| 619 | 5 | 60 |
| 下载次数 | 被引频次 | 阅读次数 |
贻贝(Mytilus)中富含各种黏附蛋白,其中贻贝足丝蛋白5(Mytilus galloprovincialis foot protein type 5,Mgfp-5)是贻贝与外界基质进行黏附的主要蛋白分子之一。贻贝黏蛋白天然提取时产量低、易凝固且纯化困难,通过化学合成和基因工程等方法重组贻贝黏蛋白解决该问题。本研究在E.coli BL21(DE3)中表达了贻贝蛋白Mgfp-5,亲和层析纯化到Mgfp-5蛋白的含量为12.25%,纯度为96.92%;酪氨酸酶修饰Mgfp-5后,其DOPA(多巴,3,4-二羟基苯丙氨酸)含量为9.60 pmol/g,是修饰前的9.32倍,生物黏性增强到1 116 n N,是Cell-Tak TM黏性的1.60倍;3.0μg/cm2的Mgfp-5蛋白的毒性级别为1级;Mgfp-5浓度≤20μg/m L时对红细胞无溶血现象,具有生物安全性。本研究可为重组Mgfp-5蛋白研究奠定基础,同时为来源于Mgfp-5黏蛋白的临床生物黏合剂的研制提供参考。
Abstract：Mytilus contains a variety of adhesive proteins, among which Mytilus galloprovincialis foot protein type 5(Mgfp-5) is one of the major proteins to stick with the substrates. Natural extraction of Mgfp-5 could obtain very little purified protein since the process is labor-intensive and inefficient. While the recombination of mussel adhesive protein through chemical synthesis and genetic engineering could overcome these limitations. In this research, the recombinant protein Mgfp-5 was expressed in E. coli BL21(DE3) and purified by affinity chromatography(12.25% production yield and 96.92% purity). After modifying by tyrosinase, the DOPA content in Mgfp-5 was 9.60 pmol/g and the adhesion enhanced to 1 116 n N, which were 9.32 times and 1.6 times as large as unmodified Mgfp-5, respectively. The toxicity level of 3.0 μg/cm2 Mgfp-5 protein was of the first level. The hemolysis of red blood cells didn't appear when the concentration of Mgfp-5 protein was no more than 20 μg/m L,which meant that it was bio-safe. This study could provide the basis for the recombination of Mgfp-5 protein and provide reference for the development of clinical bioadhesive of Mgfp-5 as well.
Bakker P.J.M.,Tukker L.J.,Stap J.,Veenhof C.H.N.,and Aten J.A.,1993,Micronuclei expression in tumours as a test for radiation sensitivity,Radiotherapy and Oncology,26(1):69-72
Dong J.P.,and Mao G.Z.,2000,Direct study of C12E5 aggregation on Mica by atomic force microscopy imaging and force Measurements,Langmuir,16(16):6641-6647
Dove J.,and Sheridan P.,1986,Adhesive protein from Mussels:possibilities for dentistry,medicine,and industry,The Journal of the American Dental Association,112(6):879
Ducker W.A.,Senden T.J.,and Pashley R.M.,1991,Direct measurement of colloidal forces using an atomic force microscope,Nature,353(6341):239-241
Higa O.Z.,Rogero S.O.,Machado L.D.B.,Mathor M.B.,Lugo A.B.,1999,Biocompatibility study for PVP wound dressing obtained in different conditions,Radiation Physics and Chemistry,55(5-6):705-707
Hwang D.S.,Zeng H.,Masic A.,Harrington M.J.,Israelachvili J.N.,and Waite J.H.,2010,Protein-and metal-dependent interactions of a prominent protein in mussel adhesive plaques,The Journal of Biological Chemistry,285(33):25850-25858
Jiang Z.,Liu J.P.,Jin L.H.,and Zhang Q.Q.,2010,Extraction and application of Perna viridis foot protein as bioadhesive,Shengwuyixue Gongchengxue Zazhi(Journal of Biomedical Engineering),27(6):1266-1273(蒋臻,刘加鹏,金利华,张其清,2010,翡翠贻贝足蛋白作为生物黏合剂的提取及应用,生物医学工程学杂志,27(6):1266-1273
Kan Y.,Danner E.W.,Israelachvili J.N.,Chen Y.,and Waite J.H.,2014,Boronate complex formation with Dopa containing Mussel adhesive protein retards p H-induced oxidation and enables adhesion to mica,PLo S One,9(10):e108869
Liao Z.,Shen W.,and Wang R.X.,2007,Adhesive proteins from marine animals,Zhejiang Haiyangxueyuan Xuebao(Ziran Kexueban)(Journal of Zhejiang Ocean University(Natural Science)),26(4):439-447(廖智,申望,王日昕,2007,海洋生物黏附蛋白研究进展,浙江海洋学院学报(自然科学报),26(4):439-447)
Le e B.P.,Messersmith P.B.,Israelachvili J.N.,and Waite J.H.,2011,Mussel-inspired adhesives and coatings,Annual Review of Materials Research,41:99-132
