微生物学报

Type	Distribution	Subunit composition	Function
I	Skin, bones, tendons, cornea, organs, and blood vessels	α1[I]₂α2[I]	Mutations can lead to osteoporosis, tooth deformities, bluish sclera, thinning skin, weak tendons, and hearing loss. It binds to bone morphogenetic protein-2 and transforming growth factor P, promoting cartilage development
II	Cartilage	α1[II]₃	Binds to bone morphogenetic protein-2 and transforming factor P, which promotes the development of cartilage
III	Reticular fibers, blood vessels, skin, uterus, and intestines	α1[III]₃	Mutations in this gene cause Ehler-Danlos syndrome
IV	Basement membrane and capillaries	α1[IV]₂α2[IV] α3[IV]α4[IV] α5[IV]α5[IV]₂α6[IV]	Support structure of cells and tissues, inhibit angiogenesis and tumor growth
V	Cells, bones, skin, placenta, cornea, and hair	α1[V]₃, α1[V]₂α2[V], α1[V]α2[V]α3[V]	Neural development and regeneration, mutations in which cause Ehler-Danlos syndrome
VI	Skin, bones, blood vessels, cartilage, and cornea	α1[VI]α2[VI]α3[VI], α1[VI]α2[VI]α4[VI]	Support structure of cells and tissues, muscle function
VII	Mucous membranes, bladder, skin, amniotic fluid, and umbilical cord	α1[VII]₂α2[VII]	Mutations in which cause epidermolysis bullosa
VIII	Heart, skin, kidneys, brain, bones, blood vessels, and cartilage	α1[VIII]₃, α2[VIII]₃, α1[VIII]₂α2[VIII]	Serves as a support structure for cells and tissues
IX	Cornea, cartilage	α1[IX]α2[IX]α3[IX]	Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen
X	Cartilage	α1[X]₃	Acts as a support structure for cells and tissues, promoting cartilage development
XI	Cartilage and intervertebral disc	α1[XI]α2[XI]α3[XI]	Promote cartilage development
XII	Cartilage, skin, tendons	α1[XII]₃	Maintain the integrity and stability of the extracellular matrix and tissues, and regulate the formation of collagen
XIII	Skeletal muscle, eyes, heart, endothelial cells, and skin	α1[XIII]₃	Transmembrane collagen associated with neuromuscular junction development
XIV	Blood vessels, nerves, eyes, bones, tendons, cartilage, and skin	α1[XIV]₃	Maintain the integrity and stability of the extracellular matrix and tissues, and regulate the formation of collagen
XV	Capillaries, heart, ovaries, skin, testicles, kidneys, and placenta	α1[XV]₃	Inhibits angiogenesis and tumor growth
XVI	Skin, heart, smooth muscle, and kidneys	α1[XVI]₃	Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen
XVII	Skin	α1[XVII]₃	Mutations in this gene cause epidermolysis bullosa, inhibit angiogenesis and tumor growth, signaling molecule receptors, and maintain kidney morphology
XVIII	Kidneys, liver, and lungs	α1[XVIII]₃	Mutations in this gene cause epidermolysis bullosa, inhibit angiogenesis and tumor growth, signaling molecule receptors, and maintain kidney morphology
XIX	Skin, liver, kidneys, spleen, placenta, and prostate	α1[XIX]₃	Regulates the collagen formation process
XX	Corneal epithelium	α1[XX]₃	Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen
XXI	Stomach, heart, kidneys, placenta, skeletal muscle, and blood vessels	α1[XXI]₃	Extracellular matrix component of the blood vessel wall, secreted by smooth muscle cells
XXII	Organizational connections	α1[XXII]₃	Structurally and functionally independent aggregates of cartilage matrix that are integrated with the extracellular matrix of cartilage fibers
XXIII	Metastatic carcinoma cells	α1[XXIII]₃	Essential for tissue proliferation, key structure of the extracellular matrix
XXIV	Bones and cornea	α1[XXIV]₃	Participates in the formation of bones, bone mineralization and regulation of bone homeostasis
XXV	Eyes, heart, brain, and testicles	α1[XXV]₃	Plays a role in neuromuscular development and cancer metastasis and has been implicated in Alzheimer’s disease
XXVI	Testicles and ovaries	α1[XXVI]₃	Associated with thyroid cancer
XXVII	Cartilage, dermis, cornea, retina, and heart arteries	α1[XXVII]₃	Involved in notochord morphogenesis, vertebral mineralization, and post-embryonic axial growth
XXVIII	Renal tubular epithelial cells	α1[XXVIII]₃	Associated with kidney disease
XXIX	Skin, lungs, stomach, and intestines	α1[XXIX]₃	Plays an important role in epidermal integrity and function

