重组抗菌肽海藻酸钠微囊制备与体外释放特征研究 1, 2, 1, 1,2, 1, 1, 1 (1., 266071; 2. 100049) 摘要 : 对重组表达的海洋生物抗菌肽对虾素 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 1.1 mm, 包封率为 83.87% 微囊在模拟胃液(pH 2.0) 中 2 h 左右释放量趋于稳定, 释放量低于 14%; 微囊在模拟肠液 (ph 7.8) 中不断释放, 5 h 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 关键词 : 重组抗菌肽 ; 海藻酸钠 ; 微囊 ; 包封率 ; 体外释放 ; 口服给药中图分类号 : S948 文献标识码 : A 文章编号 : 1000-3096(2012)04-0001-06 (Antimicrobial peptides, AMP),, [1-4],,,,,, [5-6],,, / [7] [8] (rcgh),, rcgh ; [9],, ; Romalde [10],, 3-2,,,,,,, 1 材料与方法 1.1 材料及主要试剂配制, 3-2(GenBank DQ308408) : 20 mmol/l, 500 mmol/l NaCl, 20 mmol/l, 0.45 μm, ph7.4 : 20 mmol/l, 500 mmol/l, 0.45 μm, ph 7.4 : 16.4 ml, 800 : 2011-03-13; : 2011-07-22 : (2006AA100311) : (1986-),,,,, : 0532-82898723, E-mail: zhangshouqing08@163.com;,,,, : 82898722, E-mail: wanglei@qdio.ac.cn Marine Sciences / Vol. 36, No. 4 / 2012 1
ml, 10 g, ph 2.0, 1000 ml : 6.8g 500 ml, 10 g,, 0.1mol/L ph 7.8, 1 000 ml 1.2 重组抗菌肽的分离纯化与纯度检测, 4, 12 000 r/min, 20 min, 0.22 μm His Ni 2+,,,,, 20 μl, Tricine-SDS-PAGE(15.5%)40V,, 1.3 微囊的制备方法 (25 ±1 ),, ( G),, ( )15 min,, 48 h [11],,,, 1.7 重组抗菌肽海藻酸钠微囊的体外释放,, 30 ml (ph 2.0) (ph 7.8), 28.0 ± 0.5, 90 r/min,, Bradford, 2 结果与分析 2.1 重组抗菌肽的纯化结果 1 5.8 kda,,,, ;, 1.4 微囊粒径的测定与形态表征 (n>30), [12], 3,, D= D i /n 1.5 微囊包封率的测定 1.3,, V, Bradford C : E= (G CV)/G 100% G, CV 1.6 微囊制备工艺的研究 (1.0%, 1.5%, 2.0%) 3 (1.0%, 1.5%, 2.0%), 3, 1 Tricine-SDS-PAGE Fig. 1 Tricine-SDS-PAGE profile of purified recombinant AMP 1. Maker; 2, 3. ; 4. 1. ultra-low molecular weight protein maker; 2,3.samples of purified recombinant AMP; 4. sample of pre-purified recombinant AMP 2.2 重组抗菌肽海藻酸钠微囊的制备工艺, 1.5%, 2 / 2012 / 36 / 4
,, ;,,, 1.5% 1.0% ; 1.5%, ;,, 2.0% 1, 1.5% 2.0% 表 1 氯化钙浓度和海藻酸钠浓度对微囊的形态和包封率的影响 ( 平均值 ± 标准误差, n=3) Tab. 1 Effects of calcium chloride concentration and sodium alginate concentration on the morphology and encapsulation rate of microcapsules(mean±sd, n=3) (%) (%) (%) 1.0 1.5 2.0 1.0 1.5, 83.59±1.14 2.0, 85.76±1.65 1.0, 78.61±2.47 1.5, 80.32±2.96 2.0, 83.87±1.73 1.0, 76.84±2.07 1.5, 78.43±0.95 2.0, 81.75±1.05 2.3 微囊的包封率和粒径,, 1.1 mm, 83.87% 2.4 微囊的体外释放情况,, 2 h, 14%, 3, (ph 2.0), 3 (ph 2.0) Fig. 3 In vitro releasing curve of sodium alginate microcapsules in simulated gastric fluid (ph 2.0) 2 Fig. 2 The size of recombinant AMP sodium alginate microcapsules A. ; B. A. fresh microcapsules; B. freeze-dried microcapsules, 5 h 98%, 4 3 4, Marine Sciences / Vol. 36, No. 4 / 2012 3
, 4 (ph 7.8) Fig. 4 In vitro releasing curve of sodium alginate microcapsules in simulated intestinal fluid (ph 7.8) 3 讨论,,,,,,,, [13] [14] [15], [16],, ; [17] 8,,, ; [18],,,, ; [19],,,,,,, [20-21], Ca 2+,,, [22] ;,,,,, [23],,, [24], [25],,,,,,, ;, [26] ;,,,,,,,,,, 参考文献 : [1],,,. [J]., 2009, 33(11): 95-99. [2] Munoz M, Vandenbulcke F, Gueguen Y, et al. Expression of penaeidin antimicrobial peptides in early larval stages of the shrimp Penaeus vannamei[j]. Developmental and Comparative Immunology, 2003, 27(4): 283-289. 4 / 2012 / 36 / 4
