聚乙二醇化南瓜蛋白的制备及生物学活性的初步研究

发布时间：2018-06-17 11:49

本文选题：南瓜蛋白（CUS） + 单甲氧基聚乙二醇丁醛（mPEG-BAD）　；参考：《福建医科大学》2014年硕士论文

【摘要】：目的：研究单甲氧基聚乙二醇丁醛(mPEG-BAD，20KD)修饰南瓜蛋白(CUS)的最适反应条件以及修饰产物的纯化方法，以目标修饰产物（单聚mPEG-CUS-BAD）为研究对象，对其体内体外抗肿瘤活性及免疫原性进行初步研究，为研制和开发南瓜蛋白的新剂型提供一定的理论依据。方法：1、用SDS-PAGE分析不同反应条件对修饰反应产物的影响；用离子交换柱进行分离纯化。 2、用MTT法检测目标修饰产物单聚mPEG-CUS-BAD对体外培养的HL-60、 PANC-1肿瘤细胞的增殖影响。 3、应用昆明小鼠H22肿瘤模型观察目标修饰产物单聚mPEG-CUS-BAD的体内抗肿瘤活性。 4、应用ELISA技术观察目标修饰产物的免疫原性变化。结果：1、通过筛选，选定CUS与mPEG-BAD的摩尔比为1：5，CUS的浓度2mg/ml，，反应时间48h，pH6.5的磷酸缓冲液为最佳修饰反应条件。在此条件下，目标产物单聚mPEG-CUS-BAD的总修饰率可达约65%，经两步离子交换柱的分离纯化，目标修饰产物的最终得率达到约40%。产物经SDS-PAGE鉴定，基本上只显示一条带，表观分子量明显增大，约70KDa左右，纯度大于97%。 2、与末修饰的CUS相比，单聚mPEG-CUS-BAD体外抗肿瘤活性大幅度下降。 3、与末修饰的CUS相比，单聚mPEG-CUS-BAD体内抗肿瘤作用增强。 4、免疫原性分析显示：与CUS相比，单聚mPEG-CUS-BAD免疫小鼠后血清内IgG和IgE水平均有不同程度降低，以IgE水平降低更显著。结论：1、CUS与mPEG-BAD的反应摩尔比为1：5、CUS的反应浓度2mg/ml，反应时间48h， pH6.5的磷酸缓冲液。 2、单聚mPEG-CUS-BAD体外抗肿瘤活性显著降低，体内抗肿瘤作用增强，免疫原性有所降低。
[Abstract]:Objective: To study the optimum reaction conditions of mPEG-BAD (20KD) modified pumpkin protein (CUS) and the purification method of the modified product. The target modified product (mono mPEG-CUS-BAD) was used as the research object, and the antitumor activity and immunogenicity in vitro were preliminarily studied in order to develop and develop pumpkin protein. The dosage forms provide a certain theoretical basis.
Methods: 1. The effects of different reaction conditions on the modified products were analyzed by SDS-PAGE.
2, MTT method was used to detect the effect of target modifier product mPEG-CUS-BAD on the proliferation of HL-60 and PANC-1 tumor cells cultured in vitro.
3, Kunming mice H22 tumor model was used to observe the in vivo antitumor activity of target modifier product mPEG-CUS-BAD.
4, ELISA technology was used to observe the immunogenicity of target modifiers.
Results: 1, through screening, the mole ratio of CUS and mPEG-BAD was selected as 1:5, the concentration of CUS was 2mg/ml, the reaction time was 48h, and the phosphoric acid buffer solution of pH6.5 was the best modification condition. Under this condition, the total modification rate of the target product mono mPEG-CUS-BAD was about 65%. The final yield of the target modified product was obtained by the separation and purification of the two step ion exchange column. About 40%. products were identified by SDS-PAGE, showing only one band, the apparent molecular weight increased obviously, about 70KDa, and the purity was more than 97%.
2, compared with the modified CUS, the antitumor activity of mPEG-CUS-BAD in vitro decreased significantly.
3, compared with the modified CUS, the antitumor activity of the monomer mPEG-CUS-BAD increased.
4, the immunogenicity analysis showed that compared with CUS, the serum levels of IgG and IgE decreased in different degrees after the immunization of mono mPEG-CUS-BAD mice, and decreased more significantly at the level of IgE.
Conclusion: 1, the molar ratio of CUS to mPEG-BAD is 1:5, CUS's reaction concentration 2mg/ml, reaction time 48h, pH6.5's phosphate buffer.
2, the antitumor activity of mono mPEG-CUS-BAD in vitro was significantly reduced, the anti-tumor effect in vivo was enhanced, and the immunogenicity was reduced.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;R96

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