鱼精蛋白包覆PLGA纳米粒用于乙肝疫苗载体的初步研究

发布时间：2018-02-08 14:52

  本文关键词： SK84-6His S19K84-6His 抑菌活性 二级结构 PLGA 鱼精蛋白 HBsAg 复乳溶剂挥发法　出处：《华中科技大学》2013年硕士论文　论文类型：学位论文

【摘要】：本论文的研究工作主要分为两部分，第一部分初步探讨了抗菌肽SK84结构与功能的关系，第二部分介绍了鱼精蛋白包覆PLGA纳米粒用于乙肝疫苗载体的初步研究。 第一部分：抗菌肽是从低等生物到包括人在内的高等生物在遭遇外源微生物入侵时，自身产生具有免疫调节和抗菌活性的生物小肽。由于杀菌效果好、抗菌谱广、种类多、耙菌株不易产生耐药性等特点，使其被认为是取代抗生素的最佳药物。SK84是2004年陆婕博士从蝇蛆中分离纯化出的具有高效杀菌作用的抗菌肽，该肽富含Gly，具有Gly二联体、Gly六联体、GxxxG等柔韧结构，对裂解细胞膜、最终杀死细胞具有重要作用。为了解抗菌肽SK84结构与功能的关系，我们截短了N端18个氨基酸（S19K84），开展的主要工作如下： （1）构建融合质粒pET32a-s19k84-6his（前期工作已构建了pET32a-sk84-6his），原核诱导表达并分离纯化获得基因重组蛋白SK84-6His、S19K84-6His； （2）抑菌圈法与微量法检测结果显示：S19K84-6His与SK84-6His相比，活性明显降低； （3）圆二色谱分析表明，S19K84-6His中的α螺旋减少53.8%，而α螺旋是抗菌肽发挥抑菌作用的结构基础。 此研究表明，SK84-6His中N端富Gly区能够影响抗菌肽α螺旋的形成，对抗菌活性有很大的影响，为进一步研究SK84结构与功能的关系提供实验依据。 第二部分：聚乳酸羟基乙酸（PLGA）包裹药物制备成纳米粒具有保持药物的活性、增加药物的摄取、控制药物的释放等作用，而用鱼精蛋白包覆PLGA纳米粒，能有效地增强树突细胞（DC）对外源性抗原的交叉递呈作用，诱导更有效的免疫反应。为开展鱼精蛋白包覆PLGA纳米粒用于乙肝疫苗的研究，我们以OVA和HBsAg为药物模型，，用复乳溶剂挥发法制备了PLGA-OVA纳米粒、PLGA-HBsAg纳米粒、PLGA-HBsAg/PS纳米粒，结果如下： （1）制备了PLGA-OVA纳米粒，并考察其在体外的释放情况，分为突释与缓释两个阶段； （2）从PVA类型、PVA浓度、超声时间、外水相体积、药物浓度5个方面优化制备纳米粒的条件，获得粒径237.9±14.4nm、分散系数PDI0.089±0.006、Zeta电位-13.2±2.1mv的PLGA-HBsAg纳米粒，在4℃能稳定保存20d。用鱼精蛋白包覆PLGA-HBsAg纳米粒后，Zeta电位由负变正，粒径、分散系数、Zeta电位分别为：268.4±10.6nm，0.162±0.015、9.6±1.3mv； （3）TEM观察，PLGA-HBsAg纳米粒与PLGA-HBsAg/PS纳米粒均为核-壳球形结构。 以上纳米粒的制备及表征为今后进一步探讨鱼精蛋白包覆PLGA纳米粒用于乙肝疫苗的研究提供实验材料。
[Abstract]:The research work of this thesis is divided into two parts. In the first part, the relationship between the structure and function of antimicrobial peptide SK84 is discussed. In the second part, the application of protamine coated PLGA nanoparticles to hepatitis B vaccine vector is introduced. Part I: antimicrobial peptides are small biological peptides with immunomodulatory and antimicrobial activities, which are produced from lower organisms to higher organisms, including human beings, when they are invaded by exogenous microbes. Because of their good bactericidal efficacy, they have a wide spectrum of antimicrobial activities, and there are many kinds of antimicrobial peptides. Harrow strain is not easy to produce drug resistance, which makes it the best drug to replace antibiotics. SK84 is a highly effective bactericidal peptide isolated from fly maggots by Dr. Lu Jie in 2004. The peptide is rich in Glycine, and has flexible structure such as GxxxG, GxxxG, GxxxG, etc. It plays an important role in lysing cell membrane and killing cells. In order to understand the relationship between structure and function of antimicrobial peptide SK84, We have truncated 18 N-terminal amino acids, S19K84, and our main work is as follows:. The fusion plasmid pET32a-s19k84-6hisis (pET32a-sk84-6hishe) was constructed. The recombinant protein SK84-6HisS19K84-6HisS19K84-6Hiswas obtained by prokaryotic induction and purification. 2) the results of bacteriostasis and microanalysis showed that the activity of SK84-6His was significantly lower than that of S19K84-6His. The circular dichroism analysis showed that the 伪 -helix in S19K84-6His was reduced by 53.8%, and the 伪 -helix was the structural basis of antimicrobial peptides. This study shows that the N-terminal Gly rich region in SK84-6His can affect the formation of 伪 -helix of antimicrobial peptide, and has a great influence on the antibacterial activity, which provides the experimental basis for further study on the relationship between the structure and function of SK84. The second part: PLGA nanoparticles were prepared by encapsulating the drug with polylactic acid hydroxy acetate (PLGA), which could maintain the activity of the drug, increase the uptake of the drug and control the release of the drug, while the nanoparticles were coated with protamine. It can effectively enhance the cross-presentation of dendritic cells to exogenous antigens and induce a more effective immune response. In order to study the application of protamine coated PLGA nanoparticles to hepatitis B vaccine, we used OVA and HBsAg as drug models. The PLGA-OVA nanoparticles, PLGA-HBsAg nanoparticles, PLGA-HBsAg / PS nanoparticles were prepared by solvent volatilization method. The results are as follows:. (1) PLGA-OVA nanoparticles were prepared and their release in vitro was investigated, which was divided into two stages: sudden release and sustained release. (2) the preparation conditions of PVA nanoparticles were optimized from five aspects: PVA concentration, ultrasonic time, volume of external water phase and drug concentration. The PLGA-HBsAg nanoparticles with particle size 237.9 卤14.4nmand dispersion coefficient PDI0.089 卤0.006G Zeta potential -13.2 卤2.1mv were obtained. The Zeta potential of PLGA-HBsAg nanoparticles coated with protamine changed from negative to positive, the particle size and the dispersion coefficient of Zeta potential were 0.162 卤0.0159.6 卤1.3 MV, respectively, at 4 鈩

本文编号：1495700