[3] Destoumieux D, Munoz M, Cosseau C, et al. Penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge[j]. Journal of Cell Science, 2000, 113(3): 461-469. [4] Destoumieux D, Bulet P, Loew D, et al. Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeus vannamei (decapoda)[j]. Journal of Biological Chemistry, 1997, 272(45): 28398-28406. [5] Schep L J, Tucker I G, Young G, et al. Controlled release opportunities for oral peptide delivery in aquaculture[j]. Journal of Controlled Release, 1999, 59(1): 1-14. [6] Sarmento B, Ribeiro A, Veiga F, et al. Oral bioavailability of insulin contained in polysaccharide nanoparticles[j]. Biomacromolecules, 2007, 8(10): 3054-3060 [7]. [J]., 2008, 12(10): 1931-1934. [8],. [J]., 2002, 23(6): 449-455. [9],. [J]., 2001, 8(2): 76-79. [10] Romalde J L, Luzardo-Alvarez A, Ravelo C, et al. Oral immunization using alginate microparticles as a useful strategy for booster vaccination against fish lactoccocosis[j]. Aquaculture, 2004, 236(1-4): 119-129. [11],,,. / [J]., 2007(2): 117-118. [12],,,. [J]., 2007, 38(2): 125-128. [13]. [J]., 2006(1): 13-14. [14],,,. [J]., 2001(2): 34-36. [15],,,. [J]., 2001(18): 13-14. [16],. [J]., 2002, 9(2): 152-156. [17],,,. [J]., 2010, 17(2): 258-266. [18],,. [J]., 2005, 26(8): 33-34. [19],,,. [J]., 2005(4): 375-381. [20]. [J]., 2008, 12(10): 1931-1934. [21],,. -tau [J]., 2007(6): 378-381. [22],,,. - [J]. ( ), 2009(5): 382-390. [23],,,. [J]., 2009(9): 1043-1045. [24],. [J]., 2005, 9(6): 369-376. [25] Zhang Y, Wei W, Lv P, et al. Preparation and evaluation of alginate-chitosan microspheres for oral delivery of insulin[j]. European Journal of Pharmaceutics and Biopharmaceutics, 2011, 77(1): 11-19. [26], 倢,. [J]., 2007(B12): 189-194. Marine Sciences / Vol. 36, No. 4 / 2012 5
Preparation and in vitro releasing characteristics of recombinant antimicrobial peptides sodium alginate microcapsules ZHANG Shou-qing 1,2, WANG Bao-jie 1, JIANG Shan 1,2, JIANG Ke-yong 1, LIU Mei 1, WANG Lei 1 (1.Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; 2.Gradutate University, Chinese Academy of Sciences, Beijing 100049, China) Received: Mar.,13,2011 Key words: recombinant antimicrobial peptides; sodium alginate; microcapsules; encapsulation rate; in vitro release; oral drug delivery system Abstract: 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 to optimize the preparation technology. The in vitro releasing characteristics of the optimal microcapsules was investigated and the results showed that these ball-shaped microcapsules with a diameter of 1.1mm and an encapsulation rate of 83.87% were prepared with 1.5% calcium chloride and 2.0% sodium alginate. The releasing rate of recombinant antimicrobial peptides after standing for 2 h in simulated gastric fluid (ph 2.0) was less than 14%. In contrast, after standing in simulated intestinal fluid (ph 7.8) for 5 h, the releasing rate of recombinant antimicrobial peptides reached 98%. These data suggested that the intestinal-lysis microcapsules have strong resistance to gastric fluid and can be used as a sustained-release and controlled-release reagent, providing the experimental basis for oral drug delivery system in aquaculture. ( 本文编辑 : 康亦兼 ) 6 / 2012 / 36 / 4