本文關(guān)鍵詞：金錢龜抗腫瘤肽的分離純化及其納米粒子的制備　出處：《華南理工大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 金錢龜 分離純化 抗腫瘤活性 多肽 納米粒子

【摘要】：本文通過酶解法制備了金錢龜抗腫瘤多肽,并以抗腫瘤活性為指標(biāo)優(yōu)化酶解條件。通過超濾和葡聚糖凝膠色譜柱對多肽成分進(jìn)行分離純化,利用質(zhì)譜技術(shù)分析了抗腫瘤活性較好組分的多肽組成和多肽序列,結(jié)合PEAKS解譜軟件得到多肽序列,并最終合成。最后用殼聚糖對活性較好的混合肽和合成肽進(jìn)行包被,制備成了復(fù)合納米粒子。本文的主要研究成果如下:(1)以粗蛋白得率為指標(biāo)對鹽提、Tris-HCl提取和酶解提取三種方法進(jìn)行比較,最終選擇得率最高的酶解法,其粗蛋白得率最高達(dá)到了84.15%。此方法的另一優(yōu)點是蛋白提取過程中伴隨著酶解可直接獲得多肽,該過程無大量鹽分的混入,為后續(xù)的分離純化提供了便利。(2)分別用胰蛋白酶、堿性蛋白酶、木瓜蛋白酶和復(fù)合蛋白酶protamex這四種酶在一定的條件下對金錢龜肉糜進(jìn)行水解得到多肽,將酶解液超濾后對得到的12種多肽組分進(jìn)行MTT試驗,活性檢測結(jié)果顯示:胰蛋白酶酶解產(chǎn)物分子量為0-3 K的組分(設(shè)為組分E)對兩種癌細(xì)胞的抑制作用最強,濃度為1mg/m L時,對Hep G-2的抑制率為92.95%,MCF-7的抑制率為67.08%,因此可選擇E進(jìn)行下一步的分離純化實驗。(3)以抗腫瘤活性為指標(biāo),用L9(34)正交實驗對酶解產(chǎn)物活性較低的堿性蛋白酶、木瓜蛋白酶和復(fù)合蛋白酶protamex酶解條件進(jìn)一步優(yōu)化。其中對乳腺癌細(xì)胞MCF-7的抑制率較高的最佳酶種和酶解條件為:木瓜蛋白酶:p H為8,T為55℃,E/S為1.5%,濃度為1 mg/m L時,對MCF-7抑制率為92.37%,對肝癌細(xì)胞Hep G-2的抑制率較高的最佳酶種和酶解條件為:復(fù)合蛋白酶:p H為8,T為40℃,E/S為2.0%,濃度為1mg/m L時,對Hep G-2抑制率為94.16%。將在最佳酶種和酶解條件下得到的酶解液進(jìn)行超濾純化,以MTT實驗為指標(biāo)選擇了活性好的復(fù)合蛋白酶酶解后小于3 K的組分(設(shè)為組分F)和木瓜蛋白酶酶解后小于3 K的組分(設(shè)為組分M)進(jìn)行下一步的分離純化。(4)經(jīng)Sephadex G-15分子篩層析E分離純化出四個組分E1、E2、E3和E4;M分離純化出三個組分M1、M2和M3;F分離純化出五個組分F1、F2、F3、F4和F5。MTT實驗結(jié)果顯示當(dāng)這些組分的濃度為500μg/m L時,E1、E2、M2、F4的抗腫瘤效果明顯。對肝癌細(xì)胞(Hep G-2)抑制率分別為81.91%、70.65%、74.70%和78.60%,其中陽性對照5-氟尿嘧啶的抑制率為76.22%。對乳腺癌細(xì)胞(MCF-7)的抑制率分別為34.70%、73.70%、62.93%和70.59%,其中陽性對照5-氟尿嘧啶的抑制率為63.60%。活性最為突出的為E1,其對Hep G-2的半抑制濃度IC50為77.29?g/m L。(5)組分E1、E2、M2、F4用MALDI-TOF-TOF/MS法進(jìn)行一級質(zhì)譜分析、二級質(zhì)譜分析和PEAKS解譜,其中荷質(zhì)比為769.490(E1-1)、852.549(E1-2)、777.399(E2-1)、827.464(M2-1)、771.574(F4-1)和861.159(F4-2)最終解出完整序列。其中E1-1的氨基酸序列為:RGVKGPR;E1-2的氨基酸序列為:KLGPKGPR;E2-1的氨基酸序列為:SSPGPPVH;M2-1的氨基酸序列為:EMLQPPL;F4-1的氨基酸序列為:PGKPLFL;F4-2的氨基酸序列為:SCCSCDED。將合成肽進(jìn)行MTT抗腫瘤實驗結(jié)果顯示:活性較為突出的為E2-1,其對MCF-7的半抑制濃度IC50為112.03?g/m L。(6)離子交聯(lián)法制備了抗腫瘤肽E1和E2-1的殼聚糖納米粒子復(fù)合物,通過單因素實驗確定最佳實驗條件為:C(TPP)為0.8 mg/m L,p H(CS)為4.5,T為50℃制得CS/TPP納米粒子的粒徑為191.2 nm,Zeta電位為31.2 mv。在最優(yōu)條件下制得的抗腫瘤肽納米粒子E1-CS/TPP的粒徑為224.8 nm;Zeta電位為38.4 mv;E2-1-CS/TPP的粒徑為210.6 nm;Zeta電位為36.5 mv;并測得其載藥量分別為15.22%和21.39%;包埋率分別為28.93%和43.15%。通過MTT抗腫瘤實驗發(fā)現(xiàn),經(jīng)殼聚糖包被后的抗腫瘤肽在給藥質(zhì)量相同的情況下抗腫瘤活性稍有降低,而將載藥量考慮在內(nèi)時,多肽含量相同時其抗腫瘤活性基本相同。將對Hep G-2抑制活性較好的E1-CS/TPP納米粒子模擬人體內(nèi)消化發(fā)現(xiàn)所制備的納米粒子在胃液中得到了一定的保護(hù),同時在腸液中得到了釋放,剩余未釋放的多肽則可能在盲腸和結(jié)腸中繼續(xù)釋放。
