重组胶原蛋白表达体系研究进展

doi:10.12211/2096-8280.2023-020

合成生物学 ›› 2023, Vol. 4 ›› Issue (4): 808-823.DOI: 10.12211/2096-8280.2023-020

重组胶原蛋白表达体系研究进展

潘家豪¹, 潘炜松², 邱健³, 谢东玲², 邹奇⁴, 吴川¹

^1.中南大学冶金与环境学院，湖南长沙 410083
^2.湖南诺合新生物科技有限公司，湖南长沙 410001
^3.中南大学湘雅医院，湖南长沙 410008
^4.华南师范大学生命科学学院，广东广州 510631

收稿日期:2023-03-06 修回日期:2023-05-17 出版日期:2023-08-31 发布日期:2023-09-14
通讯作者: 吴川
作者简介:潘家豪（2000—），男，硕士研究生。研究方向为重组胶原蛋白在植物表达及其应用。E-mail：thekid123go@163.com
吴川（1983—），女，教授，博士生导师，湖湘青年英才，香港裘槎访问学者。研究方向为植物生物反应器、重金属污染生物修复等方面的研究。E-mail：wuchuan@csu.edu.cn
基金资助:
国家自然科学基金(31670955);中南大学前沿交叉项目(2023QYJC035)

Research progress on recombinant collagen expression system

Jiahao PAN¹, Weisong PAN², Jian QIU³, Donling XIE², Qi ZOU⁴, Chuan WU¹

^1.School of Metallurgy and Environment，Central South University，Changsha 410083，Hunan，China
^2.Hunan Novomore Biotechnology Corporation，Changsha 410001，Hunan，China
^3.Xiangya Hospital of Central South University，Changsha 410008，Hunan，China
^4.School of Life Sciences，South China Normal University，Guangzhou 510631，Guangdong，China

Received:2023-03-06 Revised:2023-05-17 Online:2023-08-31 Published:2023-09-14
Contact: Chuan WU

摘要/Abstract

摘要：

胶原蛋白是哺乳动物中含量最多的蛋白质，至今已发现28种类型，主要分为纤维性胶原蛋白、网状胶原蛋白、珠状丝状胶原蛋白、锚定纤维蛋白、膜蛋白以及multiplexins胶原蛋白，其中纤维性胶原蛋白中Ⅰ型、Ⅱ型、Ⅲ型胶原蛋白占人体胶原蛋白的80%~90%。目前，根据来源，胶原蛋白大致分为动物源胶原蛋白和重组胶原蛋白。动物源胶原蛋白主要来源于陆生动物以及海洋动物，而重组胶原蛋白是指将人胶原蛋白基因克隆到选定的表达载体并转化到表达细胞内，最后通过纯化技术所获得的蛋白质。本文简述了胶原蛋白的结构、类别和生物合成机制，重点阐述了重组胶原蛋白表达体系及特点，包括原核生物、酵母、植物、杆状病毒以及哺乳动物细胞等表达体系及其优势与局限性，介绍了重组胶原蛋白市场前景及在眼科、软骨工程、皮肤治疗等生物医药方面的实际应用，并对重组胶原蛋白的研究和产业发展进行了展望。

关键词: 胶原蛋白, 基因工程, 重组胶原蛋白表达体系

Abstract:

Collagen is the most abundant protein in mammals, and its production has been widely used in biomedicine, cosmetics, leather, biotechnology, etc. At present, collagen is generally divided into animal collagen and recombinant collagen. Although animal collagen is the main source of collagen, most of it comes from animal carcasses, and its collagen has been cross-linked and embedded in native tissues, which is more demanding on extraction and purification technology. In addition, pathogen contamination and allergy risks have become unavoidable problems for animal collagen. Recombinant collagen is a protein obtained by using human collagen cDNA fragments as the backbone gene, cloning the gene to the selected expression vector and converting it into an expression cell, and finally achieved by purification technology. Due to its single molecule, clear structure and easy control, recombinant collagen is the best alternative to replace animal collagen in biomedicine and tissue engineering. In this paper, the structure, category, biosynthesis mechanism and market scale of collagen are briefly described. Emphasis is placed on the construction strategies, advantages and limitations of different expression systems of recombinant collagen, including prokaryotic, yeast, plant, baculovirus and mammalian or human cell expression systems. Prokaryotes and yeast have a short cycle of producing recombinant collagen, but do not have a triple helix structure. The plant expression system produces recombinant collagen with a moderate cycle and a certain triple helix structure. The baculovirus-insect expression system and the mammalian expression system have a long cycle of recombinant collagen production and a complete triple helix structure. The practical application of recombinant collagen in ophthalmology, cartilage engineering, skin treatment and other biological medicine is described. Currently, the most commercially valuable use of collagen is subcutaneous injection of soluble protein to repair damaged skin. At the same time, collagen, as the main component of animal skin, can cross-link collagen in raw hides through chemical processes such as tanning, so that collagen becomes harder, more durable, and corrosion-resistant leather. By designing collagen scaffolds that are familiar with the natural cytoplasmic matrix environment, it can effectively reveal the pathogenesis of cell behavior and disease etiology. It is expected to provide suggestions on the research of recombinant collagen and future industrial development. {L-End}

Key words: collagen, gene engineering, recombinant collagen expression system

中图分类号:

Q816

潘家豪, 潘炜松, 邱健, 谢东玲, 邹奇, 吴川. 重组胶原蛋白表达体系研究进展[J]. 合成生物学, 2023, 4(4): 808-823.

