CN116606485A - 一种黄原胶填料及制备方法和应用 - Google Patents
一种黄原胶填料及制备方法和应用 Download PDFInfo
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- xanthan gum
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Abstract
本发明涉及蛋白质纯化填料的制备技术领域,尤其涉及一种黄原胶微球填料及制备方法和应用，其由质量比为(1:1‑1:4)的聚乙烯醇与黄原胶组成。所制备的黄原胶基微球绿色环保、无毒、可降解、生物相容性好，得到的微球粒径均一，表面光滑；其原料黄原胶价格低廉，性能稳定。
Description
技术领域
本发明涉及蛋白质纯化填料的制备技术领域,尤其涉及一种黄原胶微球的制备方法。
背景技术
天然多糖具有良好的亲水性和生物相容性，能够维持蛋白的构相，保持蛋白的活性。多糖基质的蛋白纯化填料是目前蛋白质分离纯化工业使用的最主要的填料之一。
黄原胶(XG)是一种价格低廉，性能优良的生物高分子酸性杂多糖，含有纤维素骨架和三糖侧链，是集增稠、悬浮、乳化、稳定于一体，性能较为优越的生物胶。以其为原料所制备的水凝胶亲水性强、无毒、可降解、生物相容性好，常作高吸水性树脂、药物载体和微胶囊等，在生物领域具有广阔的应用前景。
随着近年国内生物医药行业和体外诊断等行业的快速发展，蛋白纯化填料的市场需求也随着变大。当前生物医药的迅速发展，人们对蛋白类药物的需求量日益增长，同时也对蛋白分离纯化的技术提出了挑战。快速、高效的分离纯化技术是必然的发展需求。其中蛋白纯化是生物药生产的核心环节，决定产品的质量，也决定着整个下游生产成本的高低。
层析技术是生物大分子分离纯化最有效的手段之一,可以实现很高的分离度,其中离子交换层析的应用最为广泛。对于离子交换层析,目标蛋白质与介质间的静电相互作用起主导作用，直接影响着蛋白质的分离纯化。近年来，离子交换层析技术已经广泛应用于蛋白质、酶、核酸、肤、寡核昔酸、病毒、噬菌体和多糖的分离和纯化。具有如下优点：(1)具有开放性支持骨架，大分子可以自由进入和迅速扩散，故吸附容量大。(2)具有亲水性，用温和条件就可以洗脱，不致引起蛋白质变性或酶的失活。(3)表面积大、交换容量大，回收率高，可用于分离和制备。
本项目将制备一种黄原胶微球填料，项目不仅能有效解决蛋白质纯化工艺瓶颈问题，可将未知得基因序列得野生菌进行纯化，也将为蛋白纯化乃至其它生物大分子高效纯化提供必要的理论前提和技术支持。
发明内容
为了实现上述目的，本发明采用了如下技术方案：
一种黄原胶微球的制备方法，包括以下步骤：
S1：将聚乙烯醇溶解在70-100℃的热水中，冷却至室温后，加入黄原胶(总聚合物浓度为1-4％，w/v)搅拌5-16h，得到均匀的无气泡溶液，作为水相；
S2：将油相和乳化剂加入水相中，用搅拌器以800rpm/min-5000rpm/min搅拌40min-4h；
S3：缓慢加入4×10-6-4×10-4％(占体系体积)戊二醛，及1-2mL 1M硫酸,继续用搅拌器以800rpm/min-5000rpm/min搅拌40min-4h；
S4：用热乙醇和蒸馏水洗涤3-5次，去除油相；
S5：将制备的微球在室温～50℃下干燥1-24h，得到黄原胶微球。
优选的，所述的S1中，黄原胶与聚乙烯醇质量比为(1:1-1:4)，优选1：2，且总聚合物(聚乙烯醇与黄原胶质量之和)浓度为1-4％，w/v，g/mL。
优选的，所述的S2中，油相为液体石蜡，乳化剂为1-8％w/w(占油相)span 80，水相在体系中的体积不超过40％(1-40％)。
优选的，所述的S4中，清洗微球的溶剂可选择苯、乙醚、氯仿、二硫化碳、热乙醇(80-95℃)中的一种或二种以上。
优选的，若所述的S4中，选用热乙醇清洗可以直接得到干燥的黄原胶微球，可省略步骤S5。
本发明具有如下优点：
所制备的黄原胶基微球绿色环保、无毒、可降解、生物相容性好，得到的微球粒径均一，表面光滑；其原料黄原胶价格低廉，性能稳定。微球制备过程简单方便，无需多步重复步骤。可将蛋白液中的葡萄糖苷酶分离纯化，纯化效果较好。
附图说明
图1为本发明提出的一种黄原胶微球的制备方法实施例1中黄原胶微球的扫描电子显微镜示意图；
图2为本发明提出的一种黄原胶微球的制备方法实施例1的应用例中SDS-PAGE验证图；
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。
实施例中相关试剂均购自生工生物工程(上海)股份有限公司；
