金钱龟抗肿瘤肽的分离纯化及其纳米粒子的制备

发布时间：2018-01-03 22:01

  本文关键词：金钱龟抗肿瘤肽的分离纯化及其纳米粒子的制备　出处：《华南理工大学》2015年硕士论文　论文类型：学位论文

  更多相关文章： 金钱龟 分离纯化 抗肿瘤活性 多肽 纳米粒子

【摘要】：本文通过酶解法制备了金钱龟抗肿瘤多肽,并以抗肿瘤活性为指标优化酶解条件。通过超滤和葡聚糖凝胶色谱柱对多肽成分进行分离纯化,利用质谱技术分析了抗肿瘤活性较好组分的多肽组成和多肽序列,结合PEAKS解谱软件得到多肽序列,并最终合成。最后用壳聚糖对活性较好的混合肽和合成肽进行包被,制备成了复合纳米粒子。本文的主要研究成果如下:(1)以粗蛋白得率为指标对盐提、Tris-HCl提取和酶解提取三种方法进行比较,最终选择得率最高的酶解法,其粗蛋白得率最高达到了84.15%。此方法的另一优点是蛋白提取过程中伴随着酶解可直接获得多肽,该过程无大量盐分的混入,为后续的分离纯化提供了便利。(2)分别用胰蛋白酶、碱性蛋白酶、木瓜蛋白酶和复合蛋白酶protamex这四种酶在一定的条件下对金钱龟肉糜进行水解得到多肽,将酶解液超滤后对得到的12种多肽组分进行MTT试验,活性检测结果显示:胰蛋白酶酶解产物分子量为0-3 K的组分(设为组分E)对两种癌细胞的抑制作用最强,浓度为1mg/m L时,对Hep G-2的抑制率为92.95%,MCF-7的抑制率为67.08%,因此可选择E进行下一步的分离纯化实验。(3)以抗肿瘤活性为指标,用L9(34)正交实验对酶解产物活性较低的碱性蛋白酶、木瓜蛋白酶和复合蛋白酶protamex酶解条件进一步优化。其中对乳腺癌细胞MCF-7的抑制率较高的最佳酶种和酶解条件为:木瓜蛋白酶:p H为8,T为55℃,E/S为1.5%,浓度为1 mg/m L时,对MCF-7抑制率为92.37%,对肝癌细胞Hep G-2的抑制率较高的最佳酶种和酶解条件为:复合蛋白酶:p H为8,T为40℃,E/S为2.0%,浓度为1mg/m L时,对Hep G-2抑制率为94.16%。将在最佳酶种和酶解条件下得到的酶解液进行超滤纯化,以MTT实验为指标选择了活性好的复合蛋白酶酶解后小于3 K的组分(设为组分F)和木瓜蛋白酶酶解后小于3 K的组分(设为组分M)进行下一步的分离纯化。(4)经Sephadex G-15分子筛层析E分离纯化出四个组分E1、E2、E3和E4;M分离纯化出三个组分M1、M2和M3;F分离纯化出五个组分F1、F2、F3、F4和F5。MTT实验结果显示当这些组分的浓度为500μg/m L时,E1、E2、M2、F4的抗肿瘤效果明显。对肝癌细胞(Hep G-2)抑制率分别为81.91%、70.65%、74.70%和78.60%,其中阳性对照5-氟尿嘧啶的抑制率为76.22%。对乳腺癌细胞(MCF-7)的抑制率分别为34.70%、73.70%、62.93%和70.59%,其中阳性对照5-氟尿嘧啶的抑制率为63.60%。活性最为突出的为E1,其对Hep G-2的半抑制浓度IC50为77.29?g/m L。(5)组分E1、E2、M2、F4用MALDI-TOF-TOF/MS法进行一级质谱分析、二级质谱分析和PEAKS解谱,其中荷质比为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)最终解出完整序列。其中E1-1的氨基酸序列为:RGVKGPR;E1-2的氨基酸序列为:KLGPKGPR;E2-1的氨基酸序列为:SSPGPPVH;M2-1的氨基酸序列为:EMLQPPL;F4-1的氨基酸序列为:PGKPLFL;F4-2的氨基酸序列为:SCCSCDED。将合成肽进行MTT抗肿瘤实验结果显示:活性较为突出的为E2-1,其对MCF-7的半抑制浓度IC50为112.03?g/m L。(6)离子交联法制备了抗肿瘤肽E1和E2-1的壳聚糖纳米粒子复合物,通过单因素实验确定最佳实验条件为:C(TPP)为0.8 mg/m L,p H(CS)为4.5,T为50℃制得CS/TPP纳米粒子的粒径为191.2 nm,Zeta电位为31.2 mv。在最优条件下制得的抗肿瘤肽纳米粒子E1-CS/TPP的粒径为224.8 nm;Zeta电位为38.4 mv;E2-1-CS/TPP的粒径为210.6 nm;Zeta电位为36.5 mv;并测得其载药量分别为15.22%和21.39%;包埋率分别为28.93%和43.15%。通过MTT抗肿瘤实验发现,经壳聚糖包被后的抗肿瘤肽在给药质量相同的情况下抗肿瘤活性稍有降低,而将载药量考虑在内时,多肽含量相同时其抗肿瘤活性基本相同。将对Hep G-2抑制活性较好的E1-CS/TPP纳米粒子模拟人体内消化发现所制备的纳米粒子在胃液中得到了一定的保护,同时在肠液中得到了释放,剩余未释放的多肽则可能在盲肠和结肠中继续释放。
[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 绉嶅拰閰惰В鏉′欢涓，

本文编号：1375746