Jiahao PAN, Weisong PAN, Jian QIU, Donling XIE, Qi ZOU, Chuan WU. Research progress on recombinant collagen expression system[J]. Synthetic Biology Journal, 2023, 4(4): 808-823.

图/表 5

参考文献 110

1	AVILA RODRÍGUEZ M I, RODRÍGUEZ BARROSO L G, SÁNCHEZ M L. Collagen: a review on its sources and potential cosmetic applications[J]. Journal of Cosmetic Dermatology, 2018, 17(1): 20-26.
2	KADLER K E, BALDOCK C, BELLA J, et al. Collagens at a glance[J]. Journal of Cell Science, 2007, 120(12): 1955-1958.
3	VEIT G, KOBBE B, KEENE D R, et al. Collagen ⅩⅩⅧ, a novel von Willebrand factor A domain-containing protein with many imperfections in the collagenous domain[J]. Journal of Biological Chemistry, 2006, 281(6): 3494-3504.
4	DI LULLO G A, SWEENEY S M, KÖRKKÖ J, et al. Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type Ⅰ collagen[J]. Journal of Biological Chemistry, 2002, 277(6): 4223-4231.
5	FRIESS W. Collagen-biomaterial for drug delivery[J]. European Journal of Pharmaceutics and Biopharmaceutics, 1998, 45(2): 113-136.
6	SIONKOWSKA A, SKRZYŃSKI S, ŚMIECHOWSKI K, et al. The review of versatile application of collagen[J]. Polymers for Advanced Technologies, 2017, 28(1): 4-9.
7	GRIFFITH M, JACKSON W B, LAGALI N, et al. Artificial corneas: a regenerative medicine approach[J]. Eye, 2009, 23(10): 1985-1989.
8	DUNN M G, DOILLON C J, BERG R A, et al. Wound healing using a collagen matrix: effect of DC electrical stimulation[J]. Journal of Biomedical Materials Research, 1988, 22(S13): 191-206.
9	RAO J R, KANTHIMATHI M, THANIKAIVELAN P, et al. Pickle-free chrome tanning using a polymeric synthetic tanning agent for cleaner leather processing[J]. Clean Technologies and Environmental Policy, 2004, 6(4): 243-249.
10	INOUE T, TODA S, NARISAWA Y, et al. Subcutaneous adipocytes promote the differentiation of squamous cell carcinoma cell line (DJM-1) in collagen gel matrix culture[J]. Journal of Investigative Dermatology, 2001, 117(2): 244-250.
11	CHE Z M, JUNG T H, CHOI J H, et al. Collagen-based co-culture for invasive study on cancer cells-fibroblasts interaction[J]. Biochemical and Biophysical Research Communications, 2006, 346(1): 268-275.
12	GINGRAS M, BEAULIEU M M, GAGNON V, et al. In vitro study of axonal migration and myelination of motor neurons in a three-dimensional tissue-engineered model[J]. Glia, 2008, 56(3): 354-364.
13	CHAMBERLAIN L J, YANNAS I V, HSU H P, et al. Collagen-GAG substrate enhances the quality of nerve regeneration through collagen tubes up to level of autograft[J]. Experimental Neurology, 1998, 154(2): 315-329.
14	SEROR J, STERN M, ZARKA R, et al. The potential use of novel plant-derived recombinant human collagen in aesthetic medicine[J]. Plastic & Reconstructive Surgery, 2021, 148(6S): 32S-38S.
15	SHOSEYOV O, POSEN Y, GRYNSPAN F. Human recombinant type Ⅰ collagen produced in plants[J]. Tissue Engineering Part A, 2013, 19(13/14): 1527-1533.
16	SHOSEYOV O, POSEN Y, GRYNSPAN F. Human collagen produced in plants: more than just another molecule[J]. Bioengineered, 2014, 5(1): 49-52.
17	XU N, PENG X L, LI H R, et al. Marine-derived collagen as biomaterials for human health[J]. Frontiers in Nutrition, 2021, 8: 702108.
18	傅容湛, 范代娣, 杨婉娟, 等. 重组胶原蛋白的产业发展历程和生物医学应用前景展望[J]. 生物工程学报, 2022, 38(9): 3228-3242.
	FU R Z, FAN D D, YANG W J, et al. Industrial development and biomedical application prospect of recombinant collagen[J]. Chinese Journal of Biotechnology, 2022, 38(9): 3228-3242.
19	中国国家药品监督管理局. 重组胶原蛋白生物材料命名指导原则 [S/OL]. 2021[2023-02-01]. .
	China National Medical Products Administration. Guidelines for the naming of recombinant collagen biomaterials [S/OL]. 2021[2023-02-01]. .
20	BERISIO R, VITAGLIANO L, MAZZARELLA L, et al. Crystal structure of the collagen triple helix model [(Pro-Pro-Gly)₁₀]₃ [J]. Protein Science, 2002, 11(2): 262-270.
21	NEUMAN R E, LOGAN M A. The determination of collagen and elastin in tissues[J]. Journal of Biological Chemistry, 1950, 186(2): 549-556.
22	BRODSKY B, RAMSHAW J A M. The collagen triple-helix structure[J]. Matrix Biology, 1997, 15(8/9): 545-554.
23	SHOULDERS M D, RAINES R T. Collagen structure and stability[J]. Annual Review of Biochemistry, 2009, 78: 929-958.
24	LEIKINA E, MERTTS M V, KUZNETSOVA N, et al. Type Ⅰ collagen is thermally unstable at body temperature[J]. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(3): 1314-1318.
25	SAXENA T, KARUMBAIAH L, VALMIKINATHAN C M. Proteins and poly(amino acids)[M/OL]//Natural and Synthetic Biomedical Polymers. Amsterdam: Elsevier, 2014: 43-65 [2023-02-01]. .
26	JONES E Y, MILLER A. Analysis of structural design features in collagen[J]. Journal of Molecular Biology, 1991, 218(1): 209-219.
27	ITO S, NAGATA K. Quality control of procollagen in cells[J]. Annual Review of Biochemistry, 2021, 90: 631-658.
28	ERDMANN R S, WENNEMERS H. Effect of sterically demanding substituents on the conformational stability of the collagen triple helix[J]. Journal of the American Chemical Society, 2012, 134(41): 17117-17124.
29	LEÓN-LÓPEZ A, MORALES-PEÑALOZA A, MARTÍNEZ-JUÁREZ V M, et al. Hydrolyzed collagen—sources and applications[J]. Molecules, 2019, 24(22): 4031.
30	RICHARDS A J, SNEAD M P. Molecular basis of pathogenic variants in the fibrillar collagens[J]. Genes, 2022, 13(7): 1199.
31	RICARD-BLUM S. The collagen family[J]. Cold Spring Harbor Perspectives in Biology, 2011, 3(1): a004978.
32	LI Y B, LIU Y Z, LI R H, et al. Collagen-based biomaterials for bone tissue engineering[J]. Materials & Design, 2021, 210: 110049.
33	SIONKOWSKA A, ADAMIAK K, MUSIAŁ K, et al. Collagen based materials in cosmetic applications: a review[J]. Materials, 2020, 13(19): 4217.
34	BRODSKY B, THIAGARAJAN G, MADHAN B, et al. Triple-helical peptides: an approach to collagen conformation, stability, and self-association[J]. Biopolymers, 2008, 89(5): 345-353.
35	SILVER F H, FREEMAN J W, SEEHRA G P. Collagen self-assembly and the development of tendon mechanical properties[J]. Journal of Biomechanics, 2003, 36(10): 1529-1553.
36	PROCKOP D J, FERTALA A. The collagen fibril: the almost crystalline structure[J]. Journal of Structural Biology, 1998, 122(1/2): 111-118.
37	SILVER D, MILLER J, HARRISON R, et al. Helical model of nucleation and propagation to account for the growth of type Ⅰ collagen fibrils from symmetrical pointed tips: a special example of self-assembly of rod-like monomers[J]. Proceedings of the National Academy of Sciences of the United States of America, 1992, 89(20): 9860-9864.
