马氏珠母贝免疫活性肽的纯化与鉴定Purification and Identification of Immunomodulating Peptides from Enzymatic Hydrolysates of Pinctada martensi
邓志程,张迪,吉宏武,曹文红,苏伟明
摘要(Abstract):
为制备马氏珠母贝免疫活性肽,以马氏珠母贝全脏器为原料,利用体外模拟消化的方法对其进行酶解,然后通过超滤系统收集分子量小于3 ku的酶解液。采用细胞培养方法,以体外免疫活性为指标,利用Sephadex G-25凝胶柱层析、Capto Q强阴离子交换色谱等对马氏珠母贝酶解液进行分离纯化与筛选,获得2个具有免疫活性的肽段,利用电喷雾静电场离子阱串联质谱分别对2个活性肽序列进行测定。结果表明:一个肽的相对分子质量为245.0,肽序列为Ala-Arg/Pro-Met;另一个别肽的相对分子质量为274.0,肽序列为Val-Arg。在浓度0.3 mg/m L时,2种肽能显著促进脾淋巴细胞的增殖与提高巨噬细胞的吞噬能力,表明这2种肽均具有较好的免疫活性。
关键词(KeyWords): 马氏珠母贝;免疫活性肽;分离纯化;氨基酸序列
基金项目(Foundation): 国家海洋公益性行业科研专项(201305018);; 广东省高等学校学科建设项目(2013CXZDA020);; 广东省部产学研合作专项资金项目(2013B090600155);; 广东海洋大学创新强校工程项目(GDOU2014050203)
作者(Author): 邓志程,张迪,吉宏武,曹文红,苏伟明
参考文献(References):
- [1]朱春华,申玉春,谢恩义,等.湛江流沙湾马氏珠母贝的养殖容量[J].热带海洋学报,2011,30(3):76-81.
- [2]谢恩义,陈秀丽,朱小江,等.流沙湾贝类资源调查[J].广东海洋大学学报,2010,30(3):39-46.
- [3]王庆恒,逯云召,邓岳文,等.马氏珠母贝生长性状与珍珠质量和珍珠层厚度的相关分析[J].广东海洋大学学报,2013,33(3):18-21.
- [4]章超桦,吴红棉,洪鹏志,等.马氏珠母贝肉的营养成分及其游离氨基酸组成[J].水产学报,2000,24(2):180-184.
- [5]隆海银,马文静,陈琳,等.马氏珠母贝肉研究进展[J].轻工科技,2015(11):3-5,8.
- [6]LóPEZ-ZABALZA M J,MARTíNEZ-LAUSíN S,BENGOECHEA-ALONSO M T,et al.Signaling pathway triggered by a short immunomodulating peptide on human monocytes[J].Archives of Biochemistry and Biophysics,1997,338(2):136-142.
- [7]GILL H S,DOULL F,RUTHERFURD K J,et al.Immunoregulatory peptides in bovine milk[J].British Journal of Nutrition,2000,84(S1):11l-117.
- [8]PARKER F,MIGLIORE-SAMOUR D,FLOC’H F,et al.Immunostimulating hexapeptide from human casein:amino acid sequence,synthesis and biological properties[J].European Journal of Biochemistry,1984,145(3):677-682.
- [9]AGYEI D,DANQUAH M K.Rethinking food-derived bioactive peptides for antimicrobial and immunomodulatory activities[J].Trends in Food Science&Technology,2012,23(2):62-69.
- [10]DUARTE J,VINDEROLA G,RITZ B,et al.Immunomodulating capacity of commercial fish protein hydrolysate for diet supplementation[J].Immunobiology,2006,211(5):341-350.
- [11]曾珍,李诚,付刚,等.猪骨免疫活性肽的分离纯[J].食品与发酵工业,2014,40(11):116-120.
- [12]LINDON J C,FARRANT R D,SANDERSON P N,et al.Separation and characterization of components of peptide libraries using on-flow coupled HPLC-NMR spectroscopy[J].Magnetic Resonance in Chemistry,1995,33(11):857-863.
- [13]陈绍农,潘远江,陈耀祖.多肽及蛋白质质谱分析新进展[J].质谱学报,1995,16(3):15-21.
- [14]邓志程,吉宏武,曹文红,等.人工神经网络优化酶法制备马氏珠母贝低聚肽的工艺研究[J].广东农业科学,2014,41(24):99-104.
- [15]邓志程.马氏珠母贝全脏器免疫活性肽的制备及其免疫活性的研究[D].湛江:广东海洋大学,2015.
- [16]何小庆.波纹巴非蛤活性肽的酶法制备及其免疫活性的研究[D].湛江:广东海洋大学,2014.
- [17]TZEHOVAL E,SEGAL S,STABINSKY Y,et al.Tuftsin(an Ig-associated tetrapeptide)triggers the immunogenic function of macrophages:implications for activation of programmed cells[J].Proceedings of the National Academy of Sciences of the United States of America,1978,75(7):3400-3404.
- [18]MIGLIORE-SAMOUR D,FLOC’H F,JOLLèS P.Biologically active casein peptides implicated in immunomodulation[J].Journal of Dairy Research,1989,56(3):357-362.
- [19]CHIBA H,TANI F,YOSHIKAWA M.Opioid antagonist peptides derived fromκ-casein[J].Journal of Dairy Research,1989,56(3):363-366.
- [20]侯银臣,吴丽,刘旺旺,等.羊胎盘免疫活性肽的制备及其活性[J].中国食品学报,2016,16(1):123-129.
- [21]纪丽娜.金枪鱼头酶解物免疫活性肽的分离及对小鼠腹腔巨噬细胞功能的影响[D].湛江:广东海洋大学,2012.
- [22]房新平.牛胎盘免疫活性肽提取与酶法制备研究[D].无锡:江南大学,2007.
- [23]QIAN B J,XING M Z,CUI L,et al.Antioxidant,antihypertensive,and immunomodulatory activities of peptide fractions from fermented skim milk with Lactobacillus delbrueckii ssp.bulgaricus LB340[J].Journal of Dairy Research,2011,78(1):72-79.
- [24]STUKNYTE M,DE NONI I,GUGLIELMETTI S,et al.Potential immunomodulatory activity of bovine casein hydrolysates produced after digestion with proteinases of lactic acid bacteria[J].International Dairy Journal,2011,21(10):763-769.
- [25]IEMURA Y,YAMADA T,TAKAHASHI T,et al.Properties of the peptides liberated from rice protein in Sokujo-moto[J].Journal of Bioscience and Bioengineering,1999,88(3):276-280.
- [26]侯虎.鳕鱼免疫活性肽的可控制备及其免疫活性研究[D].青岛:中国海洋大学,2011.