Type	Distribution	Subunit composition	Function
I	Skin, bones, tendons, cornea, organs, and blood vessels	α1[I]₂α2[I]	Mutations can lead to osteoporosis, tooth deformities, bluish sclera, thinning skin, weak tendons, and hearing loss. It binds to bone morphogenetic protein-2 and transforming growth factor P, promoting cartilage development
II	Cartilage	α1[II]₃	Binds to bone morphogenetic protein-2 and transforming factor P, which promotes the development of cartilage
III	Reticular fibers, blood vessels, skin, uterus, and intestines	α1[III]₃	Mutations in this gene cause Ehler-Danlos syndrome
IV	Basement membrane and capillaries	α1[IV]₂α2[IV] α3[IV]α4[IV] α5[IV]α5[IV]₂α6[IV]	Support structure of cells and tissues, inhibit angiogenesis and tumor growth
V	Cells, bones, skin, placenta, cornea, and hair	α1[V]₃, α1[V]₂α2[V], α1[V]α2[V]α3[V]	Neural development and regeneration, mutations in which cause Ehler-Danlos syndrome
VI	Skin, bones, blood vessels, cartilage, and cornea	α1[VI]α2[VI]α3[VI], α1[VI]α2[VI]α4[VI]	Support structure of cells and tissues, muscle function
VII	Mucous membranes, bladder, skin, amniotic fluid, and umbilical cord	α1[VII]₂α2[VII]	Mutations in which cause epidermolysis bullosa
VIII	Heart, skin, kidneys, brain, bones, blood vessels, and cartilage	α1[VIII]₃, α2[VIII]₃, α1[VIII]₂α2[VIII]	Serves as a support structure for cells and tissues
IX	Cornea, cartilage	α1[IX]α2[IX]α3[IX]	Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen
X	Cartilage	α1[X]₃	Acts as a support structure for cells and tissues, promoting cartilage development
XI	Cartilage and intervertebral disc	α1[XI]α2[XI]α3[XI]	Promote cartilage development
XII	Cartilage, skin, tendons	α1[XII]₃	Maintain the integrity and stability of the extracellular matrix and tissues, and regulate the formation of collagen
XIII	Skeletal muscle, eyes, heart, endothelial cells, and skin	α1[XIII]₃	Transmembrane collagen associated with neuromuscular junction development
XIV	Blood vessels, nerves, eyes, bones, tendons, cartilage, and skin	α1[XIV]₃	Maintain the integrity and stability of the extracellular matrix and tissues, and regulate the formation of collagen
XV	Capillaries, heart, ovaries, skin, testicles, kidneys, and placenta	α1[XV]₃	Inhibits angiogenesis and tumor growth
XVI	Skin, heart, smooth muscle, and kidneys	α1[XVI]₃	Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen
XVII	Skin	α1[XVII]₃	Mutations in this gene cause epidermolysis bullosa, inhibit angiogenesis and tumor growth, signaling molecule receptors, and maintain kidney morphology
XVIII	Kidneys, liver, and lungs	α1[XVIII]₃	Mutations in this gene cause epidermolysis bullosa, inhibit angiogenesis and tumor growth, signaling molecule receptors, and maintain kidney morphology
XIX	Skin, liver, kidneys, spleen, placenta, and prostate	α1[XIX]₃	Regulates the collagen formation process
XX	Corneal epithelium	α1[XX]₃	Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen
XXI	Stomach, heart, kidneys, placenta, skeletal muscle, and blood vessels	α1[XXI]₃	Extracellular matrix component of the blood vessel wall, secreted by smooth muscle cells
XXII	Organizational connections	α1[XXII]₃	Structurally and functionally independent aggregates of cartilage matrix that are integrated with the extracellular matrix of cartilage fibers
XXIII	Metastatic carcinoma cells	α1[XXIII]₃	Essential for tissue proliferation, key structure of the extracellular matrix
XXIV	Bones and cornea	α1[XXIV]₃	Participates in the formation of bones, bone mineralization and regulation of bone homeostasis
XXV	Eyes, heart, brain, and testicles	α1[XXV]₃	Plays a role in neuromuscular development and cancer metastasis and has been implicated in Alzheimer’s disease
XXVI	Testicles and ovaries	α1[XXVI]₃	Associated with thyroid cancer
XXVII	Cartilage, dermis, cornea, retina, and heart arteries	α1[XXVII]₃	Involved in notochord morphogenesis, vertebral mineralization, and post-embryonic axial growth
XXVIII	Renal tubular epithelial cells	α1[XXVIII]₃	Associated with kidney disease
XXIX	Skin, lungs, stomach, and intestines	α1[XXIX]₃	Plays an important role in epidermal integrity and function