实施例1
一种黄原胶微球的制备方法，包括以下步骤：
1)将聚乙烯醇溶解在95℃的热水中，冷却至室温后，加入黄原胶(黄原胶与聚乙烯醇质量比1：4，总聚合物(聚乙烯醇和黄原胶之和)浓度为4％，(w/v，g/mL)搅拌14h，得到均匀的无气泡溶液，作为水相；
2)将油相液体石蜡和4％w/w(液体石蜡质量的4％w/w)的乳化剂span80加入水相中(油相在水相油相混合物的体系中的体积30％)，将制备的油相加入水相中，用搅拌器以900rpm/min搅拌1h，缓慢加入0.15μL戊二醛(交联剂)和1mL 1M H2SO4用搅拌器以900rpm/min搅拌4h；
3)用热乙醇洗涤5次(使用95℃热乙醇，每次100mL)，去除油相，得到干燥的黄原胶微球。
使用扫描电镜对微球进行表面形貌表征，见图1，结果显示直径为20μm-50μm的黄原胶微球成功制备。
将黄原胶基微球作为填料(经过离子交换剂预处理)对蛋白进行纯化并验证纯化效果(应用例)：
蛋白溶液的获取：pET28a-BGL3质粒由吉林省库美生物科技有限公司全基因合成，合成方式为pET28a载体多克隆位点Nco I、Xho I两处连接BGL3(β-葡萄糖苷酶)，(该蛋白在NCBI数据库中可查全基因序列，编号：4840204；，并委托吉林省库美生物科技有限公司将质粒转入大肠杆菌制备表达菌株BL21-pET28a-BGL3。
将BL21-pET28a-BGL3表达菌株于LB固体平板(含卡那霉素抗性，30μg/mL)划线，37℃培养箱培养12h，取出挑取单菌落接种于10mL LB液体培养基(LB液体培养基配方：蛋白胨1％(w/v),酵母粉0.5％(w/v),氯化钠1.0％(w/v).pH7.0-7.2，此处7.0，于水中)(含卡那霉素抗性，30μg/mL)锥形瓶中，37℃，200rpm摇床培养至OD600nm＝0.6作为种子液，以种子液：发酵培养基等于1:50(v/v)的比例向200mL LLB液体培养基(LLB液体培养基配方：蛋白胨1％(w/v),酵母粉0.5％(w/v),氯化钠0.5％(w/v).pH7.0-7.2，此处7.0，于水中)(含有30μg/mL卡那霉素Kan)中加入4mL种子液，在37℃，200rpm的条件下扩大发酵培养至OD600nm＝0.6时，取出发酵液，向其中添加终浓度为0.5mmol/L的IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactoside)进行诱导蛋白质的表达，并将菌液放到16℃，200rpm的条件下发酵16h；将200mL发酵液从摇床中取出后装入250mL的离心杯中，在8000g的条件下离心5min以收集菌体，完成后弃上清。再以相同离心条件进行水洗离心，取0.02mol/L，pH7.3的Tris-HCl缓冲液50mL进行重悬并进行功率为500W，工作时间5s，间歇时间2s，总时间5-15min(在此为12min)的超声破碎(工作和间歇交替进行)；破碎后10,000rpm，4℃，离心30min，收集上清液，得到含有目的蛋白的粗酶液；沉淀以0.02mol/L，pH7.3的Tris-HCl缓冲液50mL进行重悬，得沉淀液。
离子交换剂的处理：称取2g黄原胶基微球干粉填料，加入0.5mol/LNaOH溶液(50mL)，轻轻搅拌，浸泡0.5h，用玻璃砂漏斗抽滤，并用去离子水洗至近中性(pH7.0)，抽干后，将微球填料放入烧杯中，加50mL 0.5mol/L HCl，搅匀,，浸泡0.5h后用去离子水洗至近中性(pH7.0)，再用50mL 0.5mol/L NaOH重复浸泡0.5h一次，用去离子水洗至近中性后(pH7.0)，抽干备用。
装柱与平衡：先将内径10mm，长度200mm的玻璃层析柱(F508001，购自生工生物工程(上海)股份有限公司)垂直装好，在烧杯内用0.02mol/L、pH7.3 Tris-HCl缓冲液洗离子交换剂处理过的黄原胶基微球填料3-5次(在此为4次)，每次200mL。吸取烧杯底部黄原胶基微球填料装柱5mL，然后使用新配制的0.02mol/L、pH7.3 Tris-HCl缓冲液洗柱至流出液的电导率与缓冲液相同时即可上样。
上样与洗脱：
本实验洗脱液采用500mL 0.02mol/L、pH7.3的Tris-HCl缓冲液和500mL含0.2mol/L浓度NaCl的0.02mol/L、pH7.3的Tris-HCl缓冲液。
