MAP30的免疫亲和层析纯化及其表征

doi:10.3877/cma.j.issn.1674-0785.2023.02.015

目的

建立一种快速纯化苦瓜毒素抗艾滋病毒蛋白（MAP30）多肽的免疫亲和层析方法。

方法

将苦瓜毒素MAP30作为抗原，免疫新西兰大白兔（雄性），制备抗血清。采用Mabselect亲和层析法纯化多克隆抗体。以Sepharose 2B作为基质与多克隆抗体相偶联合成的抗体-Sepharose 2B亲和层析介质用于纯化苦瓜毒素MAP30。

结果

用自制的免疫亲和介质纯化的MAP30毒素，经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），在还原条件下均显示单一蛋白着色带，对应的相对分子量均为30 kDa；酸性PAGE分离后，经高碘酸氧化法，显示糖蛋白特征；采用硫酸-蒽酮法测得总糖含量是1.54%；PC12细胞作为测试对象，MAP30抗肿瘤活性的最大抑制率为90%。

结论

取得的实验结果表明，纯化的目标蛋白-MAP30的结构、性质以及活性特性均与文献报道相一致，表明本文建立的免疫亲和层析方法用于分离和纯化MAP30可行。

关键词: 免疫亲和层析, 苦瓜毒素抗艾滋病毒蛋白, 核糖体失活蛋白, 多克隆抗体

Objective

To establish an immunoaffinity chromatography method for rapid purification of MAP30 polypeptide (a momordica toxin).

Methods

New Zealand white rabbits (male) were immunized with the toxin MAP30 as an antigen to prepare antiserum. Polyclonal antibodies were purified by Mabselect affinity chromatography. Synthesized antibody-Sepharose 2B affinity chromatography medium using Sepharose 2B as a matrix coupled with polyclonal antibody was used to purify the momordica toxin MAP30.

Results

The MAP30 toxin purified with the self-made immunoaffinity medium showed a single color band under reducing conditions on SDS-PAGE, and the corresponding relative molecular weights were all 30 kDa. The total sugar content measured by the sulfate-anthrone method was 1.54%. PC12 cells were used as the test cell type, and the maximum inhibition rate of MAP30 (anti-tumor activity) was 90%.

Conclusion

The structure, properties, and activity characteristics of the purified target protein-MAP30 in this study are consistent with those reported in the literature, which suggests that the immunoaffinity chromatography method established in this study is feasible for the separation and purification of MAP30.

Key words: Immunoaffinity chromatography, MAP30, RIP, Polyclonal antibody

图1 Mabselect亲和层析纯化抗MAP30多克隆抗体注：P1为杂蛋白峰；P2为目的蛋白峰

图2 还原和非还原条件下抗MAP30多克隆抗体的SDS-PAGE。图a为还原性条件下抗MAP30多克隆抗体的SDS-PAGE电泳图（M为低分子量标准蛋白；泳道1为20 μg MAP30-PcAb；泳道2为15 μg MAP30-PcAb；泳道3为10 μg MAP30-PcAb）；图b为非还原性条件下抗MAP30多克隆抗体的SDS-PAGE电泳图（M为高分子量标准蛋白；泳道1为10 μg MAP30-PcAb；泳道2为15 μg MAP30-PcAb；泳道3为20 μg MAP30-PcAb）注：MAP30为苦瓜毒素抗艾滋病毒蛋白；SDS-PAGE为十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；IgG为免疫球蛋白G

图3 IgG-Sepharose 2B纯化苦瓜毒素MAP30的层析图谱注：F1为杂蛋白峰；F2为MAP30目的蛋白峰

图4 IgG-Sepharose 2B纯化MAP30的还原和非还原SDS-PAGE。图a为还原性条件下抗MAP30 SDS-PAGE电泳图（M为标准蛋白；泳道1为20 μg纯化的MAP30；泳道2为15 μg纯化的MAP30；泳道3为10 μg标准MAP30）；图b为非还原性条件下MAP30 SDS-PAGE电泳图（M为标准蛋白；泳道1为15 μg标准MAP30；泳道2为15 μg纯化的MAP30；泳道3为20 μg纯化的MAP30）注：MAP30为苦瓜毒素抗艾滋病毒蛋白；SDS-PAGE为十二烷基硫酸钠-聚丙烯酰胺凝胶电泳

图5 MAP30的酸性电泳注：MAP30为苦瓜毒素抗艾滋病毒蛋白；泳道1为20 μg纯化的MAP30；泳道2为20 μg标准MAP30；泳道3为10 μg纯化的MAP30；泳道4为10 μg标准MAP30；泳道5为15 μg纯化的MAP30；泳道6为15 μg标准MAP30

图6 MAP30的糖蛋白分析注：MAP30为苦瓜毒素抗艾滋病毒蛋白；泳道1为20 μg纯化的MAP30；泳道2为20 μg标准MAP30；泳道3为25 μg纯化的MAP30；泳道4为25 μg标准MAP30；泳道5为30 μg纯化的MAP30；泳道6为30 μg标准MAP30

图7 IgG-Sepharose 2B纯化的MAP30抑制PC12细胞增殖效果注：^****P<0.05；MAP30为苦瓜毒素抗艾滋病毒蛋白；PC12为肾上腺嗜铬细胞瘤细胞

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Purification and characterization of MAP30 by immunoaffinity chromatography

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Purification and characterization of MAP30 by immunoaffinity chromatography