38	SUNDARAMOORTHY M, MEIYAPPAN M, TODD P, et al. Crystal structure of NC1 domains: structural basis for type Ⅳ collagen assembly in basement membranes[J]. Journal of Biological Chemistry, 2002, 277(34): 31142-31153.
39	BOUDKO S P, ENGEL J, BäCHINGER H P. The crucial role of trimerization domains in collagen folding[J]. The International Journal of Biochemistry & Cell Biology, 2012, 44(1): 21-32.
40	ORTEGA N, WERB Z. New functional roles for non-collagenous domains of basement membrane collagens[J]. Journal of Cell Science, 2002, 115(22): 4201-4214.
41	FERTALA A. Three decades of research on recombinant collagens: reinventing the wheel or developing new biomedical products?[J]. Bioengineering, 2020, 7(4): 155.
42	HINTZE V, STEPLEWSKI A, ITO H, et al. Cells expressing partially unfolded R789C/p.R989C type Ⅱ procollagen mutant associated with spondyloepiphyseal dysplasia undergo apoptosis[J]. Human Mutation, 2008, 29(6): 841-851.
43	BACHINGER H P, BRUCKNER P, TIMPL R, et al. Folding mechanism of the triple helix in type-Ⅲ collagen and type-Ⅲ pN-collagen. role of disulfide bridges and peptide bond isomerization[J]. European Journal of Biochemistry, 1980, 106(2): 619-632.
44	VAN DIJK F S, NESBITT I M, ZWIKSTRA E H, et al. PPIB mutations cause severe osteogenesis imperfecta[J]. The American Journal of Human Genetics, 2009, 85(4): 521-527.
45	PLOTKIN H. Syndromes with congenital brittle bones[J].BMC Pediatrics, 2004, 4(1): 16.
46	SEVIER C S, KAISER C A. Formation and transfer of disulphide bonds in living cells[J]. Nature Reviews Molecular Cell Biology, 2002, 3(11): 836-847.
47	SEVIER C S, KAISER C A. Conservation and diversity of the cellular disulfide bond formation pathways[J]. Antioxidants & Redox Signaling, 2006, 8(5/6): 797-811.
48	THORPE C, COPPOCK D L. Generating disulfides in multicellular organisms: emerging roles for a new flavoprotein family[J]. Journal of Biological Chemistry, 2007, 282(19): 13929-13933.
49	HERAS B, KURZ M, SHOULDICE S R, et al. The name's bond……disulfide bond[J]. Current Opinion in Structural Biology, 2007, 17(6): 691-698.
50	BRINCKMANN J, NOTBOHM H, MÜLLER P K. Collagen: primer in structure, processing, and assembly[M/OL]. Berlin: Springer, 2005[2023-02-01]. .
51	GEDDIS A E, PROCKOP D J. Expression of human COL1A1 gene in stably transfected HT1080 cells: the production of a thermostable homotrimer of type Ⅰ collagen in a recombinant system[J]. Matrix, 1993, 13(5): 399-405.
52	BULLEID N J, JOHN D C A, KADLER K E. Recombinant expression systems for the production of collagen[J]. Biochemical Society Transactions, 2000, 28(4): 350-353.
53	OLSEN D, YANG C L, BODO M, et al. Recombinant collagen and gelatin for drug delivery[J]. Advanced Drug Delivery Reviews, 2003, 55(12): 1547-1567.
54	BOYDSTON J A, CHEN P, STEICHEN C T, et al. Orientation within the exosporium and structural stability of the collagen-like glycoprotein BclA of Bacillus anthracis [J]. Journal of Bacteriology, 2005, 187(15): 5310-5317.
55	RUGGIERO F, KOCH M. Making recombinant extracellular matrix proteins[J]. Methods, 2008, 45(1): 75-85.
56	WERKMEISTER J A, RAMSHAW J A M. Recombinant protein scaffolds for tissue engineering[J]. Biomedical Materials, 2012, 7(1): 012002.
57	RAMSHAW J A M, WERKMEISTER J A, DUMSDAY G J. Bioengineered collagens: emerging directions for biomedical materials[J]. Bioengineered, 2014, 5(4): 227-233.
58	YIN J, LIN J H, LI W T, et al. Evaluation of different promoters and host strains for the high-level expression of collagen-like polymer in Escherichia coli [J]. Journal of Biotechnology, 2003, 100(3): 181-191.
59	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.
60	杨晶, 余洁莹, 王蒙, 等. 重组类人Ⅰ型胶原蛋白肽在大肠杆菌中的表达纯化及功能鉴定[J]. 现代食品科技, 2016, 32(2): 60-65.
	YANG J, YU J Y, WANG M, et al. Expression, purification, and functional characterization of recombinant human-like type Ⅰ collagen peptide in E.coli [J]. Modern Food Science and Technology, 2016, 32(2): 60-65.
61	RUTSCHMANN C, BAUMANN S, CABALZAR J, et al. Recombinant expression of hydroxylated human collagen in Escherichia coli [J]. Applied Microbiology and Biotechnology, 2014, 98(10): 4445-4455.
62	LIU S, LI Y M, WANG M, et al. Efficient coexpression of recombinant human fusion collagen with prolyl 4-hydroxylase from Bacillus anthracis in Escherichia coli [J]. Biotechnology and Applied Biochemistry, 2023, 70(2): 761-772.
63	VUORELA A. Assembly of human prolyl 4-hydroxylase and type Ⅲ collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase[J]. The EMBO Journal, 1997, 16(22): 6702-6712.
64	高力虎. 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 repetitive human-like collagen expression vector and its secretory expression in Pichia pastoris [D]. Nanjing: Nanjing University of Science and Technology, 2007.
65	WANG L N, FAN D D, HE J, et al. A new strategy for secretory expression and mixed fermentation of recombinant human collagen α1 (Ⅲ) chain in Pichia pastoris [J]. Biotechnology and Bioprocess Engineering, 2014, 19(5): 916-924.
66	李伟娜, 尚子方, 段志广, 等. 毕赤酵母高密度发酵产Ⅲ型类人胶原蛋白及其胃粘膜修复功能[J]. 生物工程学报, 2017, 33(4): 672-682.
	LI W N, SHANG Z F, DUAN Z G, et al. Production of gastric-mucosa protective collagen Ⅲ by Pichia pastoris [J]. Chinese Journal of Biotechnology, 2017, 33(4): 672-682.
67	侯增淼, 李晓颖, 李敏, 等. 重组人源性胶原蛋白的制备及表征[J]. 生物工程学报, 2019, 35(2): 319-326.
	HOU Z M, LI X Y, LI M, et al. Preparation and characterization of recombinant human-source collagen[J]. Chinese Journal of Biotechnology, 2019, 35(2): 319-326.
68	RUGGIERO F, EXPOSITO J Y, BOURNAT P, et al. Triple helix assembly and processing of human collagen produced in transgenic tobacco plants[J]. FEBS Letters, 2000, 469(1): 132-136.
69	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(1/2/3): 114-118.
70	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(9): 2640-2645.
71	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): 69.
72	LAMBERG A, HELAAKOSKI T, MYLLYHARJU J, et al. Characterization of human type Ⅲ collagen expressed in a baculovirus system. Production of a protein with a stable triple helix requires coexpression with the two types of recombinant prolyl 4-hydroxylase subunit[J]. Journal of Biological Chemistry, 1996, 271(20): 11988-11995.
73	MYLLYHARJU J, KIVIRIKKO K I. Collagens and collagen-related diseases[J]. Annals of Medicine, 2001, 33(1): 7-21.
74	TOMITA M, MUNETSUNA H, SATO T, et al. Transgenic silkworms produce recombinant human type Ⅲ procollagen in cocoons[J]. Nature Biotechnology, 2003, 21(1): 52-56.
75	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(6): 2189-2198.
