弹性蛋白样生物材料的制备及性质鉴定 <sup>*</sup>

doi:10.13523/j.cb.2003055

中国生物工程杂志

2020, Vol. 40

Issue (8): 33-40 DOI: 10.13523/j.cb.2003055

技术与方法

弹性蛋白样生物材料的制备及性质鉴定 ^*

张潇航¹,李媛媛²,贾敏晅^2,³,顾奇^2,^4,^**(

)

1 中国科学院大学存济医学院北京 101408
2 中国科学院动物研究所膜生物学国家重点实验室北京 100101
3 东北农业大学生命科学学院哈尔滨 150030
4 中国科学院干细胞与再生医学创新研究院北京 100101

Identification and Expression of Elastin-like Polypeptides

ZHANG Xiao-hang¹,LI Yuan-yuan²,JIA Min-xuan^2,³,GU Qi^2,^4,^**(

)

1 Savaid Medical School, University of Chinese Academy of Sciences, Beijing 101408, China
2 State Key Laboratory of Membrane Biology, Institute of Zoology, CAS, Beijing 100101, China
3 College of Life Science, Northeast Agricultural University, Harbin 150030, China
4 Stem Cell and Regenerative Medicine Institute, CAS, Beijing 100101, China

全文: PDF(2172 KB) HTML

摘要：

组织工程器官重建因不同的用途而对材料的弹性、刚度、生物活性等有一系列的需求,但当前所应用的材料多难以同时满足,例如弹性材料聚乙二醇双丙烯酸酯等高分子材料并不具备较高的生物活性,而生物材料胶原等生物活性好而弹性较差。弹性蛋白(elastin)作为广泛存在于动物体内的一种弹性极佳的功能蛋白,因其可承受较大形变而不破坏本身结构,故应用于弹性器官的组织工程重建中。为获得高生物活性的弹性蛋白样多肽(elastin-like polypeptides,ELP),根据其基本重复单元Val-Pro-Gly-Xaa-Gly设计了氨基酸序列,并进行优化,构建成原核表达质粒,通过大肠杆菌BL21(DE3)菌株进行诱导表达并收集,SDS-PAGE鉴定。通过流变检测、扫描电镜检测、细胞活性检测等方法鉴定材料的弹性性能、物理结构及生物活性,为下一步通过调整交联方法提高其弹性提供了材料,也为ELP作为生物材料应用于组织工程器官重建奠定了基础。

关键词： 弹性蛋白样多肽; 原核表达; 生物材料

Abstract:

Organ reconstruction may have severals of requirements for the elasticity, stiffness, and biological activity of the materials due to different applications, but currently many materials are challenging to meet these requirements at the same time. For example, polyethylene glycol diacrylate, a kind of widely used elastic material, do not have high biological activity, while bio-materials such as collagen have poor elasticity. As an elastic functional protein which widely existing in animals, elastin is valued in tissue engineering reconstruction of elastic organs for its special properties that can withstand large deformation without destroying its structure. The amino acid sequence of the elastin-like polypeptide (ELP) designed in this article meets the requirements in the project, excellent biological activity and elasticity, according to the basic repeat unit Val-Pro-Gly-Xaa-Gly and preference and degeneracy of the E coli. condon. After the plasmid was constructed, the ELP was expressed and collected in E coli. BL21 (DE3), and then identified the protein by SDS-PAGE. The modulus data of the protein was tested by rheology, the microstructure of the material system was tested by SEM, the biological activity of the material was tested by cell culture. These methods identified the elastic properties, physical structure and biological activity of materials. They contributed the basis for enhancing the elasticity by crosslinking and its application in tissue engineering and organs reconstruction.

Key words: Elastin-like polypeptides Prokaryotic expression Bio-materials

收稿日期: 2020-03-23 出版日期: 2020-09-10

ZTFLH:

Q819

基金资助: *中国科学院器官重建与制造战略性先导科技专项(XDA16020802)

通讯作者: 顾奇 E-mail: qgu@ioz.ac.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	张潇航
	李媛媛
	贾敏晅
	顾奇

引用本文:

张潇航,李媛媛,贾敏晅,顾奇. 弹性蛋白样生物材料的制备及性质鉴定 ^*[J]. 中国生物工程杂志, 2020, 40(8): 33-40.

