摘要
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。
Abstract
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.
基金项目
国家自然科学基金(31600269)
四川省科技厅应用基础研究项目(2019YJ0369)
四川省医学会研究项目(S18018)
成都医学院学科创新团队(CMC-XK-2103)
成都医学院-郫都区人民医院联合基金(2021LHZD-04)
成都医学院研究生创新基金(YCX2021-36)
成都医学院研究生创新基金(YCX2022-03-06)
成都医学院-新都区人民医院联合基金(2022LHXD-01)
万方数据