【作者】 张守庆；

【导师】 王雷；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 水产养殖， 2011， 硕士

【摘要】 重组抗菌肽是一类利用基因工程的方法生产的小分子多肽,具有分子量小、热稳定好、广谱抗菌活性,被认为是解决病原微生物对抗生素不断增强的抗性问题的很好选择。水产动物的养殖特点决定了该类物质的应用一般只能进行口服给药,由于蛋白类药物普遍存在半衰期短、易被体内酶降解等问题,在口服给药体系中受到极大的限制,人们通常采用可以对药物进行控/缓释作用的微胶囊化技术解决上述问题。本研究对重组表达的对虾素3-2进行亲和层析纯化,以海藻酸钠为壁材,采用凝聚法制备了重组抗菌肽-海藻酸钠微囊,以微囊的形态和包封率为指标优化制备工艺,对制备的微囊进行体外释放特征的初步研究。结果显示,在氯化钙浓度为1.5%,海藻酸钠浓度为2.0%时,制备的微囊为完整的球形,冷冻干燥后的直径约为1.1mm,包封率为83.87%。微囊在模拟胃液(pH2.0)中2h左右释放量趋于稳定,释放量低于14%;微囊在模拟肠液(pH7.8)中不断释放,5h时释放量达98%,表明微囊具有良好的肠溶性而可以抵抗胃液的破坏,可以用作重组抗菌肽缓释/控释制剂,为抗菌肽在水产病害防治过程的口服给药提供实验基础。将制备的重组抗菌肽-海藻酸钠微囊口服给药罗非鱼,另设特异性饲料组和空白对照组,连续投喂20d后取其肝脏、胃、肠道、血清和肌肉组织,利用Western Blot技术检测样品中是否含有重组抗菌肽。结果发现,空白对照组各样品均未检测到目的蛋白,而特异性饲料组和海藻酸钠微囊组也未发现重组抗菌肽。重组抗菌肽的口服导入及检测技术还有待于深入研究。以纯化的重组抗菌肽为模型蛋白,乳酸/羟乙酸共聚物(PLGA)为载体,采用复乳溶剂挥发法制备重组抗菌肽-PLGA微球,通过正交化设计法对微球制备的7种因素进行优化,以粒径、包封率及24h释放率为观察指标。结果发现,在重组抗菌肽的浓度为10mg,PLGA用量200mg,PVA浓度为1%,初乳超声功率100W,复乳超声功率为100W,PEG用量为5%,氯化钠的浓度为5%的条件下制备的重组抗菌肽-PLGA微球,SEM下具有光滑的表面且形态规整,大部分微球粒径集中在3μm左右,但微囊的包封率都比较低。更多还原

【Abstract】 Recombinant antimicrobial peptide is a kind of minor polypeptides produced through genetic engineering method. Due to their small molecular weight, heat stability, broad-spectrum of antimicrobial activities, the AMP were considered to be excellent candidates for potential novel antibiotic agents. This protein drugs should be generally delivered by oral approach for the aquaculture characteristics. However the clinic applications through oral delivery systems are still not successful for most of them because of their short half-life resulting from acid-catalytic or proteolytic degradation in the gastrointestinal tracts. A potential solution is microencapsulation through which the protein drugs are encapsulated, preventing from the degradation, and releasing in sustained and controlled manners.In this paper, the recombinant antimicrobial peptides (penaeidin 3-2) were purified with affinity chromatography, and recombinant antimicrobial peptides sodium alginate microcapsules were prepared by using coacervation technology. The morphology and encapsulation rate were introduced as indexes of microcapsules formulation. Afterward, in vitro releasing characteristics of the optimal microcapsules was investigated. The results showed that the microcapsules with a diameter of 1.1mm and an encapsulation rate of 83.87% were prepared when the concentrations of calcium chloride and sodium alginate were 1.5% and 2.0%, respectively. The releasing rate of recombinant AMP after standing for 2 h in simulated gastric fluid (pH2.0) was less than 14%. However, after standing in simulated intestinal fluid (PH7.8) for 5 h, the releasing rate of recombinant AMP reached 98%.Which indicated that the intestinal-lysis microcapsules have the potential to be used as sustained-release and controlled-release preparations, providing the experimental basis for oral drug delivery system in aquaculture.Recombinant antimicrobial peptides-sodium alginate microcapsules were orally given to Tilapia and fishes in the control groups were given with special and ordinary forage.20d after that liver, stomach, intestine, sera and muscle were analyzed by Western Blot. The recombinant AMP was not discovered in simples of all groups. In this case, the oral administration of recombinant AMP still need to be further researched.Using purified recombinant AMP as model protein and PLGA as controlled-release carrier, recombinant AMP-PLGA microspheres were prepared by W/O/W multiple emulsion volatilizing method. Using entrapment efficiency, particle size, and 24h-release amount as the evaluating indicators, we optimized the 7 factors that influenced the preparation technique for microspheres by orthogonal factorization method. The morphology was investigated using scanning electron microscope(SEM) .The results showed that the microspheres seemed to be smooth and uniform with mean particle size of 3μm under conditions of recombinant AMP of 10 mg, PLGA of 200 mg, 1%PVA, ultrasonic power of 100 W, 5%PEG, 10%NaCl. However, the encapsulation efficiency of microspheres was only 10%.更多还原