[Abstract]:This paper through the hydrolysis of legal turtle anti-tumor peptide synthesis and antitumor activity index for the optimization of hydrolysis conditions. The polypeptide components were purified by ultrafiltration and Sephadex column chromatography, analyzed the antitumor activity of the group with better polypeptide composition and peptide sequence divided by mass spectrometry, combined with PEAKS spectrum analysis software get polypeptide sequence and finally, synthetic. Finally with chitosan on the activity of better mixed peptides and synthetic peptides for coating, preparation of the composite nanoparticles. The main results are as follows: (1) the crude protein rate as the index of salt extraction, compare three different extraction methods of Tris-HCl extraction and enzymatic hydrolysis, the final choice the yield of enzyme with the highest crude protein yield reached 84.15%. another advantage of this method is in the process of protein extraction with enzyme solution can be obtained directly into the process of polypeptide, without a lot of salt, for The following provides a convenient separation and purification. (2) respectively with trypsin, alkaline protease, papain and protamex protamex these four enzymes under certain conditions of meat was hydrolyzed by turtle polypeptide, enzymatic ultrafiltration to get 12 polypeptide components of MTT test, assay: trypsin hydrolysates with molecular weight of 0-3 K components (for component E) had the strongest inhibitory effect on two kinds of cancer cells, the concentration of 1mg/m L, Hep G-2 on the inhibition rate was 92.95%, the inhibition rate of MCF-7 was 67.08%, the optional E separation and purification test of the next step. (3) with antitumor activity as index, L9 (34) orthogonal experimental solution of alkaline protease activity was lower in the product of enzyme, further optimize the hydrolysis conditions of papain and protamex protamex enzyme. The optimum enzyme inhibition rate is higher for the MCF-7 on breast cancer cells 縐嶅拰閰惰В鏉′歡涓，

本文編號：1375746