發(fā)布時(shí)間：2018-08-24 18:48

【摘要】：探討雙齒圍沙蠶酶解多肽制備的關(guān)鍵技術(shù)及其對肺癌A549細(xì)胞的作用。以增殖抑制率為指標(biāo),確定最佳酶種并根據(jù)單因素試驗(yàn)和正交試驗(yàn)確定其最佳酶解工藝。經(jīng)超濾獲得1~3 ku的酶解液,通過陰離子色譜、凝膠過濾色譜和制備色譜對其進(jìn)行進(jìn)一步的分離純化。采用四甲基偶氮唑藍(lán)法測定沙蠶多肽PAP對肺癌A549細(xì)胞的增殖抑制率并通過倒置顯微鏡、吖啶橙/溴化乙錠熒光染色及Hoechst熒光染色觀察其細(xì)胞形態(tài)的變化。實(shí)驗(yàn)結(jié)果表明最佳酶種是堿性蛋白酶,其最佳酶解條件為:酶解溫度50℃、pH 11、料液比1∶1(g/mL)、酶解時(shí)間6 h、加酶量300 U/g。經(jīng)純化獲得的多肽命名為PAP,其氨基酸序列為Ile-Glu-Pro-Gly-Thr-Val-Gly-Met-Met-Phe,且PAP對A549細(xì)胞的作用呈時(shí)間與劑量依賴關(guān)系,作用后細(xì)胞出現(xiàn)了凋亡的形態(tài)學(xué)特征。因此,PAP能明顯抑制肺癌細(xì)胞A549增殖,可以誘導(dǎo)其發(fā)生凋亡而發(fā)揮抗腫瘤作用。
[Abstract]:To study the key technology of preparation of polypeptide by enzymatic hydrolysis and its effect on lung cancer A549 cells. The optimum enzyme species was determined by using the inhibition rate of proliferation as the index, and the optimum enzymatic hydrolysis process was determined according to the single factor experiment and orthogonal experiment. The enzymatic hydrolysis solution of 1 ~ 3 ku was obtained by ultrafiltration. It was further separated and purified by anionic chromatography, gel filtration chromatography and preparation chromatography. The proliferation inhibition rate of silkworm polypeptide PAP on lung cancer A549 cells was determined by tetramethyl azolium blue method. The morphological changes of A549 cells were observed by inverted microscope, acridine orange / ethidium bromide fluorescence staining and Hoechst fluorescence staining. The results showed that the best enzyme species was alkaline protease. The optimum conditions of enzymatic hydrolysis were as follows: hydrolysis temperature 50 鈩，

本文編號：2201713