CN115561341B - 一种利用高效液相色谱制备眼镜王蛇抗菌肽oh-cath30标准品的方法 - Google Patents
一种利用高效液相色谱制备眼镜王蛇抗菌肽oh-cath30标准品的方法 Download PDFInfo
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Abstract
本发明旨涉及一种利用高效液相色谱制备眼镜王蛇抗菌肽OH‑CATH30标准品的方法，其特征在于，本发明采用反相高效液相色谱法，通过不断优化制备条件，最终以Zorbax SB300‑C8色谱柱（9.4mm*250mm，5μm），流动相A为0.05%水溶液TFA，流动相B为0.05% TFA的乙腈溶液，先进行洗脱，测得眼镜王蛇抗菌肽OH‑CATH30产物占粗样品蛋白含量的百分比，再对粗样品进行分离纯化，制备眼镜王蛇抗菌肽OH‑CATH30标准品，分离纯化洗脱梯度条件为0‑12min，线性A:B梯度比例98:2‑76:24；12‑160min，线性A:B梯度比例76:24‑60:40；160‑165min，线性A:B梯度比例60:40‑40:60；165‑170min，A:B梯度比例40:60。最终制得的眼镜王蛇抗菌肽OH‑CATH30标准品纯度达到95%，为眼镜王蛇抗菌肽OH‑CATH30基因工程表达后续研究提供了基础。
Description
技术领域
本发明涉及微生物制备方法，具体涉及一种利用高效液相色谱制备眼镜王蛇抗菌肽OH-CATH30标准品的方法。
背景技术
cathelicidins是一种在先天免疫系统中发挥重要作用的阳离子宿主防御肽，眼镜王蛇抗菌肽OH-CATH30为cathelicidins的截短肽，由30个氨基酸序列组成，目前研究发现其对大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌和产气肠杆菌等多种革兰氏阴性菌和革兰氏阳性菌均具有较好的抑菌效果。
目前还没有基因工程表达眼镜王蛇抗菌肽OH-CATH30标准品，基因工程表达眼镜王蛇抗菌肽OH-CATH30标准品是眼镜王蛇抗菌肽OH-CATH30基因工程表达研究的基础。标准品的制得有以下好处：一是可以使用标准品作为参照，利用液相色谱仪绘制标准曲线，检测基因工程表达的抗菌肽浓度；二是通过比较基因工程表达的重组蛋白和标准品的抑菌实验结果，比较分析表达获得的重组蛋白抑菌活性；三是可以通过对标准品进行耐酸碱，高温和低温等实验，确定所要基因工程表达的抗菌肽样品在极端条件下的稳定性。因此亟需一种有效的分离方法对眼镜王蛇抗菌肽OH-CATH30进行分离纯化方法，能够制得眼镜王蛇抗菌肽OH-CATH30标准品。
发明内容
基于上述背景技术，本发明旨在提供一种利用高效液相色谱制备眼镜王蛇抗菌肽OH-CATH30标准品的方法，通过不断优化制备条件，对基因工程表达制备的眼镜王蛇抗菌肽OH-CATH30样品进行分离纯化，制备眼镜王蛇抗菌肽OH-CATH30标准品，可以在短时间内完成分离目的，缩短分离时间,提高分离效率，最终标准品纯度达到95%。
具体地，本发明提供一种利用高效液相色谱制备眼镜王蛇抗菌肽OH-CATH30标准品的方法，所述制备方法包括以下步骤：
S01：眼镜王蛇抗菌肽OH-CATH30上清液的制备。
S02：眼镜王蛇抗菌肽OH-CATH30上清液的透析。
S03：利用反相高效液相色谱对透析后的眼镜王蛇抗菌肽OH-CATH30上清液进行洗脱，测得眼镜王蛇抗菌肽OH-CATH30产物占粗样品蛋白含量的百分比。
S04：利用反相高效液相色谱对眼镜王蛇抗菌肽OH-CATH30粗样品进行分离纯化，制备眼镜王蛇抗菌肽OH-CATH30标准品。
优选的，所述S03步骤及S04步骤中的色谱柱为Zorbax SB300-C8，9.4mm×250mm，粒径5μm。
优选的，所述S03步骤及S04步骤中的流速为0.3 mL/min。
优选的，所述S03步骤及S04步骤中的上样量为4mL。
优选的，所述S03步骤及S04步骤中的洗脱液 A 为 0.05% 水溶液TFA，pH 2.0；洗脱液 B 为含 0.05% TFA的乙腈溶液。
优选的，所述S03步骤及S04步骤中的柱温为室温，即25℃。
优选的，所述S03步骤中的洗脱梯度为0-10min，20%洗脱液 B；10-40min，50%洗脱液 B；40-50min，20%洗脱液 B。
优选的，所述S04步骤中的洗脱梯度为0-12min，线性A:B梯度比例98:2-76:24；12-160min，线性A:B梯度比例76:24-60:40；160-165min，线性A:B梯度比例60:40-40:60；165-170min，A:B梯度比例40:60。
本发明优点：通过不断优化高效液相色谱流动相、流速、洗脱梯度等制备条件，对基因工程表达制备的眼镜王蛇抗菌肽OH-CATH30样品进行分离纯化，最终制得的眼镜王蛇抗菌肽OH-CATH30标准品纯度达到95%，为眼镜王蛇抗菌肽OH-CATH30基因工程表达后续研究提供了基础，同时该分离方法具有分离效率高、操作简单，安全性强，污染小等优点。
