本文關鍵詞： SK84-6His S19K84-6His 抑菌活性 二級結構 PLGA 魚精蛋白 HBsAg 復乳溶劑揮發(fā)法　出處：《華中科技大學》2013年碩士論文　論文類型：學位論文

【摘要】：本論文的研究工作主要分為兩部分，第一部分初步探討了抗菌肽SK84結構與功能的關系，第二部分介紹了魚精蛋白包覆PLGA納米粒用于乙肝疫苗載體的初步研究。 第一部分：抗菌肽是從低等生物到包括人在內的高等生物在遭遇外源微生物入侵時，自身產生具有免疫調節(jié)和抗菌活性的生物小肽。由于殺菌效果好、抗菌譜廣、種類多、耙菌株不易產生耐藥性等特點，使其被認為是取代抗生素的最佳藥物。SK84是2004年陸婕博士從蠅蛆中分離純化出的具有高效殺菌作用的抗菌肽，該肽富含Gly，具有Gly二聯體、Gly六聯體、GxxxG等柔韌結構，對裂解細胞膜、最終殺死細胞具有重要作用。為了解抗菌肽SK84結構與功能的關系，我們截短了N端18個氨基酸（S19K84），開展的主要工作如下： （1）構建融合質粒pET32a-s19k84-6his（前期工作已構建了pET32a-sk84-6his），原核誘導表達并分離純化獲得基因重組蛋白SK84-6His、S19K84-6His； （2）抑菌圈法與微量法檢測結果顯示：S19K84-6His與SK84-6His相比，活性明顯降低； （3）圓二色譜分析表明，S19K84-6His中的α螺旋減少53.8%，而α螺旋是抗菌肽發(fā)揮抑菌作用的結構基礎。 此研究表明，SK84-6His中N端富Gly區(qū)能夠影響抗菌肽α螺旋的形成，對抗菌活性有很大的影響，為進一步研究SK84結構與功能的關系提供實驗依據。 第二部分：聚乳酸羥基乙酸（PLGA）包裹藥物制備成納米粒具有保持藥物的活性、增加藥物的攝取、控制藥物的釋放等作用，而用魚精蛋白包覆PLGA納米粒，能有效地增強樹突細胞（DC）對外源性抗原的交叉遞呈作用，誘導更有效的免疫反應。為開展魚精蛋白包覆PLGA納米粒用于乙肝疫苗的研究，我們以OVA和HBsAg為藥物模型，，用復乳溶劑揮發(fā)法制備了PLGA-OVA納米粒、PLGA-HBsAg納米粒、PLGA-HBsAg/PS納米粒，結果如下： （1）制備了PLGA-OVA納米粒，并考察其在體外的釋放情況，分為突釋與緩釋兩個階段； （2）從PVA類型、PVA濃度、超聲時間、外水相體積、藥物濃度5個方面優(yōu)化制備納米粒的條件，獲得粒徑237.9±14.4nm、分散系數PDI0.089±0.006、Zeta電位-13.2±2.1mv的PLGA-HBsAg納米粒，在4℃能穩(wěn)定保存20d。用魚精蛋白包覆PLGA-HBsAg納米粒后，Zeta電位由負變正，粒徑、分散系數、Zeta電位分別為：268.4±10.6nm，0.162±0.015、9.6±1.3mv； （3）TEM觀察，PLGA-HBsAg納米粒與PLGA-HBsAg/PS納米粒均為核-殼球形結構。 以上納米粒的制備及表征為今后進一步探討魚精蛋白包覆PLGA納米粒用于乙肝疫苗的研究提供實驗材料。
[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 偽 -helix in S19K84-6His was reduced by 53.8%, and the 偽 -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 偽 -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 鹵14.4nmand dispersion coefficient PDI0.089 鹵0.006G Zeta potential -13.2 鹵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 鹵0.0159.6 鹵1.3 MV, respectively, at 4 鈩

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