76	FRISCHHOLZ S, BEIER F, GIRKONTAITE I, et al. Characterization of human type Ⅹ procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells[J]. Journal of Biological Chemistry, 1998, 273(8): 4547-4555.
77	TOMAN P D, PIEPER F, SAKAI N M, et al. Production of recombinant human type Ⅰ procollagen homotrimer in the mammary gland of transgenic mice[J]. Transgenic Research, 1999, 8(6): 415-427.
78	曾斐鸿. 人胚胎肾细胞HEK293F表达重组胶原蛋白Ⅳ-α1[D]. 天津: 天津科技大学, 2020.
	ZENG F H. Expression of recombinant collagen Ⅳ-α1 in human embryonic kidney 293 cells[D]. Tianjin: Tianjin University of Science & Technology, 2020.
79	YU Z, AN B, RAMSHAW J A M, et al. Bacterial collagen-like proteins that form triple-helical structures[J]. Journal of Structural Biology, 2014, 186(3): 451-461.
80	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]. The Protein Journal, 2017, 36(4): 322-331.
81	GOFFEAU A, BARRELL B G, BUSSEY H, et al. Life with 6000 genes[J]. Science, 1996, 274(5287): 546-567.
82	高云. 真核表达系统的研究进展[J]. 中华男科学杂志, 2002, 8(4): 292-294, 298.
	GAO Y. Advances in eukaryotic expression systems[J]. National Journal of Andrology, 2002, 8(4): 292-294, 298.
83	BUYEL J F. Plant molecular farming - integration and exploitation of side streams to achieve sustainable biomanufacturing[J]. Frontiers in Plant Science, 2019, 9: 1893.
84	CEREGHINO J L, CREGG J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris [J]. FEMS Microbiology Reviews, 2000, 24(1): 45-66.
85	邹奇, 潘炜松, 邱健, 等. 植物生物反应器优化策略与最新应用[J]. 中国生物工程杂志, 2023, 43(1): 71-86.
	ZOU Q, PAN W S, QIU J, et al. Recent advances in optimization strategies and applications of plant bioreactors[J]. China Biotechnology, 2023, 43(1): 71-86.
86	NOKELAINEN M, HELAAKOSKI T, MYLLYHARJU J, et al. Expression and characterization of recombinant human type Ⅱ collagens with low and high contents of hydroxylysine and its glycosylated forms[J]. Matrix Biology, 1998, 16(6): 329-338.
87	IKONOMOU L, SCHNEIDER J Y, AGATHOS S N. Insect cell culture for industrial production of recombinant proteins[J]. Applied Microbiology and Biotechnology, 2003, 62(1): 1-20.
88	WURM F M. Human therapeutic proteins from silkworms[J]. Nature Biotechnology, 2003, 21(1): 34-35.
89	MYLLYHARJU J, LAMBERG A, NOTBOHM H, et al. Expression of wild-type and modified proα chains of human type Ⅰ procollagen in insect cells leads to the formation of stable [α1(Ⅰ)]₂α2(Ⅰ) collagen heterotrimers and[α1(Ⅰ)]₃ homotrimers but not[α2(Ⅰ)]₃ homotrimers[J]. Journal of Biological Chemistry, 1997, 272(35): 21824-21830.
90	WOODLEY D T, KEENE D R, ATHA T, et al. Injection of recombinant human type Ⅶ collagen restores collagen function in dystrophic epidermolysis bullosa[J]. Nature Medicine, 2004, 10(7): 693-695.
91	FICHARD A, TILLET E, DELACOUX F, et al. Human recombinant α1(Ⅴ) collagen chain. Homotrimeric assembly and subsequent processing[J]. Journal of Biological Chemistry, 1997, 272(48): 30083-30087.
92	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(4): 385-389.
93	张智武, 夏伟, 胡张捷. 胶原蛋白产业现状及其发展趋势的研究[J]. 中国食品, 2021(18): 126-128.
	ZHANG Z W, XIA W, HU Z J. Study on the present situation and development trend of collagen industry[J]. China Food, 2021(18): 126-128.
94	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(4): 353.
95	BÁEZ J, OLSEN D, POLAREK J W. Recombinant microbial systems for the production of human collagen and gelatin[J]. Applied Microbiology and Biotechnology, 2005, 69(3): 245-252.
96	HUA C, ZHU Y, XU W, et al. Characterization by high-resolution crystal structure analysis of a triple-helix region of human collagen type Ⅲ with potent cell adhesion activity[J]. Biochemical and Biophysical Research Communications, 2019, 508(4): 1018-1023.
97	QUE R A, ARULMOLI J, SILVA N A DA, et al. Recombinant collagen scaffolds as substrates for human neural stem/progenitor cells[J]. Journal of Biomedical Materials Research Part A, 2018, 106(5): 1363-1372.
98	WOODLEY D T, WANG X Y, AMIR M, et al. Intravenously injected recombinant human type Ⅶ collagen homes to skin wounds and restores skin integrity of dystrophic epidermolysis bullosa[J]. Journal of Investigative Dermatology, 2013, 133(7): 1910-1913.
99	PODREBARAC J. Development of recombinant human collagen type Ⅰ and type Ⅲ injectable hydrogels for cardiac therapy[EB/OL]. 2017[2023-02-01]. .
100	FARKASH U, AVISAR E, VOLK I, et al. First clinical experience with a new injectable recombinant human collagen scaffold combined with autologous platelet-rich plasma for the treatment of lateral epicondylar tendinopathy (tennis elbow)[J]. Journal of Shoulder and Elbow Surgery, 2019, 28(3): 503-509.
101	MIRAZUL ISLAM M, CĖPLA V, HE C L, et al. Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels for regenerative medicine applications[J]. Acta Biomaterialia, 2015, 12: 70-80.
102	ZHAO L L, LI X, ZHAO J Q, et al. A novel smart injectable hydrogel prepared by microbial transglutaminase and human-like collagen: Its characterization and biocompatibility[J]. Materials Science and Engineering: C, 2016, 68: 317-326.
103	PAN H, FAN D D, CAO W, et al. Preparation and characterization of breathable hemostatic hydrogel dressings and determination of their effects on full-thickness defects[J]. Polymers, 2017, 9(12): 727.
104	DONG Y Q, ZHU W D, LEI X X, et al. Treatment of acute wounds with recombinant human-like collagen and recombinant human-like fibronectin in C57BL/6 mice individually or in combination[J]. Frontiers in Bioengineering and Biotechnology, 2022, 10: 908585.
105	MCLAUGHLIN S, MCNEILL B, PODREBARAC J, et al. Injectable human recombinant collagen matrices limit adverse remodeling and improve cardiac function after myocardial infarction[J]. Nature Communications, 2019, 10: 4866.
106	FAGERHOLM P, LAGALI N S, ONG J A, et al. Stable corneal regeneration four years after implantation of a cell-free recombinant human collagen scaffold[J]. Biomaterials, 2014, 35(8): 2420-2427.
107	FAGERHOLM P, LAGALI N S, MERRETT K, et al. A biosynthetic alternative to human donor tissue for inducing corneal regeneration: 24-month follow-up of a phase 1 clinical study[J]. Science Translational Medicine, 2010, 2(46): 46ra61.
108	SONG X, ZHU C H, FAN D D, et al. A novel human-like collagen hydrogel scaffold with porous structure and sponge-like properties[J]. Polymers, 2017, 9(12): 638.
109	CHEN Z Y, ZHANG Z, MA X X, et al. Newly designed human-like collagen to maximize sensitive release of BMP-2 for remarkable repairing of bone defects[J]. Biomolecules, 2019, 9(9): 450.
110	FAN H, HUI J F, DUAN Z G, et al. Novel scaffolds fabricated using oleuropein for bone tissue engineering[J]. BioMed Research International, 2014, 2014: 652432.