表达系统 Expression system	表达重组胶原蛋白类型 Recombinant collagen types	重组胶原蛋白表达水平 Recombinant collagen expression level (g/L)	参考文献 References
细菌(大肠杆菌) Bacteria (E. coli)	羟基化胶原蛋白Hydroxylated collagen	0.80	[90]
人类I型胶原蛋白Human type I collagen	0.50	[91]
人类I型胶原蛋白Human type I collagen	1.43	[92]
人类I型胶原蛋白Human type I collagen	1.88	[93]
真核细胞(酵母) Eukaryotic cells (yeast)	I、II、III型胶原蛋白Type I, II, III collagen	0.20-0.60	[94]
I型胶原蛋白Type I collagen	0.50	[95]
重组类人胶原蛋白 Recombinant human collagen	4.50	[96]
III型胶原蛋白Type III collagen	0.70	[97]
	人类III型胶原蛋白Human type III collagen	8.00	[98]
	III型胶原蛋白Type III collagen	0.20	[99]
植物(烟草) Plant (Tobacco)	I型胶原蛋白Type I collagen	20.00	[100]
昆虫Insect	II型胶原蛋白Type II collagen	0.05	[101]

表达系统 Expression system	表达重组胶原蛋白类型 Recombinant collagen types	重组胶原蛋白表达水平 Recombinant collagen expression level (g/L)	参考文献 References
细菌(大肠杆菌) Bacteria (E. coli)	羟基化胶原蛋白Hydroxylated collagen	0.80	[90]
人类I型胶原蛋白Human type I collagen	0.50	[91]
人类I型胶原蛋白Human type I collagen	1.43	[92]
人类I型胶原蛋白Human type I collagen	1.88	[93]
真核细胞(酵母) Eukaryotic cells (yeast)	I、II、III型胶原蛋白Type I, II, III collagen	0.20-0.60	[94]
I型胶原蛋白Type I collagen	0.50	[95]
重组类人胶原蛋白 Recombinant human collagen	4.50	[96]
III型胶原蛋白Type III collagen	0.70	[97]
	人类III型胶原蛋白Human type III collagen	8.00	[98]
	III型胶原蛋白Type III collagen	0.20	[99]
植物(烟草) Plant (Tobacco)	I型胶原蛋白Type I collagen	20.00	[100]
昆虫Insect	II型胶原蛋白Type II collagen	0.05	[101]