ZHANG Xiao-hang,LI Yuan-yuan,JIA Min-xuan,GU Qi. Identification and Expression of Elastin-like Polypeptides. China Biotechnology, 2020, 40(8): 33-40.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2003055 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I8/33

图1 重组表达载体pET-28b-RGD-ELP构建策略图及检测

图2 SDS-PAGE鉴定RGD-ELP的原核表达

图3 10% RGD-ELP交联前后状态及与5% PEG-NHS等体积混合后体系的流变测试

图4 扫描电镜样品制备、RGD-ELP与PEG-NHS交联后断面结构及空隙面积统计

图5 实验组与对照组材料分别与PEG-NHS交联后用作hMSC细胞3D培养的结果

[1]	Kim M S, Lee D W, Park K, et al. Temperature-triggered tumor-specific delivery of anticancer agents by cRGD-conjugated thermosensitive liposomes. Colloids Surf B Biointerfaces, 2014,116:17-25. doi: 10.1016/j.colsurfb.2013.12.045 pmid: 24441178
[2]	Liu H, Liu H, Deng X, et al. CXCR4 antagonist delivery on decellularized skin scaffold facilitates impaired wound healing in diabetic mice by increasing expression of SDF-1 and enhancing migration of CXCR4-positive cells. Wound Repair Regen, 2017,25(4):652-664. doi: 10.1111/wrr.12552 pmid: 28783870
[3]	Tort S, Acarturk F, Besikci A. Evaluation of three-layered doxycycline-collagen loaded nanofiber wound dressing. Int J Pharm, 2017,529(1-2):642-653. doi: 10.1016/j.ijpharm.2017.07.027 pmid: 28705624
[4]	Zhong S P, Zhang Y Z, Lim C T. Tissue scaffolds for skin wound healing and dermal reconstruction. Wiley Interdiscip Rev Nanomed Nanobiotechnol, 2010,2(5):510-525. doi: 10.1002/wnan.100 pmid: 20607703
[5]	Lee A, Hudson A R, Shiwarski D J, et al. 3D bioprinting of collagen to rebuild components of the human heart. Science, 2019,365(6452):482-487. doi: 10.1126/science.aav9051 pmid: 31371612
[6]	Ji S, Almeida E, Guvendiren M. 3D bioprinting of complex channels within cell-laden hydrogels. Acta Biomater, 2019,95:214-224.
[7]	Lu Y, Aimetti A A, Langer R, et al. Bioresponsive materials. Nat Rev Mater, 2016,2(1):16075.
[8]	Datta L P, Manchineella S, Govindaraju T. Biomolecules-derived biomaterials. Biomaterials, 2020,230:119633. doi: 10.1016/j.biomaterials.2019.119633 pmid: 31831221
[9]	Raub C B, Suresh V, Krasieva T, et al. Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy. Biophys J, 2007,92(6):2212-2222. doi: 10.1529/biophysj.106.097998 pmid: 17172303
[10]	Abdulghani S, Morouco P G. Biofabrication for osteochondral tissue regeneration: bioink printability requirements. J Mater Sci Mater Med, 2019,30(2):20. pmid: 30689057
[11]	Zhang Z, Ortiz O, Goyal R, et al. Principles of tissue engineering. 4th ed. Boston,USA: Academic Press, 2014: 441-473.
[12]	Vindin H, Mithieux S M, Weiss A S. Elastin architecture. Matrix Biol, 2019,84:4-16. pmid: 31301399
[13]	Urry D W, Pattanaik A, Xu J, et al. Elastic protein-based polymers in soft tissue augmentation and generation. J Biomater Sci Polym Ed, 1998,9(10):1015-1048.
[14]	Morelli M A, DeBiasi M, DeStradis A, et al. An aggregating elastin-like pentapeptide. J Biomol Struct Dyn, 1993,11(1):181-190. doi: 10.1080/07391102.1993.10508716 pmid: 8216943
[15]	Meyer D E, Chilkoti A. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol, 1999,17(11):1112-1115. doi: 10.1038/15100 pmid: 10545920
[16]	Zeng Q, Desai M S, Jin H E, et al. Self-healing elastin-bioglass hydrogels. Biomacromolecules, 2016,17(8):2619-2625.
[17]	Lin C Y, Liu J C. Incorporation of short, charged peptide tags affects the temperature responsiveness of positively-charged elastin-like polypeptides. J Mater Chem B, 2019,7(34):5245-5256. doi: 10.1039/c9tb00821g pmid: 31384872
[18]	Aladini F, Araman C, Becker C F. Chemical synthesis and characterization of elastin-like polypeptides (ELPs) with variable guest residues. J Pept Sci, 2016,22(5):334-342. pmid: 27005861
