Green biological manufacture and application of recombinant collagen

doi:10.16085/j.issn.1000-6613.2020-2109

Abstract

Abstract:

Collagen exists in all tissues and organs. Compared with animal collagen, recombinant collagen has a more single component, higher safety, and more controllable production process. This review briefly introduces the construction of different expression systems of recombinant collagen, including the expression systems of animals, plants, and microorganisms, and their advantages and disadvantages are compared. The regulation of different fermentation parameters affecting the expression of the product in the microbial system, the separation and purification process of the product, and the application of recombinant collagen in the medical field were introduced emphatically. It is pointed out that the microbial fermentation system has a lower cost, simple operation, and easier to expand production than the animal and plant system. Temperature, pH, dissolved oxygen, glucose, and acetic acid concentration affect the protein expression in Escherichia coli fermentation. In yeast fermentation, the addition amount of methanol, temperature, pH, and dissolved oxygen are the main influencing parameters. Microbial fermentation systems need to obtain high purity products through different crude and precision technologies. Meanwhile, recombinant collagen plays an important role in the field of biomedicine.

Key words: recombinant collagen, Escherichia coli, yeast, fermentation, purification, application

摘要：

胶原蛋白存在于各个组织器官，与动物胶原蛋白相比，重组胶原蛋白组分单一、安全性高、生产过程可控。本篇综述简述了重组胶原蛋白不同表达体系的构建，包括动植物以及微生物表达体系，比较了不同体系的优缺点。着重介绍了微生物体系中影响产物表达的不同发酵参数的调控，产物的分离纯化工艺以及重组胶原蛋白在医学领域的应用。提出微生物发酵体系较动植物体系成本低，操作简单，易于扩大生产；温度、pH、溶解氧、葡萄糖、乙酸浓度等影响大肠杆菌发酵中的蛋白表达量；酵母发酵中，甲醇添加量、温度、pH和溶解氧是主要影响参数；微生物发酵体系均需通过不同的粗纯及精纯技术获得纯度较高的产物。同时，重组胶原蛋白在生物医学领域发挥着重要作用。

关键词: 重组胶原蛋白, 大肠杆菌, 酵母, 发酵, 纯化, 应用

CLC Number:

Q815

LI Yang, ZHU Chenhui, FAN Daidi. Green biological manufacture and application of recombinant collagen[J]. Chemical Industry and Engineering Progress, 2021, 40(3): 1262-1275.

李阳, 朱晨辉, 范代娣. 重组胶原蛋白的绿色生物制造及其应用[J]. 化工进展, 2021, 40(3): 1262-1275.