类型	组成	分布	功能
Ⅰ	α1[Ⅰ]₂α2[Ⅰ]	真皮、骨骼、肌腱、韧带	其变异会造成骨质疏松、牙齿异常、蓝色巩膜、皮肤薄、肌腱无力、听力丧失
Ⅱ	α1[Ⅱ]₃	软骨、玻璃体	与骨形态形成蛋白-2和转化因子P结合，有利于软骨的发育
Ⅲ	α1[Ⅲ]₃	皮肤、血管、肠	其突变会造成埃勒-丹洛斯综合征
Ⅳ	α1[Ⅳ]₂α2[Ⅳ] α3[Ⅳ]α4[Ⅳ] α5[Ⅳ]α5[Ⅳ]₂α6[Ⅳ]	基底膜	作为细胞和组织的支撑结构，细胞选择型过滤器，抑制血管生成与肿瘤生长
Ⅴ	α1[Ⅴ]₃ α1[Ⅴ]₂α2[Ⅴ] α1[Ⅴ]α2[Ⅴ]α3[Ⅴ]	骨、真皮、角膜、胎盘	神经发育与再生，其突变会造成埃勒-丹洛斯综合征
Ⅵ	α1[Ⅵ]α2[Ⅵ] α3[Ⅵ] α1[Ⅵ]α2[Ⅵ] α4[Ⅵ]	骨头、软骨、角膜、真皮层	作为细胞和组织的支撑结构，细胞选择型过滤器，肌功能主要贡献值
Ⅶ	α1[Ⅶ]₂α2[Ⅶ]	真皮、膀胱	其突变会造成大疱性表皮松解症
Ⅷ	α1[Ⅷ]₃ α2[Ⅷ]₃ α1[Ⅷ]₂α2[Ⅷ]	真皮、大脑、心脏、肾脏	作为细胞和组织的支撑结构、细胞选择型过滤器
Ⅸ	α1[Ⅸ]α2[Ⅸ]α3[Ⅸ]	软骨、角膜、玻璃体	参与细胞外基质的完整性和稳定性，调节胶原蛋白形成过程
Ⅹ	α1[Ⅹ]₃	软骨	作为细胞和组织的支撑结构，细胞选择型过滤器，有利于软骨发育
Ⅺ	α1[Ⅺ]α2[Ⅺ]α3[Ⅺ]	软骨、椎间盘	有利于软骨发育
Ⅻ	α1[Ⅻ]₃	真皮、肌腱	参与细胞外基质的完整性和稳定性，调节胶原蛋白形成过程，维持组织的完整性与机械性
ⅩⅢ	α1[ⅩⅢ]₃	内皮细胞、真皮、心脏、眼睛	—
ⅩⅣ	α1[ⅩⅣ]₃	骨、真皮、软骨	参与细胞外基质的完整性和稳定性，调节胶原蛋白形成过程，维持组织的完整性与机械性
ⅩⅤ	α1[ⅩⅤ]₃	毛细血管、肾脏、睾丸、心脏	抑制血管生成与肿瘤生长
ⅩⅥ	α1[ⅩⅥ]₃	真皮、肾	参与细胞外基质的完整性和稳定性，调节胶原蛋白形成过程
ⅩⅦ	α1[ⅩⅦ]₃	上皮细胞中半脂小体	其突变会造成大疱性表皮松解症
ⅩⅧ	α1[ⅩⅧ]₃	基底膜、肝脏	抑制血管生成与肿瘤生长，信息分子受体，维护肾脏形态
ⅩⅨ	α1[ⅩⅨ]₃	基底膜	调节胶原蛋白形成过程
ⅩⅩ	α1[ⅩⅩ]₃	角膜（小鸡）	参与细胞外基质的完整性和稳定性，调节胶原蛋白形成过程
ⅩⅪ	α1[ⅩⅪ]₃	胃、肾	—
ⅩⅫ	α1[ⅩⅫ]₃	组织连接	软骨中结构和功能分离的基质聚集体，与软骨纤维外基质结合
ⅩⅩⅢ	α1[ⅩⅩⅢ]₃	心、视网膜	增殖区组织所必需，细胞外基质关键的结构作用
ⅩⅩⅣ	α1[ⅩⅩⅣ]₃	骨头、角膜	—
ⅩⅩⅤ	α1[ⅩⅩⅤ]₃	大脑、心脏、睾丸	—
ⅩⅩⅥ	α1[ⅩⅩⅥ]₃	睾丸、卵巢	—
ⅩⅩⅦ	α1[ⅩⅩⅦ]₃	软骨	—
ⅩⅩⅧ	α1[ⅩⅩⅧ]₃	真皮、坐骨神经	—