Technology	Principle	Advantage	Shortcoming	Application
Ultrafiltration	Use a semi-permeable membrane to filter collagen based on molecular weight, separating it from impurities	Simple operation, low cost, and efficient removal of macromolecular impurities	Less efficient at separating small molecules or dissolved salts; membranes require regular replacement	Protein purification, macromolecular impurity removal, and collagen concentration
Chromatography	Separate collagen based on properties such as molecular size, hydrophilicity, or charge	Good separation effect; enables targeted separation of different collagen types	Complex, time-consuming process with high equipment costs	Different types of collagen separation and purification
Magnetic adsorption	Collagen is adsorbed onto magnetic particles and separated using a magnetic field	Fast, reusable, with high collagen adsorption efficiency	Magnetic particles may impact collagen, requiring careful control during operation	Separation and purification of collagen, loaded drug delivery system, etc.

Technology	Principle	Advantage	Shortcoming	Application
Ultrafiltration	Use a semi-permeable membrane to filter collagen based on molecular weight, separating it from impurities	Simple operation, low cost, and efficient removal of macromolecular impurities	Less efficient at separating small molecules or dissolved salts; membranes require regular replacement	Protein purification, macromolecular impurity removal, and collagen concentration
Chromatography	Separate collagen based on properties such as molecular size, hydrophilicity, or charge	Good separation effect; enables targeted separation of different collagen types	Complex, time-consuming process with high equipment costs	Different types of collagen separation and purification
Magnetic adsorption	Collagen is adsorbed onto magnetic particles and separated using a magnetic field	Fast, reusable, with high collagen adsorption efficiency	Magnetic particles may impact collagen, requiring careful control during operation	Separation and purification of collagen, loaded drug delivery system, etc.

重组胶原蛋白的生物合成研究进展

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夏煌慧 , 黄建忠

微生物学报 | 综述 2025,65(5): 1939-1957

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微生物学报 | 综述 2025, 65(5): 1939-1957

重组胶原蛋白的生物合成研究进展

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夏煌慧, 黄建忠

作者信息

福建师范大学生命科学学院，工业微生物发酵技术国家地方联合工程研究中心，工业微生物教育部工程中心，福建福州

Research progress in the biosynthesis of recombinant collagen

Huanghui XIA, Jianzhong HUANG

Affiliations

Engineering Research Center of Industrial Microbiology, Ministry of Education, National and Local United Engineering Research Center of Industrial Microbiology and Fermentation Technology, College of Life Science, Fujian Normal University, Fuzhou, Fujian, China

出版时间: 2025-05-04 doi: 10.13343/j.cnki.wsxb.20240758

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摘要

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胶原蛋白是哺乳动物体内最丰富的蛋白质，约占人体蛋白质的1/3，是结缔组织和细胞外基质的重要成分，对维持生理功能和损伤修复至关重要，在医药、食品和美容领域也有着广泛应用。胶原蛋白的生产方法主要有天然提取法、化学合成法和生物合成法。天然提取法通常从动物结缔组织中获取，但存在伦理问题、质量不稳定以及传染病风险；化学合成法成本高且难以合成复杂的胶原蛋白结构；生物合成法则通过基因工程技术，根据不同用途生产重组胶原蛋白，能够提供更可控、更安全、更精确的生产方式。然而，由于胶原蛋白结构复杂，其生物合成依赖于特定的分子伴侣和修饰酶，因此重组胶原蛋白的生产仍具挑战性。此外，不同类型的胶原蛋白需要形成特定的组织结构，如原纤维、网状或跨膜结构，这进一步增加了生产的难度。本综述旨在阐明重组人源性胶原蛋白的多功能性，分析其生物合成研究的最新进展和面临的挑战，并展望未来的发展方向。希望借此帮助科研人员、工程师和行业从业者更好地理解重组胶原蛋白的研究趋势，推动其在不同应用领域的进一步开发和商业化。