[19]	Wang E, Lee S H, Lee S W. Elastin-like polypeptide based hydroxyapatite bionanocomposites. Biomacromolecules, 2011,12(3):672-680. doi: 10.1021/bm101322m pmid: 21218767
[20]	Raphel J, Karlsson J, Galli S, et al. Engineered protein coatings to improve the osseointegration of dental and orthopaedic implants. Biomaterials, 2016,83:269-282. doi: 10.1016/j.biomaterials.2015.12.030 pmid: 26790146
[21]	Shmidov Y, Zhou M, Yosefi G, et al. Hydrogels composed of hyaluronic acid and dendritic ELPs: hierarchical structure and physical properties. Soft Matter, 2019,15(5):917-925.
[22]	Desai M S, Wang E, Joyner K, et al. Elastin-based rubber-like hydrogels. Biomacromolecules, 2016,17(7):2409-2416. doi: 10.1021/acs.biomac.6b00515 pmid: 27257908
[23]	Huettner N, Dargaville T R, Forget A. Discovering cell-adhesion peptides in tissue engineering: beyond RGD. Trends Biotechnol, 2018,36(4):372-383. doi: 10.1016/j.tibtech.2018.01.008 pmid: 29422411
[24]	Wang H, Cai L, Paul A, et al. Hybrid elastin-like polypeptide-polyethylene glycol (ELP-PEG) hydrogels with improved transparency and independent control of matrix mechanics and cell ligand density. Biomacromolecules, 2014,15(9):3421-3428. doi: 10.1021/bm500969d pmid: 25111283
[25]	Sun F, Bu Y, Chen Y, et al. An injectable and instant self-healing medical adhesive for wound sealing. ACS Appl Mater Interfaces, 2020,12(8):9132-9140. doi: 10.1021/acsami.0c01022 pmid: 32058692
[26]	Matai I, Kaur G, Seyedsalehi A, et al. Progress in 3D bioprinting technology for tissue/organ regenerative engineering. Biomaterials, 2020,226:119536. doi: 10.1016/j.biomaterials.2019.119536 pmid: 31648135
[27]	Croce S, Peloso A, Zoro T, et al. A hepatic scaffold from decellularized liver tissue: food for thought. Biomolecules, 2019,9(12):813.
[28]	Valot L, Martinez J, Mehdi A, et al. Chemical insights into bioinks for 3D printing. Chem Soc Rev, 2019,48(15):4049-4086. doi: 10.1039/c7cs00718c pmid: 31271159
[29]	Tsuchiya T, Doi R, Obata T, et al. Lung microvascular niche, repair, and engineering. Front Bioeng Biotechnol, 2020, 8: 105. doi: 10.3389/fbioe.2020.00105 pmid: 32154234
[30]	Wei L, Wu S, Kuss M, et al. 3D printing of silk fibroin-based hybrid scaffold treated with platelet rich plasma for bone tissue engineering. Bioact Mater, 2019,4:256-260. doi: 10.1016/j.bioactmat.2019.09.001 pmid: 31667442
[31]	Sykes M, Sachs D H. Transplanting organs from pigs to humans. Sci. Immunol, 2019,4(41):12.
[32]	Chimene D, Kaunas R, Gaharwar A K. Hydrogel bioink reinforcement for additive manufacturing: a focused review of emerging strategies. Adv Mater, 2020,32(1):e1902026. doi: 10.1002/adma.201902026 pmid: 31599073
[33]	Daamen W F, Veerkamp J H, van Hest J C, et al. Elastin as a biomaterial for tissue engineering. Biomaterials, 2007,28(30):4378-4398. doi: 10.1016/j.biomaterials.2007.06.025 pmid: 17631957
[34]	Dhandhukia J P, Shi P, Peddi S, et al. Bifunctional elastin-like polypeptide nanoparticles bind rapamycin and integrins and suppress tumor growth in vivo. Bioconjug Chem, 2017,28(11):2715-2728. doi: 10.1021/acs.bioconjchem.7b00469 pmid: 28937754
[35]	Samourides A, Browning L, Hearnden V, et al. The effect of porous structure on the cell proliferation, tissue ingrowth and angiogenic properties of poly(glycerol sebacate urethane) scaffolds. Mater Sci Eng C Mater Biol Appl, 2020,108:110384. pmid: 31924046
[36]	Veneti E, Tu R S, Auguste D T. RGD-targeted liposome binding and uptake on breast cancer cells is dependent on elastin linker secondary structure. Bioconjug Chem, 2016,27(8):1813-1821. doi: 10.1021/acs.bioconjchem.6b00205 pmid: 27463763

