本文選題：南瓜蛋白（CUS） + 單甲氧基聚乙二醇丁醛（mPEG-BAD）　； 參考：《福建醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：研究單甲氧基聚乙二醇丁醛(mPEG-BAD，20KD)修飾南瓜蛋白(CUS)的最適反應(yīng)條件以及修飾產(chǎn)物的純化方法，以目標(biāo)修飾產(chǎn)物（單聚mPEG-CUS-BAD）為研究對象，對其體內(nèi)體外抗腫瘤活性及免疫原性進行初步研究，為研制和開發(fā)南瓜蛋白的新劑型提供一定的理論依據(jù)。 方法：1、用SDS-PAGE分析不同反應(yīng)條件對修飾反應(yīng)產(chǎn)物的影響；用離子交換柱進行分離純化。 2、用MTT法檢測目標(biāo)修飾產(chǎn)物單聚mPEG-CUS-BAD對體外培養(yǎng)的HL-60、 PANC-1腫瘤細胞的增殖影響。 3、應(yīng)用昆明小鼠H22腫瘤模型觀察目標(biāo)修飾產(chǎn)物單聚mPEG-CUS-BAD的體內(nèi)抗腫瘤活性。 4、應(yīng)用ELISA技術(shù)觀察目標(biāo)修飾產(chǎn)物的免疫原性變化。 結(jié)果：1、通過篩選，選定CUS與mPEG-BAD的摩爾比為1：5，CUS的濃度2mg/ml，，反應(yīng)時間48h，pH6.5的磷酸緩沖液為最佳修飾反應(yīng)條件。在此條件下，目標(biāo)產(chǎn)物單聚mPEG-CUS-BAD的總修飾率可達約65%，經(jīng)兩步離子交換柱的分離純化，目標(biāo)修飾產(chǎn)物的最終得率達到約40%。產(chǎn)物經(jīng)SDS-PAGE鑒定，基本上只顯示一條帶，表觀分子量明顯增大，約70KDa左右，純度大于97%。 2、與末修飾的CUS相比，單聚mPEG-CUS-BAD體外抗腫瘤活性大幅度下降。 3、與末修飾的CUS相比，單聚mPEG-CUS-BAD體內(nèi)抗腫瘤作用增強。 4、免疫原性分析顯示：與CUS相比，單聚mPEG-CUS-BAD免疫小鼠后血清內(nèi)IgG和IgE水平均有不同程度降低，以IgE水平降低更顯著。 結(jié)論：1、CUS與mPEG-BAD的反應(yīng)摩爾比為1：5、CUS的反應(yīng)濃度2mg/ml，反應(yīng)時間48h， pH6.5的磷酸緩沖液。 2、單聚mPEG-CUS-BAD體外抗腫瘤活性顯著降低，體內(nèi)抗腫瘤作用增強，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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R943;R96

【參考文獻】

相關(guān)期刊論文 前6條

1 吳影新;翟艷琴;雷建都;馬光輝;蘇志國;;干擾素α-2b的聚乙二醇修飾[J];生物工程學(xué)報;2008年09期

2 惠希武;陳虹;黃秉仁;;蛋白質(zhì)、多肽類藥物聚乙二醇修飾的研究進展[J];中國生物制品學(xué)雜志;2012年04期

3 姜忠義,高蓉,許松偉,王艷強;蛋白質(zhì)的化學(xué)修飾研究進展[J];現(xiàn)代化工;2001年08期

4 謝捷明;俞昌喜;陳明晃;許盈;趙蓉;吳國華;周琳瑛;;南瓜蛋白對B16細胞的誘導(dǎo)分化作用[J];中國藥理學(xué)通報;2006年03期

5 張永明;劉盛邦;黃凱;聶宇;孟延發(fā);;苦瓜籽MAP30的聚乙二醇化學(xué)修飾條件優(yōu)化及純化研究[J];中草藥;2008年11期

6 印春華,張敏;蛋白質(zhì)和多肽類藥物的聚乙二醇結(jié)合物——一種新型給藥系統(tǒng)[J];中國藥學(xué)雜志;2001年05期

本文編號：2030936