Li N.N.,Shi G.,Yang X.X.,Wang Z.P.,and Liao Z.,2011,Detection of dihydroxyphenylalanine in native foot proteins from Mytilus coruscus byssus,Zhongguo Shengwuhuaxue Yu Fenzishengwu Xuebao(Chinese Journal Biochemistry and Molecular Biology),27(2):148-153(李楠楠,石戈,杨宵旭,王智平,廖智,2011,厚壳贻贝Mytilus coruscus足丝盘及足丝的多巴分析,中国生物化学与分子生物学报,27(2):148-153)
Li W.J.,Zhou J.,and Xu Y.Y.,2015,Study of the in vitro cytotoxicity testing of medical devices(review),Biomedical Reports,3(5):617-620
Li L.,Gao M.,Song B.,Zhang H.,and Wang Y.,2016,Effects of RECQ1 helicase silencing on non-small cell lung cancer cells,Biomed Pharmacother,83:1227-1232
Lv Y.W.,Lv Y.W.,Yang Z.S.,Wang R.J.,Zhang Y.J.,and Wang YJ.,2016,Prokaryotic expression and purification of Mytilus galloprovincialis foot protein-5,Xibeidaxue Xuebao(Ziran Kexueban)(Journal of Northwest University(Natural Science Edition)),46(3):390-396(吕玉伟,吕亚维,杨泽熵,王睿劼张雨靖,王英娟,2016,贻贝蛋白Mgfp-5的原核表达与纯化,西北大学学报(自然科学版),46(3):390-396)
Lv Y.W.,Zhang YJ..,Lv Y.W.,Wang R.J.,Yang Z.S.,and Wang YJ.,2016,Transformation of Mytilus galloprovincialis foot protein type-5(Mgfp-5)gene in tobacco,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(1):172-182(吕玉伟,张雨靖,吕亚维,王睿劼,杨泽熵,王英娟2016,贻贝粘蛋白基因(Mgfp-5)在烟草中的转化,基因组学与应用生物学,35(1):172-182)
Silverman H.G.,and Roberto F.F.,2007,Understanding marine mussel adhesion,Marine Biotechnology,9(6):661-681
Tatehata H.,Mochizuki A.,Kawashima T.,Yamashita S.,and Yamamoto H.,2000,Model polypeptide of mussel adhesive protein.I.Synthesis and adhesive studies of sequentia polypeptides(X-Tyr-Lys)n and(Y-Lys)n,Journal of Applied Polymer Science,76(6):929-937
Biological evaluation of medical devices,part 5:test for in vitro cytotoxicity,2003,the General Administration for Quality Supervision,Inspection and Quarantine,GB/T16886.5,pp.80-88(医疗器械生物学评价,第5部分:体外细胞毒性试验,2003,国家质量监督检验检疫总局,GB/T16886.5,pp.80-88)
Waite J.H.,and Tanzer M.L.,1981,Polyphenolic substance of Mytilus edulis:novel adhesive containing L-Dopa and hydroxyproline,Science,212(4498):1038-1040
Waite J.H.,2002,Adhesion a la moule,Integrative and Comparative Biology,42(6):1172-1180
Wang F.,2013,Biodistribution and safety assessment of bladder cancer specific recombinant oncolytic adenovirus in nude mice,Dissertation for Ph.D.,Lanzhou University,Supervisor:Wang Z.P.,pp.51-66(王芳,2013,膀胱组织特异性溶瘤腺病毒在动物体内的生物分布及安全性评价,博士学位论文,兰州大学,导师:王志平,pp.51-66)
Wang X.Q.,Wang Y.M.,Xu H.,Shan H.,and Lu J.R.,2008,Dynamic adsorption of monoclonal antibody layers on hydrophilic silica surface:a combined study by spectroscopic ellipsometry and AFM,Journal of Colloid And Interface Science,323(1):18-25
Wu C.T.,Han P.P.,Liu X.G.,Xu M.C.,Tian T.,Chang J.,and Xiao Y.,2014,Mussel-inspired bioceramics with self-assembled Ca-P/polydopamine composite nanolayer:preparation,formation mechanism,improved cellular bioactivity and osteogenic differentiation of bone marrow stromal cells,Acta Biomaterialia,10(1):428-438
Yu M.,Hwang J.,and Deming T.J.,1999,Role of L-3,4-dihydroxyphenylalanine in mussel adhesive proteins,Journal of the American Chemical Society,121(24):5825-5826
Zheng X.,and Wang Y.J.,2002,Transforming to Pichia pastoris gene encoding mussel adhesive protein,Dalian Qinggongye Xueyuan Xuebao(Journal of Dalian Institute of Light Industry),21(2):100-102(郑昕,王运吉,2002,贻贝粘合蛋白基因转化巴斯德毕赤酵母,大连轻工业学院学报,21(2):100-102)
基本信息:
DOI：10.13417/j.gab.036.004108
中图分类号:S917.4
引用信息:
[1]吕亚维,王睿劼,张雨靖,等.重组贻贝黏蛋白Mgfp-5的表达及功能评价[J].基因组学与应用生物学,2017,36(10):4108-4115.DOI:10.13417/j.gab.036.004108.
基金信息:
国家自然科学基金资助项目(31270411,31572665);; 陕西省科技厅社会发展攻关计划资助项目(2012K12-01-02);; 陕西省教育厅专项资金资助项目(12JK0827)共同资助
2016-12-07
2016-12-07
2016-12-07