[1]	马宁,王汉杰. 光遗传学在细菌生产调控中的应用进展[J]. 中国生物工程杂志, 2021, 41(9): 101-109.
[2]	乔圣泰,王曼琦,徐慧妮. 番茄SlTpx原核表达蛋白的体外功能分析^*[J]. 中国生物工程杂志, 2021, 41(8): 25-32.
[3]	张磊,唐永凯,李红霞,李建林,徐逾鑫,李迎宾,俞菊华. 促进原核表达蛋白可溶性的研究进展 ^*[J]. 中国生物工程杂志, 2021, 41(2/3): 138-149.
[4]	吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生. **LbCpf1基因的原核表达、纯化与体外切割检测 ^***[J]. 中国生物工程杂志, 2020, 40(8): 41-48.
[5]	李彤彤,宋彩玲,杨凯越,王文静,陈慧宇,刘明. 抗犬细小病毒VP2蛋白单链抗体的制备与中和活性研究 ^*[J]. 中国生物工程杂志, 2020, 40(4): 10-16.
[6]	程平,张洋子,马翾,陈旭,朱保庆,许文涛. 刺激响应型DNA水凝胶的性质及其应用 ^*[J]. 中国生物工程杂志, 2020, 40(3): 132-143.
[7]	陈秋利,杨丽超,李辉,温莎,李刚,何敏. 人Nek2蛋白原核表达纯化及其多克隆抗体制备 ^*[J]. 中国生物工程杂志, 2020, 40(3): 31-37.
[8]	杨隆兵,国果,马慧玲,李妍,赵欣宇,苏佩佩,张勇. 家蝇抗菌肽AMPs17蛋白原核表达条件的优化及其抗真菌活性检测 ^*[J]. 中国生物工程杂志, 2019, 39(4): 24-31.
[9]	李明英,王仁军,张帆,迟彦. β2糖蛋白Ⅰ第五结构域及其突变体、短肽片段的原核表达及活性分析 ^*[J]. 中国生物工程杂志, 2018, 38(8): 1-9.
[10]	陈远侨,龙定沛,豆晓雪,祁润,赵爱春. ELP₃₀-tag蛋白纯化能力的原核表达研究[J]. 中国生物工程杂志, 2018, 38(2): 54-60.
[11]	何亚南,孙钰椋,任雅坤,梁盛英,杨芬,刘彦礼,林俊堂. 金黄色葡萄球菌类肠毒素K与GFP融合蛋白工程菌的构建及其表达蛋白生物学活性分析 ^*[J]. 中国生物工程杂志, 2018, 38(12): 14-20.
[12]	任建委,李军,李尚泽. 人源CT55蛋白原核表达及单克隆抗体的制备 ^*[J]. 中国生物工程杂志, 2018, 38(11): 1-8.
[13]	孙文佳, 姚宇峰, 杨旭, 黄惟巍, 刘存宝, 龙琼, 褚晓杰, 马雁冰. 乙肝核心抗原病毒样颗粒呈现HPV 16L1抗原表位及特异抗体诱导[J]. 中国生物工程杂志, 2017, 37(3): 58-64.
[14]	吴剑荣,彭星桥,詹晓北. 聚唾液酸,一种非GAGs、非免疫原性生物材料的应用研究进展 ^*[J]. 中国生物工程杂志, 2017, 37(12): 96-102.
[15]	祖力皮也·吐尔逊, 曹春宝, 温浩, 丁剑冰, 德力夏提·依米提. 细粒棘球蚴EgG1Y162基因进化分析、表达及鉴定[J]. 中国生物工程杂志, 2016, 36(4): 78-87.

Viewed

Full text

Abstract

Cited

Shared

Discussed