385 390 395 400
Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys
405 410 415
Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu
420 425 430
Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val
435 440 445
Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His
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Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val
465 470 475 480
Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg
485 490 495
Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe
500 505 510
Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala
515 520 525
Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu
530 535 540
Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys
545 550 555 560
Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala
565 570 575
Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe
580 585 590
Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly
595 600 605
Leu
Claims (9)
1.重组人血白蛋白，其特征在于：所述重组人血白蛋白的氨基酸序列为序列表SEQ IDNo.2。
2.一种重组人血白蛋白表达载体的构建方法，其特征在于：步骤如下：
（1）对人血白蛋白序列进行优化，上游插入Kpn I酶切位点，下游插入Xho I酶切位点，并在Kpn I酶切位点后加入Kozak序列，目的基因全长1845bp，核苷酸序列为序列表SEQ IDNo.1；
（2）进行PCR扩增，之后与pUC57载体连接，转化大肠杆菌DH5α感受态细胞，诱导表达后，得到质粒pUC57-HSA；
（3）用Kpn I+Xho I分别双酶切pUC57-HSA质粒和pcDNA3.1载体片段并连接，将连接产物转化BL21感受态细胞，诱导表达后提取质粒，得到pcDNA3.1-HSA；
（4）利用脂质体转染或电转染将pcDNA3.1-HSA质粒转染CHO-K1-S2细胞。
3.应用权利要求2的方法制备得到的重组人血白蛋白表达载体分泌人血白蛋白。
4.一种分泌表达人血白蛋白的方法，其特征在于：是将权利要求2制备得到的重组人血白蛋白表达载体接种至培养液中，在一定条件下进行培养。
5.根据权利要求4所述的方法，其特征在于：是将权利要求2制备得到的重组人血白蛋白表达载体用G418进行持续的筛选和细胞传代，提高细胞活力至85%后，进行单克隆，选择人血白蛋白表达量最佳的细胞株，将该细胞株接种至培养液中，在一定条件下进行培养。
6.根据权利要求5所述的方法，其特征在于：是将中国仓鼠卵巢细胞CHO-K1-S2-HSACCTCC C2015171接种至培养液中，在一定条件下进行培养。
7.根据权利要求5或6所述的方法，其特征在于：所述培养液为SFM4CHO培养基。
8.根据权利要求7所述的方法，其特征在于：所述在一定条件下培养是于37±1℃、50mL/L CO2、120±30rmp条件下培养3-6天后，再于32±2℃、50mL/L CO2、120±20rmp继续培养。
9.根据权利要求7所述的方法，其特征在于：所述在一定条件下培养是将CHO-K1-S2-HSA CCTCC C2015171以5×105cells/mL～8×105cells/mL的细胞量接种，培养基为Hyclone公司的SFM4CHO培养基，培养条件为溶氧量50%、pH7.2及转速100rpm，培养温度为37±1℃，采用流加的方式进行培养。
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Application publication date: 20161214 |