向层析柱中加入蛋白溶液(上述制备的含有目的蛋白的粗酶液，含25mg蛋白，其体积为2mL)结合3h后通过AKTA仪器(美国GE Healthcare Life Science公司：AkTa primeplus蛋白质纯化仪)以2mL/min的流速将上述缓冲液分别对蛋白进行洗脱(先使用0.02mol/L、pH7.3的Tris-HCl缓冲液,再使用含0.2mol/L浓度NaCl的0.02mol/L、pH7.3的Tris-HCl缓冲液)，每个洗脱液(上述两种)均分别洗脱至电脑软件中紫外吸收检测值回到基线时(达到平稳)停止仪器自动收集装置，机器设置每管收集5mL,将收集管中使用0.2mol/L浓度NaCl的0.02mol/L、pH7.3的Tris-HCl缓冲液洗脱的流出液汇集，6管流出液，共30mL。
SDS-PAGE验证：将两种缓冲液洗脱的流出液进行SDS-PAGE验证，泳道1为洗脱液，泳道2为粗酶液，泳道3为沉淀液，如图2所示，目的蛋白BGL3(58.2KDa)经此黄原胶基微球填料后得以初步纯化，蛋白纯度达70％，收率达70％，该结果表明，本发明制备的黄原胶基微球填料可以应用于分离大肠杆菌表达的葡萄糖苷酶。
本发明制备黄原胶微球的过程简单，无需多步骤或重复，主要原料黄原胶价格低廉，微球性能稳定且可用于蛋白质层析填料，本发明不仅能有效解决蛋白质纯化工艺瓶颈问题，也将为蛋白纯化乃至其它生物大分子高效纯化提供必要的理论前提和技术支持。
Claims (4)
1.一种黄原胶填料，其特征在于：由质量比为(1:1-1:4，优选1：1-2)的黄原胶与聚乙烯醇组成。
2.一种权利要求1所述填料的制备方法，其特征在于：
包括以下步骤：
S1：将聚乙烯醇溶解在70-100℃的热水中，冷却至室温后，加入黄原胶搅拌5-16h，作为水相；
黄原胶与聚乙烯醇质量比为(1:1-1:4)(优选1：1-1：2)；水相中聚乙烯醇与黄原胶质量之和的浓度为1-4％(优选3-4％)，w/v，g/mL；
S2：将油相和乳化剂加入水相中，用搅拌器以800rpm/min-5000rpm/min搅拌40min-4h；
油相为液体石蜡、甲苯、环己烷中的一种或二种以上；乳化剂为span80，其为油相质量的1-8％(优选7-8％)；水相在搅拌后的体系中的体积(1-40％，优选28-30％)；
S3：加入4×10-6-4×10-4％(占S2获得体系体积)戊二醛，及1-2mL1-2M硫酸，继续以800rpm/min-5000rpm/min搅拌40min-4h；
S4：用80-95℃热乙醇洗涤，去除油相，得到黄原胶微球填料；
或，用溶剂和蒸馏水分别洗涤，去除油相；然后将制备的微球在室温～50℃(优选22-25℃)下干燥1-24h(优选1-2h)，得到黄原胶微球填料。
3.按照权利要求2所述填料的制备方法，其特征在于：
所述清洗微球的溶剂可选择苯、乙醚、氯仿、二硫化碳中的一种或二种以上。
4.一种权利要求1所述填料在蛋白酶液中初步分离大肠杆菌表达的葡萄糖苷酶和/或纤维素酶过程的应用。
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| CN1485094A (zh) * | 2002-09-24 | 2004-03-31 | 中国科学院过程工程研究所 | 一种天然高分子多孔微球及其制备方法和用途 |
| CN109293997A (zh) * | 2018-09-05 | 2019-02-01 | 安徽新翔包装材料有限公司 | 一种可防水的降解环保袋 |
| CN109289081A (zh) * | 2018-09-30 | 2019-02-01 | 华中科技大学鄂州工业技术研究院 | 一种抗粘连的聚乙烯醇栓塞微球及其制备方法和应用 |
| CN113355313A (zh) * | 2021-04-27 | 2021-09-07 | 浙江工业大学 | 一种聚合物微球及其制备与应用 |
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| CN1485094A (zh) * | 2002-09-24 | 2004-03-31 | 中国科学院过程工程研究所 | 一种天然高分子多孔微球及其制备方法和用途 |
| CN109293997A (zh) * | 2018-09-05 | 2019-02-01 | 安徽新翔包装材料有限公司 | 一种可防水的降解环保袋 |
| CN109289081A (zh) * | 2018-09-30 | 2019-02-01 | 华中科技大学鄂州工业技术研究院 | 一种抗粘连的聚乙烯醇栓塞微球及其制备方法和应用 |
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