Figures/Tables 12

References 93

1	LIU X H，ZHENG C，LUO X M，et al. Recent advances of collagen-based biomaterials: multi-hierarchical structure, modification and biomedical applications[J]. Materials Science and Engineering C, 2019, 99: 1509-1522.
2	LI J, WANG M C, QIAO Y Y, et al. Extraction and characterization of type Ⅰ collagen from skin of tilapia (Oreochromis niloticus) and its potential application in biomedical scaffold material for tissue engineering[J]. Process Biochemistry, 2018, 74: 156-163.
3	ALAGHA A, NOURALLAH A, HARIRI S. Characterization of dexamethasone loaded collagen-chitosan sponge and invitro release study[J]. Journal of Drug Delivery Science and Technology, 2020, 55: 101449.
4	ADAMIAK K, SIONKOWSKA A. Current methods of collagen cross-linking: review[J]. International Journal of Biological Macromolecules, 2020, 161: 550-560.
5	MIRANDA-NIEVES D, CHAIKOF E L. Collagen and elastin biomaterials for the fabrication of engineered living tissues[J]. ACS Biomaterials Science and Engineering, 2017, 3: 694-711.
6	URELLO M A, KIICK K L, SULLIVAN M O. ECM turnover-stimulated gene delivery through collagen-mimetic peptide-plasmid integration in collagen[J]. Acta Biomaterialia, 2017, 62: 167-178.
7	HORMOZI-MOGHADDAM Z, MOKHTARI-DIZAJI M, NILFOROSHZADEH M A, et al. Low-intensity ultrasound to induce proliferation and collagen Ⅰ expression of adipose-derived mesenchymal stem cells and fibroblast cells in co-culture[J]. Measurement: Journal of the International Measurement Confederation, 2021, 167: 108280.
8	GUILLOT-FERRIOLS M, RODRÍGUEZ-HERNÁNDEZ J C, CORREIA D M, et al. Poly(vinylidene) fluoride membranes coated by heparin/collagen layer-by-layer, smart biomimetic approaches for mesenchymal stem cell culture[J]. Materials Science and Engineering C, 2020, 117: 111281.
9	KOENS M J W, FARAJ K A, WISMANS R G, et al. Controlled fabrication of triple layered and molecularly defined collagen/elastin vascular grafts resembling the native blood vessel[J]. Acta Biomaterialia, 2010, 6: 4666-4674.
10	SARKAR M R, AUGAT P, SHEFELBINE S J, et al. Bone formation in a long bone defect model using a platelet-rich plasma-loaded collagen scaffold[J]. Biomaterials, 2006, 27: 1817-1823.
11	CHIU L H, LAI W F T, CHANG S F, et al. The effect of type Ⅱ collagen on MSC osteogenic differentiation and bone defect repair[J]. Biomaterials, 2014, 35: 2680-2691.
12	THAPA R K, KIICK K L, SULLIVAN M O. Encapsulation of collagen mimetic peptide-tethered vancomycin liposomes in collagen-based scaffolds for infection control in wounds[J]. Acta Biomaterialia, 2020, 103: 115-128.
13	SUN L L, LI B F, JIANG D D, et al. Nile tilapia skin collagen sponge modified with chemical cross-linkers as a biomedical hemostatic material[J]. Colloids and Surfaces B: Biointerfaces, 2017, 159: 89-96.
14	RAMSHAW J A M, WERKMEISTER J A, GLATTAUER V. Recent progress with recombinant collagens produced in Escherichia coli[J]. Current Opinion in Biomedical Engineering, 2019, 10: 149-155.
15	STEIN H, WILENSKY M, TSAFRIR Y, et al. Production of bioactive, post-translationally modified, heterotrimeric, human recombinant type-Ⅰ collagen in transgenic tobacco[J]. Biomacromolecules, 2009, 10: 2640-2645.
16	XU X, GAN Q L, CLOUGH R C, et al. Hydroxylation of recombinant human collagen type Ⅰ alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase[J]. BMC Biotechnology, 2011, 11: 1-12.
17	ZHANG C, BAEZ J, GLATZ C E. Purification and characterization of a 44-kDa recombinant collagen Ⅰ α1 fragment from corn grain[J]. Journal of Agricultural and Food Chemistry, 2009, 57: 880-887.
18	DAVID TOMAN P, PIEPER F, SAKAI N, et al. Production of recombinant human type Ⅰ procollagen homotrimer in the mammary gland of transgenic mice[J]. Transgenic Research, 1999, 8: 415-427.