类型	组成	分布	功能
Ⅰ	α1[Ⅰ]₂α2[Ⅰ]	真皮、骨骼、肌腱、韧带	其变异会造成骨质疏松、牙齿异常、蓝色巩膜、皮肤薄、肌腱无力、听力丧失
Ⅱ	α1[Ⅱ]₃	软骨、玻璃体	与骨形态形成蛋白-2和转化因子P结合，有利于软骨的发育
Ⅲ	α1[Ⅲ]₃	皮肤、血管、肠	其突变会造成埃勒-丹洛斯综合征
Ⅳ	α1[Ⅳ]₂α2[Ⅳ] α3[Ⅳ]α4[Ⅳ] α5[Ⅳ]α5[Ⅳ]₂α6[Ⅳ]	基底膜	作为细胞和组织的支撑结构，细胞选择型过滤器，抑制血管生成与肿瘤生长
Ⅴ	α1[Ⅴ]₃ α1[Ⅴ]₂α2[Ⅴ] α1[Ⅴ]α2[Ⅴ]α3[Ⅴ]	骨、真皮、角膜、胎盘	神经发育与再生，其突变会造成埃勒-丹洛斯综合征
Ⅵ	α1[Ⅵ]α2[Ⅵ] α3[Ⅵ] α1[Ⅵ]α2[Ⅵ] α4[Ⅵ]	骨头、软骨、角膜、真皮层	作为细胞和组织的支撑结构，细胞选择型过滤器，肌功能主要贡献值
Ⅶ	α1[Ⅶ]₂α2[Ⅶ]	真皮、膀胱	其突变会造成大疱性表皮松解症
Ⅷ	α1[Ⅷ]₃ α2[Ⅷ]₃ α1[Ⅷ]₂α2[Ⅷ]	真皮、大脑、心脏、肾脏	作为细胞和组织的支撑结构、细胞选择型过滤器
Ⅸ	α1[Ⅸ]α2[Ⅸ]α3[Ⅸ]	软骨、角膜、玻璃体	参与细胞外基质的完整性和稳定性，调节胶原蛋白形成过程
Ⅹ	α1[Ⅹ]₃	软骨	作为细胞和组织的支撑结构，细胞选择型过滤器，有利于软骨发育
Ⅺ	α1[Ⅺ]α2[Ⅺ]α3[Ⅺ]	软骨、椎间盘	有利于软骨发育
Ⅻ	α1[Ⅻ]₃	真皮、肌腱	参与细胞外基质的完整性和稳定性，调节胶原蛋白形成过程，维持组织的完整性与机械性
ⅩⅢ	α1[ⅩⅢ]₃	内皮细胞、真皮、心脏、眼睛	—
ⅩⅣ	α1[ⅩⅣ]₃	骨、真皮、软骨	参与细胞外基质的完整性和稳定性，调节胶原蛋白形成过程，维持组织的完整性与机械性
ⅩⅤ	α1[ⅩⅤ]₃	毛细血管、肾脏、睾丸、心脏	抑制血管生成与肿瘤生长
ⅩⅥ	α1[ⅩⅥ]₃	真皮、肾	参与细胞外基质的完整性和稳定性，调节胶原蛋白形成过程
ⅩⅦ	α1[ⅩⅦ]₃	上皮细胞中半脂小体	其突变会造成大疱性表皮松解症
ⅩⅧ	α1[ⅩⅧ]₃	基底膜、肝脏	抑制血管生成与肿瘤生长，信息分子受体，维护肾脏形态
ⅩⅨ	α1[ⅩⅨ]₃	基底膜	调节胶原蛋白形成过程
ⅩⅩ	α1[ⅩⅩ]₃	角膜（小鸡）	参与细胞外基质的完整性和稳定性，调节胶原蛋白形成过程
ⅩⅪ	α1[ⅩⅪ]₃	胃、肾	—
ⅩⅫ	α1[ⅩⅫ]₃	组织连接	软骨中结构和功能分离的基质聚集体，与软骨纤维外基质结合
ⅩⅩⅢ	α1[ⅩⅩⅢ]₃	心、视网膜	增殖区组织所必需，细胞外基质关键的结构作用
ⅩⅩⅣ	α1[ⅩⅩⅣ]₃	骨头、角膜	—
ⅩⅩⅤ	α1[ⅩⅩⅤ]₃	大脑、心脏、睾丸	—
ⅩⅩⅥ	α1[ⅩⅩⅥ]₃	睾丸、卵巢	—
ⅩⅩⅦ	α1[ⅩⅩⅦ]₃	软骨	—
ⅩⅩⅧ	α1[ⅩⅩⅧ]₃	真皮、坐骨神经	—