关键词

胶原蛋白 / 重组胶原蛋白 / 功能 / 生物合成

Abstract

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Collagen is the most abundant protein in mammals, accounting for about one-third of human protein. As an important component of the connective tissue and extracellular matrix, collagen is essential for maintaining physiological functions and repairing injuries and has important applications in the fields of medicine, food, and beauty. The main methods for producing collagen are natural extraction, chemical synthesis, and biosynthesis. Natural extraction from animal connective tissue has ethical issues, unstable quality, and infectious disease risks. Chemical synthesis is costly and it is not easy to synthesize complex collagen structures. Biosynthesis enables the production of recombinant collagen for different purposes by genetic engineering in a more controllable, safer, and more precise manner. However, due to the complex structure of collagen, its biosynthesis depends on specific molecular chaperones and modifying enzymes, and thus the production of recombinant collagen is challenging. In addition, different types of collagen need to form particular tissue structures, such as fibril, reticular, or transmembrane structures, which further increases the difficulty of production. This article clarifies the multifunctionality of recombinant human collagen, reviews the latest progress and challenges in its biosynthesis, and looks forward to future development directions. This review aims to help researchers, engineers, and industry practitioners understand the research trends of recombinant collagen and promote its further development and commercialization in different application fields.

Key words

collagen / recombinant collagen / function / biosynthesis

引用本文

夏煌慧, 黄建忠. 重组胶原蛋白的生物合成研究进展. 微生物学报, 2025 , 65 (5) : 1939 -1957 . DOI: 10.13343/j.cnki.wsxb.20240758

Huanghui XIA, Jianzhong HUANG. Research progress in the biosynthesis of recombinant collagen[J]. Acta Microbiologica Sinica, 2025 , 65 (5) : 1939 -1957 . DOI: 10.13343/j.cnki.wsxb.20240758

正文

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胶原蛋白是哺乳动物中含量最多的蛋白质，占人体蛋白质总量的1/3，是结缔组织中最主要的结构蛋白^[1]。作为细胞外基质(extracellular matrix, ECM)的主要成分，胶原蛋白在维持细胞、组织和器官的正常生理功能及损伤修复中发挥着重要作用^[2]。其结构极为复杂，每条胶原肽链由Gly-X-Y (X、Y为除Gly外的任意氨基酸残基)三联体重复构成，通常X为脯氨酸，Y为羟脯氨酸或羟赖氨酸^[3]。这2种不常见的氨基酸分别参与肽链间氢键的形成^[4]，以及糖基化修饰的构建^[5]。每个胶原分子由3条左旋α-肽链组成，形成右旋三股螺旋结构。

动物源胶原蛋白主要来源于陆生动物及海洋动物^[6]。鉴于其在生物体中的重要功能，成熟胶原蛋白在工业和医学领域具有广泛的应用价值。然而，传统的胶原蛋白生产方法(如动物提取法)存在生产周期长、成本高等问题。因此，近年来，重组胶原蛋白的生产成为了研究的焦点。重组胶原蛋白是通过基因工程技术在大肠杆菌(Escherichia coli)、酵母、哺乳动物细胞或昆虫细胞等宿主细胞中合成的，不依赖动物组织，从而降低了病原体传播的风险，且生产过程更加灵活、可控，成本逐渐降低^[7-10]。重组胶原蛋白在功能上与天然胶原蛋白相近，它在组织工程、再生医学和药物递送等领域有着广泛的应用，近年来，重组胶原蛋白的研究在基因重组、蛋白质表达及材料制备等领域取得了显著的进展^[11]。此外，通过结构优化可以设计出具有特定功能的胶原蛋白，以满足医学和生物材料等领域对高质量胶原蛋白的需求，这具有重要意义。本文将首先介绍胶原蛋白的分类、分布及其结构特征，接着探讨其功能特性和应用领域，最后重点分析现有的人源性胶原蛋白生物合成表达体系，并对重组胶原蛋白的产业前景进行展望。