19	ADACHI T, WANG X B, MURATA T, et al. Production of a non-triple helical collagen α chain in transgenic silkworms and its evaluation as a gelatin substitute for cell culture[J]. Biotechnology and Bioengineering, 2010, 106: 860-870.
20	OLSEN D, JIANG J, CHANG R, et al. Expression and characterization of a low molecular weight recombinant human gelatin: development of a substitute for animal-derived gelatin with superior features[J]. Protein Expression and Purification, 2005, 40: 346-357.
21	RUTSCHMANN C, BAUMANN S, CABALZAR J, et al. Recombinant expression of hydroxylated human collagen in Escherichia coli[J]. Applied Microbiology and Biotechnology, 2014, 98: 4445-4455.
22	张驰. 重组大肠杆菌生产类人胶原蛋白III发酵条件优化与分离纯化研究[D]. 西安: 西北大学, 2009.
	ZHANG C. Optimization of fermentation conditions and isolation and purification of recombinant Escherichia coli to produce human collagen Ⅲ[D]. Xi’an: Northwest University, 2009.
23	李俐. 类人胶原蛋白Ⅰ的分离纯化及其功能特性的研究[D]. 西安: 西北大学, 2007.
	LI L. Isolation and purification of humanoid collagen Ⅰ and its functional properties[D]. Xi’an: Northwest University, 2007.
24	LUO Y E, MU T Z, FAN D D. Preparation of a low-cost minimal medium for engineered Escherichia coli with high yield of human-like collagen Ⅱ[J]. Pakistan Journal of Pharmaceutical Sciences, 2014, 27: 663-669.
25	SHI J J, MA X X, GAO Y, et al. Hydroxylation of human type Ⅲ collagen alpha chain by recombinant coexpression with a viral prolyl 4-hydroxylase in Escherichia coli[J]. Protein Journal, 2017, 36: 322-331.
26	MI Y, GAO Y, FAN D D, et al. Stability improvement of human collagen α1(Ⅰ) chain using insulin as a fusion partner[J]. Chinese Journal of Chemical Engineering, 2018, 26: 2607-2614.
27	HE J, MA X X, ZHANG F L, et al. New strategy for expression of recombinant hydroxylated human collagen α1(Ⅲ) chains in Pichia pastoris GS115[J]. Biotechnology and Applied Biochemistry, 2015, 62: 293-299.
28	SANCHEZ-GARCIA L, MARTÍN L, MANGUES R, et al. Recombinant pharmaceuticals from microbial cells: a 2015 update[J]. Microbial Cell Factories, 2016, 15: 1-7.
29	MADURAWE R D, CHASE T E, TSAO E I, et al. A recombinant lipoprotein antigen against Lyme disease expressed in E. coli: fermentor operating strategies for improved yield[J]. Biotechnology Progress, 2000, 16: 571-576.
30	张珊珊. 重组胶原蛋白及其生物材料的制备和性质研究[D]. 兰州: 兰州交通大学, 2017.
	ZHANG S S. Preparation and properties of recombinant collagen and its biomaterials[D]. Lanzhou: Lanzhou Jiaotong University, 2017.
31	王皓. 类人胶原蛋白在大肠杆菌中的高效表达及其抗氧化活性研究[D]. 长春: 吉林农业大学, 2013.
	WANG H. Highly expression of human-like collagen in E.coli and research of oxidation resistance[D]. Changchun: Jilin Agricultural University, 2013.
32	常海燕. 重组大肠杆菌高密度发酵生产类人胶原蛋白Ⅱ工艺研究[D]. 西安: 西北大学, 2009.
	CHANG H Y. Study on the process of recombinant E.coli high-density fermentation for expressing human-like collagen Ⅱ[D]. Xi’an: Northwest University, 2009.
33	ZHU C H, FAN D D, WANG Y Y. Human-like collagen/hyaluronic acid 3D scaffolds for vascular tissue engineering[J]. Materials Science and Engineering C, 2014, 34: 393-401.
34	段志广. 类人胶原蛋白止血海绵的性能研究[D]. 西安: 西北大学, 2008.
	DUAN Z G. The study on the properties of human-like collagen hematischesis sponge[D]. Xi’an: Northwest University, 2008.
35	ZHU C H, LEI H, FAN D D, et al. Novel enzymatic crosslinked hydrogels that mimic extracellular matrix for skin wound healing[J]. Journal of Materials Science, 2018, 53: 5909-5928.
36	SALLACH R E, CONTICELLO V P, CHAIKOF E L. Expression of a recombinant elastin-like protein in Pichia pastoris[J]. Biotechnology Progress, 2009, 25: 1810-1818.
37	ROMANOS M A, SCORER C A, CLARE J J. Foreign gene expression in yeast: a review[J]. Yeast, 1992, 8: 423-488.
38	CREGG J M, VEDVICK T S, RASCHKE W C. Recent advances in the expression of foreign genes in Pichia pastoris[J]. Biotechnology, 1993, 11: 905-910.
39	XI C X, LIU N, LIANG F, et al. Molecular assembly of recombinant chicken type Ⅱ collagen in the yeast Pichia pastoris[J]. Science China Life Sciences, 2018, 61: 815-825.