重组胶原蛋白表达体系	表达量	类型	三螺旋结构	来源
大肠杆菌	100～200 mg/L	胶原蛋白聚合物	无	Yin et al.^[58]
大肠杆菌	13.2 g/L	重组类人Ⅱ型胶原蛋白	无	常海燕^[59]
大肠杆菌	500 mg/L	重组类人Ⅰ型胶原蛋白	无	杨晶等^[60]
大肠杆菌	—	重组人Ⅲ型胶原蛋白	有	Rutschman et al.^[61]
大肠杆菌	0.8 g/L	融合胶原蛋白	无	Liu et al.^[62]
酵母	2.15 g/kg	重组人Ⅲ型胶原蛋白	有	Vuorela et al.^[63]
酵母	60.1 mg/L	重组类人Ⅲ型胶原蛋白	无	高力虎 ^[64]
酵母	1.27 g/L	重组人胶原Ⅲ型胶原蛋白	有	Wang et al.^[65]
酵母	4.7 g/L	重组类人Ⅲ型胶原蛋白	无	李伟娜等^[66]
酵母	4.5 g/L	重组人Ⅰ型胶原蛋白	有	侯增淼等^[67]
植物	100 mg/kg	重组人Ⅰ型胶原蛋白	有	Ruggiero et al.^[68]
植物	50~100 mg/kg	重组人Ⅰ型胶原蛋白	有	Merle et al.^[69]
植物	200 mg/kg	重组人Ⅰ型胶原蛋白	有	CollPlant公司^[70]
植物	羟基化：4 mg/kg；未羟基化：12 mg/kg	重组人Ⅰ型胶原蛋白	有	Xu et al.^[71]
昆虫杆状病毒	前胶原蛋白：60 mg/L 胶原蛋白：40 mg/L	重组人Ⅲ型胶原蛋白	有	Lamberg et al.^[72]
昆虫杆状病毒	10～20 mg/L	重组人Ⅰ型胶原蛋白	有	Myllyharju et al.^[73]
昆虫杆状病毒	70 mg融合蛋白质	前胶原蛋白Ⅲ型	无	Tomita et al.^[74]
昆虫杆状病毒	1 mg/幼虫	重组人Ⅱ型胶原蛋白	有	Qi et al.^[75]
哺乳动物细胞	50 g/L	重组人Ⅹ型胶原蛋白	有	Frischholz et al.^[76]
哺乳动物细胞	8 g/L	重组人胶原蛋Ⅰ型	有	Toman et al.^[77]
哺乳动物细胞	6.28 mg/L	重组Ⅳ-α胶原蛋白	无	曾斐鸿 ^[78]