附图说明
图1：透析后的眼镜王蛇抗菌肽OH-CATH30上清液的反相高效液相色谱分析。
图2：反相高效液相色谱法纯化眼镜王蛇抗菌肽OH-CATH30后所得亲水杂质的反相高效液相色谱分析图。
图3：反相高效液相色谱法纯化眼镜王蛇抗菌肽OH-CATH30后所得纯化产物的反相高效液相色谱分析图。
图4：反相高效液相色谱法纯化眼镜王蛇抗菌肽OH-CATH30后所得疏水杂质的反相高效液相色谱分析图。
具体实施方式
下面对本发明进一步详细说明。但下述的实例仅仅是本发明的简易例子，并不代表或限制本发明的权利保护范围，本发明的保护范围以权利要求书为准。
S01：眼镜王蛇抗菌肽OH-CATH30上清液的制备。
①.将OH-CATH基因片段与pPIC9K载体连接，得到pPIC9K-OH-CATH重组载体；随后将重组载体转化入毕赤酵母GS115菌株中，对毕赤酵母进行诱导表达，离心后获得表达上清液。
S02：眼镜王蛇抗菌肽OH-CATH30上清液的透析。
①.将获得的眼镜王蛇抗菌肽OH-CATH30上清液放入PBS透析液中透析2-3次。
S03：利用反相高效液相色谱对透析后的眼镜王蛇抗菌肽OH-CATH30上清液进行洗脱，测得眼镜王蛇抗菌肽OH-CATH30产物占粗样品蛋白含量的百分比。
利用反相高效液相色谱对透析后的眼镜王蛇抗菌肽OH-CATH30上清液进行洗脱，色谱条件为：色谱柱为 Zorbax SB300-C8，9.4mm×250mm，粒径5μm；流速0.3 mL/min；柱温为室温25℃条件；上样量4ml；流动相A：0.05% 水溶液TFA，pH 2.0； 流动相B：含 0.05% TFA的乙腈溶液；洗脱梯度见表1；检测波长：280nm（214nm为参考波长）。
获得色谱图，如图1，测得眼镜王蛇抗菌肽OH-CATH30产物占粗样品蛋白含量的3.2%。
表1 透析后的眼镜王蛇抗菌肽OH-CATH30上清液洗脱梯度
S04：利用反相高效液相色谱对眼镜王蛇抗菌肽OH-CATH30粗样品进行分离纯化，制备眼镜王蛇抗菌肽OH-CATH30标准品。
①利用反相高效液相色谱对眼镜王蛇抗菌肽OH-CATH30粗样品分离纯化，色谱条件：色谱柱为 Zorbax SB300-C8，9.4mm×250mm，粒径5μm；流速0.3 mL/min；柱温为室温25℃条件；上样量4ml；流动相A：0.05% 水溶液TFA，pH 2.0； 流动相B：含 0.05% TFA的乙腈溶液；洗脱梯度见表2；检测波长：280nm（214nm为参考波长）。
表2眼镜王蛇抗菌肽OH-CATH30粗样品分离纯化洗脱梯度
②每2分钟收集一次馏分，并在与眼镜王蛇抗菌肽OH-CATH30上清液分析图相同的条件下，在 Zorbax SB300-C8 柱上进行反相高效液相色谱（RP-HPLC）分析。
③馏分分为亲水杂质、纯化产物和疏水杂质，三种馏分分析曲线是在与眼镜王蛇抗菌肽OH-CATH30上清液曲线相同的色谱柱和条件下获得的。
④.制得眼镜王蛇抗菌肽OH-CATH30标准品，标准品纯度>95%，见图1、图2、图3及图4。
Claims (1)
1.一种利用高效液相色谱制备眼镜王蛇抗菌肽OH-CATH30标准品的方法，其特征在于所述制备步骤如下：
S01：眼镜王蛇抗菌肽OH-CATH30上清液的制备；
S02：眼镜王蛇抗菌肽OH-CATH30上清液的透析；
S03：利用反相高效液相色谱对所述S02的眼镜王蛇抗菌肽OH-CATH30上清液进行洗脱，测得眼镜王蛇抗菌肽OH-CATH30产物占粗样品蛋白含量的百分比；具体洗脱色谱条件为：
色谱柱为 Zorbax SB300-C8，9.4mm×250mm，粒径5μm；流速0.3 mL/min；柱温条件为室温25℃；上样量4ml；
洗脱液A为0.05% 水溶液TFA，pH 2 .0；
洗脱液B为含0.05% TFA的乙腈溶液；
具体洗脱梯度为0-10min，20%洗脱液B；10-40min，50%洗脱液B；40-50min，20%洗脱液B；
S04：利用反相高效液相色谱对眼镜王蛇抗菌肽OH-CATH30粗样品进行分离纯化，制备眼镜王蛇抗菌肽OH-CATH30标准品；具体分离纯化色谱条件为：色谱柱为 Zorbax SB300-C8，9 .4mm×250mm，粒径5μm；
流速0 .3 mL/min；柱温条件为室温25℃；上样量 4ml；
洗脱液A为0.05%水溶液TFA，pH 2.0；
洗脱液B为含0.05% TFA的乙腈溶液；
具体洗脱梯度为0-12min，线性A:B梯度比例98:2-76:24；12-160min，线性A:B梯度比例76:24-60:40；160-165min，线性A:B梯度比例60:40-40:60；165-170min，A:B梯度比例40:60。
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