40	MYLLYHARJU J, NOKELAINEN M, VUORELA A, et al. Expression of recombinant human type Ⅰ-Ⅲ collagens in the yeast Pichia pastoris[J]. Biochemical Society Transactions, 2000, 28: 353-357.
41	OLSEN D R, LEIGH S D, CHANG R, et al. Production of human type Ⅰ collagen in yeast reveals unexpected new insights into the molecular assembly of collagen trimers[J]. Journal of Biological Chemistry, 2001, 276: 24038-24043.
42	徐立群. 类人胶原蛋白真核表达载体的构建及在毕赤酵母中的分泌表达[D]. 长春: 吉林农业大学, 2013.
	XU L Q. Construction of human-like collagen expression vector and its expression in Pichia pastoris[D]. Changchun: Jilin Agricultural University, 2013.
43	刘斌. 巴氏毕赤酵母基因工程菌高密度发酵表达重组人源胶原蛋白[D]. 南京: 南京理工大学, 2012.
	LIU B. High-density fermentation of genetically engineered Pichia pastoris expressing recombinant human-source collagen[D]. Nanjing: Nanjing University of Science and Technology, 2012.
44	EGELKROUT E, RAJAN V, HOWARD J A. Overproduction of recombinant proteins in plants[J]. Plant Science, 2012, 184: 83-101.
45	ZHANG C, BAEZ J, PAPPU K M, et al. Purification and characterization of a transgenic corn grain-derived recombinant collagen type Ⅰ alpha 1[J]. Biotechnology Progress, 2009, 25: 1660-1668.
46	MERLE C, PERRET S, LACOUR T, et al. Hydroxylated human homotrimeric collagen Ⅰ in Agrobacterium tumefaciens-mediated transient expression and in transgenic tobacco plant[J]. FEBS Letters, 2002, 515: 114-118.
47	ESKELIN K, RITALA A, SUNTIO T, et al. Production of a recombinant full-length collagen type Ⅰ α-1 and of a 45-kDa collagen type Ⅰ α-1 fragment in barley seeds[J]. Plant Biotechnology Journal, 2009, 7: 657-672.
48	JOHN D C A, WATSON R, KIND A J, et al. Expression of an engineered form of recombinant procollagen in mouse milk[J]. Nature Biotechnology, 1999, 17: 385-389.
49	QI Q, YAO L G, LIANG Z S, et al. Production of human type Ⅱ collagen using an efficient baculovirus-silkworm multigene expression system[J]. Molecular Genetics and Genomics, 2016, 291: 2189-2198.
50	TOMITAL M, MUNETSUNA H, SATO T, et al. Transgenic silkworms produce recombinant human type Ⅲ procollagen in cocoons[J]. Nature Biotechnology, 2003, 21: 52-56.
51	魏玮. 重组大肠杆菌高密度发酵生产Ⅵ型胶原蛋白的工艺研究[D]. 长春: 吉林农业大学, 2012.
	WEI W. Study on high cell density cultivation in recombinant Escherichia for production of collagen Ⅵ[D]. Changchun: Jilin Agricultural University, 2012.
52	XUE W J, FAN D D, SHANG L A, et al. Effects of acetic acid and its assimilation in fed-batch cultures of recombinant Escherichia coli containing human-like collagen cDNA[J]. Journal of Bioscience and Bioengineering, 2010, 109: 257-261.
53	XUE W J, FAN D D, SHANG L A, et al. Erratum: Production of biomass and recombinant human-like collagen in Escherichia coli processes with different CO₂ pulses[J]. Biotechnology Letters, 2009, 31: 1111-1112.
54	郭红丽. 重组类人胶原蛋白基因工程菌发酵动力学[D]. 南京: 南京理工大学, 2009.
	GUO H L. Fermentation kinetics of producing recombinant human-like collagen in Pichia pastoris[D]. Nanjing: Nanjing University of Science and Technology, 2009.
55	WANG L N, FAN D D, HE J, et al. A new strategy for secretory expression and mixed fermentation of recombinant human collagen α1 () chain in Pichia pastoris[J]. Biotechnology and Bioprocess Engineering, 2014, 19: 916-924.
56	梁鑫, 梁波, 张仁怀, 等. 甲醇浓度对毕赤酵母发酵表达重组人Ⅲ型胶原蛋白的影响[J]. 四川生理科学杂志, 2020, 42(3): 247-251.
	LIANG X, LIANG B, ZHANG H R, et al. Effects of methanol concentration on expression of recombinant human type Ⅲ collagen in Pichia pastoris[J]. Sichuan Journal of Physiological Sciences, 2020, 42(3): 247-251.
57	李林波. 毕赤酵母高密度发酵表达重组人Ⅲ型胶原α1链蛋白[D]. 西安: 西北大学, 2014.
	LI L B. Expression of Recombinant human-like collagen Ⅲ α1 chain protein by high density fermentation in Pichia pastoris[D]. Xi’an: Northwest University, 2014.
58	侯文洁. 重组类人胶原蛋白Ⅱ分离纯化的工艺研究[D]. 西安: 西北大学, 2006.