重组胶原蛋白表达体系	表达量	类型	三螺旋结构	来源
大肠杆菌	100～200 mg/L	胶原蛋白聚合物	无	Yin et al.^[58]
大肠杆菌	13.2 g/L	重组类人Ⅱ型胶原蛋白	无	常海燕^[59]
大肠杆菌	500 mg/L	重组类人Ⅰ型胶原蛋白	无	杨晶等^[60]
大肠杆菌	—	重组人Ⅲ型胶原蛋白	有	Rutschman et al.^[61]
大肠杆菌	0.8 g/L	融合胶原蛋白	无	Liu et al.^[62]
酵母	2.15 g/kg	重组人Ⅲ型胶原蛋白	有	Vuorela et al.^[63]
酵母	60.1 mg/L	重组类人Ⅲ型胶原蛋白	无	高力虎 ^[64]
酵母	1.27 g/L	重组人胶原Ⅲ型胶原蛋白	有	Wang et al.^[65]
酵母	4.7 g/L	重组类人Ⅲ型胶原蛋白	无	李伟娜等^[66]
酵母	4.5 g/L	重组人Ⅰ型胶原蛋白	有	侯增淼等^[67]
植物	100 mg/kg	重组人Ⅰ型胶原蛋白	有	Ruggiero et al.^[68]
植物	50~100 mg/kg	重组人Ⅰ型胶原蛋白	有	Merle et al.^[69]
植物	200 mg/kg	重组人Ⅰ型胶原蛋白	有	CollPlant公司^[70]
植物	羟基化：4 mg/kg；未羟基化：12 mg/kg	重组人Ⅰ型胶原蛋白	有	Xu et al.^[71]
昆虫杆状病毒	前胶原蛋白：60 mg/L 胶原蛋白：40 mg/L	重组人Ⅲ型胶原蛋白	有	Lamberg et al.^[72]
昆虫杆状病毒	10～20 mg/L	重组人Ⅰ型胶原蛋白	有	Myllyharju et al.^[73]
昆虫杆状病毒	70 mg融合蛋白质	前胶原蛋白Ⅲ型	无	Tomita et al.^[74]
昆虫杆状病毒	1 mg/幼虫	重组人Ⅱ型胶原蛋白	有	Qi et al.^[75]
哺乳动物细胞	50 g/L	重组人Ⅹ型胶原蛋白	有	Frischholz et al.^[76]
哺乳动物细胞	8 g/L	重组人胶原蛋Ⅰ型	有	Toman et al.^[77]
哺乳动物细胞	6.28 mg/L	重组Ⅳ-α胶原蛋白	无	曾斐鸿 ^[78]

重组胶原蛋白表达体系	成本	优点	缺点
植物表达体系	较低	可规模化，生产周期短，成本低，安全性高	产量较低，产能不足
大肠杆菌表达体系	较低	遗传背景清晰，发酵成本较低，生产周期短，效率高	产生胶原蛋白缺乏羟基化
酵母表达体系	较低	较高的安全性，发酵成本较低，产量高	多为同源性胶原蛋白，生产异源性胶原蛋白较为困难
昆虫杆状病毒表达体系	较高	背景干扰低，进行翻译后处理能力更强	周期较长、产量较低
哺乳动物细胞表达体系	较高	表达稳定，产量稳定	表达周期长，成本高，有病毒感染风险

重组胶原蛋白表达体系研究进展

Research progress on recombinant collagen expression system

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