	HOU W J. Purification of the recombinant human-like collagen (Ⅱ) in E.coli[D]. Xi’an: Northwest University, 2006.
59	蒋世强, 潘能庆, 鄢航. 重组蛋白纯化研究进展[J]. 现代农业科技, 2017, 4: 250-252.
	JIANG S Q, PAN N Q, YAN H. Research progress on purification of recombinant protein[J]. Modern Agricultural Science and Technology, 2017, 4: 250-252.
60	WINGFIELD P. Protein precipitation using ammonium sulfate[J]. Current Protocols in Protein Science, 2016, 84(1): A.3F.1-A.3F.8.
61	CHEN M, COSTA F K, LINDVAY C R, et al. The recombinant expression of full-length type Ⅶ collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa[J]. Journal of Biological Chemistry, 2002, 277: 2118-2124.
62	FERTALA A, SIERON A L, GANGULY A, et al. Synthesis of recombinant human procollagen Ⅱ in a stably transfected tumour cell line (HT1080)[J]. Biochemical Journal, 1994, 298: 31-37.
63	高立虎. 重复序列类人胶原蛋白表达载体的构建及在毕赤酵母中的分泌表达[D]. 南京: 南京理工大学, 2007.
	GAO L H. Construction of human-like collagen repeat sequence collagen expression vector and its secretory expression in Pichia pastoris[D]. Nanjing: Nanjing University of Science and Technology, 2007.
64	FAHIMIPOUR F, BASTAMI F, KHOSHZABAN A, et al. Critical-sized bone defects regeneration using a bone-inspired 3D bilayer collagen membrane in combination with leukocyte and platelet-rich fibrin membrane (L-PRF): an in vivo study[J]. Tissue and Cell, 2020, 63: 101326.
65	PULKKINEN H J, TIITU V, VALONEN P, et al. Engineering of cartilage in recombinant human type Ⅱ collagen gel in nude mouse model in vivo[J]. Osteoarthritis and Cartilage, 2010, 18: 1077-1087.
66	HAMZAH M S A, NG C, ZULKARNAIN N I S, et al. Entrapment of collagen on polylactic acid 3D scaffold surface as a potential artificial bone replacement[J]. Materials Today: Proceedings, 2020, .
67	JAMES B D, GUERIN P, IVERSON Z, et al. Mineralized DNA-collagen complex-based biomaterials for bone tissue engineering[J]. International Journal of Biological Macromolecules, 2020, 161: 1127-1139.
68	ZHENG X Y, HUI J F, LI H, et al. Fabrication of novel biodegradable porous bone scaffolds based on amphiphilic hydroxyapatite nanorods[J]. Materials Science and Engineering C, 2017, 75: 699-705.
69	LIU K Q, LIU Y N, DUAN Z G. A biomimetic bi-layered tissue engineering scaffolds for osteochondral defects repair[J]. Science China Technological Sciences, DOI: http://doi.org/10.1007/S11431-020-1597-4.
70	FENG X L, ZHANG X F, LI S Q, et al. Preparation of aminated fish scale collagen and oxidized sodium alginate hybrid hydrogel for enhanced full-thickness wound healing[J]. International Journal of Biological Macromolecules, 2020, 164: 626-637.
71	REZAII M, ORYAN S, JAVERI A. Curcumin nanoparticles incorporated collagen-chitosan scaffold promotes cutaneous wound healing through regulation of TGF-β1/Smad7 gene expression[J]. Materials Science and Engineering C, 2019, 98: 347-357.
72	CAO J, WANG P, LIU Y N, et al. Double crosslinked HLC-CCS hydrogel tissue engineering scaffold for skin wound healing[J]. International Journal of Biological Macromolecules, 2020, 155: 625-635.
73	PAN H, FAN D D, DUAN Z G, et al. Non-stick hemostasis hydrogels as dressings with bacterial barrier activity for cutaneous wound healing[J]. Materials Science and Engineering C, 2019, 105: 110118.
74	LEI H, ZHU C H, FAN D D. Optimization of human-like collagen composite polysaccharide hydrogel dressing preparation using response surface for burn repair[J]. Carbohydrate Polymers, 2020, 239: 116249.
75	ZHANG B Y, LUO Q, DENG B, et al. Construction of tendon replacement tissue based on collagen sponge and mesenchymal stem cells by coupled mechano-chemical induction and evaluation of its tendon repair abilities[J]. Acta Biomaterialia, 2018, 74: 247-259.
76	YANG S, SHI X X, LI X M, et al. Oriented collagen fiber membranes formed through counter-rotating extrusion and their application in tendon regeneration[J]. Biomaterials, 2019, 207: 61-75.
77	SUN J, MOU C C, SHI Q, et al. Controlled release of collagen-binding SDF-1α from the collagen scaffold promoted tendon regeneration in a rat Achilles tendon defect model[J]. Biomaterials, 2018, 162: 22-33.
78	MUMCUOGLU D, DE MIGUEL L, JEKHMANE S, et al. Collagen Ⅰ derived recombinant protein microspheres as novel delivery vehicles for bone morphogenetic protein-2[J]. Materials Science and Engineering C, 2018, 84: 271-280.
79	ZHANG H, LIU Y X, CHEN C W, et al. Responsive drug-delivery microcarriers based on the silk fibroin inverse opal scaffolds for controllable drug release[J]. Applied Materials Today, 2020, 19: 100540.
80	FAROKHI M, MOTTAGHITALAB F, REIS R L, et al. Functionalized silk fibroin nanofibers as drug carriers: advantages and challenges[J]. Journal of Controlled Release, 2020, 321: 324-347.
81	LU J N, CHEN Q W, DING X C, et al. BSA modified, disulfide-bridged mesoporous silica with low biotoxicity for dual-responsive drug delivery[J]. Microporous and Mesoporous Materials, 2019, 278: 257-266.
82	MONTERO N, PÉREZ E, BENITO M, et al. Biocompatibility studies of intravenously administered ionic-crosslinked chitosan-BSA nanoparticles as vehicles for antitumour drugs[J]. International Journal of Pharmaceutics, 2019, 554: 337-351.
83	ADITYA A, KIM B, KOYANI R D, et al. Kinetics of collagen microneedle drug delivery system[J]. Journal of Drug Delivery Science and Technology, 2019, 52: 618-623.
84	BREY GIL V S, BREY GIL C S, GOULART G A C, et al. Multi-drug hybrid delivery systems with distinct release profiles based on gelatin/collagen containing vesicles derived from block copolymers[J]. International Journal of Biological Macromolecules, 2019, 139: 967-974.
85	SUN C Y, CHE Y J, LU S J. Preparation and application of collagen scaffold-encapsulated silver nanoparticles and bone morphogenetic protein 2 for enhancing the repair of infected bone[J]. Biotechnology Letters, 2015, 37: 467-473.
86	KOZLOWSKA J, SIONKOWSKA A, SKOPINSKA-WISNIEWSKA J, et al. Northern pike (Esox lucius) collagen: extraction, characterization and potential application[J]. International Journal of Biological Macromolecules, 2015, 81: 220-227.
87	TAMILMOZHI S, VEERURAJ A, ARUMUGAM M. Isolation and characterization of acid and pepsin-solubilized collagen from the skin of sailfish (Istiophorus platypterus)[J]. Food Research International, 2013, 54: 1499-1505.
88	张卉. 重组类人胶原蛋白的表达纯化及在化妆品中的应用[D]. 广州: 暨南大学, 2017.
	ZHANG H. Expression and purification of recombinant human-like collagen and its application in cosmetics[D]. Guangzhou: Jinan University, 2017.
89	宋晓娟. 类人胶原蛋白治疗颜面再发性皮炎疗效观察及研究[J]. 吉林医学, 2019, 37: 2259-2260.
	SONG X J. Observation and study on the efficacy of humanoid collagen in the treatment of recurrent facial dermatitis[J]. Jilin Medical Journal, 2019, 37: 2259-2260.
90	杨光强, 牟宽厚, 郭爱琴. 地氯雷他定干混悬剂联合外用糠酸莫米松乳膏及类人胶原蛋白修复敷料治疗特应性皮炎35例疗效研究[J]. 陕西医学杂志, 2018, 47: 1477-1479.
	YANG G Q, MOU K H, GUO A Q. The investigation of the effect of desloradine for suspension combined with mometasone furoate cream and human-like collagen repair dressing on 35 cases with atopic dermatitis[J]. Shaanxi Medical Journal, 2018, 47: 1477-1479.
91	张春阳, 李云飞, 宋静卉. 类人胶原蛋白疤痕修复硅凝胶(可痕TM)治疗增生性瘢痕的研究[J]. 医药论坛杂志, 2019, 40: 11-13.
	ZHANG C Y, LI Y F, SONG J H. Study of human-like collagen scar repair gel in treatment of hypertrophic scars[J]. Journal of Medical Forum, 2019, 40: 11-13.
92	刘丽红, 韩悦, 郗金鹏. 微针导入类人胶原蛋白对面部年轻化的作用[J]. 中国医疗美容, 2012, 21: 1549-1551.
	LIU L H, HAN Y, XI J P. The role of microneedles roller importing human-like collagen into skin for facial rejuvenation[J]. Medical Cosmetology in China, 2012, 21: 1549-1551.
93	丰秋婧, 聂玉洁. 外用类人胶原蛋白治疗复发性口腔溃疡的疗效观察[J]. 全科口腔医学杂志, 2016, 3: 108.
	FENG Q J, NIE Y J. Clinical observation of human collagen in the treatment of recurrent oral ulcer[J]. General Journal of Stomatology, 2016, 3: 108.

项目	天然胶原蛋白	重组类人胶原蛋白
与人体亲和性	异体胶原、弱人体亲和性	同质胶原、高人体亲和性
生产工艺	化学提取、可控性差	基因工程、品质可控
安全性	易携带动物病毒（疯牛病等）	酵母发酵无病毒
致敏性	异源蛋白、易过敏	同质蛋白、不易过敏
生物活性	无胶原空间结构、无生物活性	有胶原空间结构、保留生物活性
纯度	混合胶原，成分复杂	单一胶原，成分固定，纯度达95%
保湿性	弱	高

项目	天然胶原蛋白	重组类人胶原蛋白
与人体亲和性	异体胶原、弱人体亲和性	同质胶原、高人体亲和性
生产工艺	化学提取、可控性差	基因工程、品质可控
安全性	易携带动物病毒（疯牛病等）	酵母发酵无病毒
致敏性	异源蛋白、易过敏	同质蛋白、不易过敏
生物活性	无胶原空间结构、无生物活性	有胶原空间结构、保留生物活性
纯度	混合胶原，成分复杂	单一胶原，成分固定，纯度达95%
保湿性	弱	高

表达体系	优点	缺点
动物	蛋白活性高，更接近于天然蛋白；正确的高级结构（如糖基化）；适合表达完整的大分子蛋白	培养基成本极高，培养周期长；培养较困难，表达量较低；操作技术要求高、产率低；不易大规模生产
植物	植物来源经济；安全性高	发酵纯化成本较高；产量低；不易于产业化生产
微生物	成本低；遗传背景清晰；生长繁殖快，培养周期短；可操作性强，易于产业化	大肠杆菌体系，纯化困难，产物活性低；毕赤酵母体系，甲醇为潜在风险

表达体系	优点	缺点
动物	蛋白活性高，更接近于天然蛋白；正确的高级结构（如糖基化）；适合表达完整的大分子蛋白	培养基成本极高，培养周期长；培养较困难，表达量较低；操作技术要求高、产率低；不易大规模生产
植物	植物来源经济；安全性高	发酵纯化成本较高；产量低；不易于产业化生产
微生物	成本低；遗传背景清晰；生长繁殖快，培养周期短；可操作性强，易于产业化	大肠杆菌体系，纯化困难，产物活性低；毕赤酵母体系，甲醇为潜在风险

微生物	发酵	纯化
原核	受pH、温度、溶解氧、乙酸浓度、碳源氮源等影响	大部分为胞内表达，需先释放胞内物质；涉及沉淀、超滤、层析等粗纯和精纯
真核	部分需要甲醇诱导受pH、温度以及甲醇含量的影响	既能胞内分泌，亦可胞外分泌，胞内释放时细胞破碎难度大，胞外释放不需细胞破碎；均涉及沉淀、超滤、层析等粗纯和精纯