1 胶原蛋白的结构、类别及分布

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胶原蛋白是一种纤维状蛋白质，它构成了人体的结缔组织，主要存在于皮肤、关节和骨骼中^[12]。由于胶原蛋白在生物结构中起着连接作用，因此它是许多生物体中最丰富的分子之一^[2]。

1.1 胶原蛋白结构

胶原蛋白的三螺旋构象首次在20世纪50年代通过皮肤中发现的胶原纤维的X射线衍射图被描述^[13]，其氨基酸序列表现出2个独特特征：(1) 甘氨酸是每3个残基中的1个形成重复的(Gly-X-Y)_n模式；(2) 大部分残基(约20%)是亚氨基酸脯氨酸和羟脯氨酸，由于这种特殊的结构基序，胶原蛋白三螺旋中发生了几种分子相互作用，从而形成了独特的沿中心轴紧密包装^[14]。胶原蛋白α链的大小各不相同，例如，人类α1(X)链和α3(VI)链分别由662-3 152个氨基酸组成^[15-16]。成熟胶原蛋白的生物合成需要进行翻译后修饰以增加胶原蛋白三螺旋结构的稳定性^[17]。

1.2 胶原蛋白家族

胶原蛋白家族是一组不同的细胞外基质分子，它们通过胶原蛋白三螺旋结构作为共同的结构元素而联系在一起^[18]。如表1所示，目前已知人体内存在29种不同的胶原蛋白亚型^[19]。胶原蛋白的命名标准尚未明确定义，在脊椎动物中以罗马数字编号(I-XXVIII)，它们分布在不同的组织结构中，发挥不同的作用^[30]。一种新型表皮胶原蛋白被称为胶原蛋白XXIX^[29]，但COL29A1基因已被证明与COL6A5基因相同，且α1(XXIX)链对应于α5(VI)链^[31]。胶原蛋白家族内部还存在更多的多样性。同一类型胶原蛋白存在多个分子亚型，例如，IV型胶原蛋白和VI型胶原蛋白各自都有不同的分子亚型^[32]。此外，还有一些胶原蛋白分子是由2种不同类型的胶原蛋白的α链组成的，这些分子被称为“混合亚型”，例如，V型和XI型胶原蛋白就属于这种混合亚型^[33]。2种替代启动子的使用产生了不同形式的α1(IX)链和α(XVIII)链，而替代剪接则导致α1(II)、α2(VI)、α3(VI)、α1(VII)、α1(XII)、α1(XIII)、α1(XIV)、α1(XIX)、α1(XXV)和α1(XXVIII)链的多个同种型的存在，而且胶原蛋白可以根据其超分子结构组装进一步细分为多个亚家族^[19]。

1.3 胶原蛋白分布

胶原蛋白原纤维由胶原蛋白II、XI和IX或胶原蛋白II和III (软骨)^[34]、胶原蛋白I和III (皮肤)以及胶原蛋白I和V组成^[35]。在哺乳动物组织中，胶原蛋白含量最高的是肌腱(80%)，其次是皮肤(70%)、骨骼(25%)和主动脉(20%)^[18,36]。

胶原纤维的形成已在肌腱中得到广泛研究，但纤维形成初始步骤的位点尚未明确定义^[37]。在肌腱发育过程中，胶原纤维形成发生在一系列细胞外区室中，纤维中间体在其中组装，成熟纤维通过中间体的沉积后融合过程生长，而未成熟的纤维中间体被纳入纤维后，会发生线性和横向纤维生长^[38]。此外，胶原纤维形成也可能发生在细胞内，前胶原蛋白加工和胶原纤维形成始于高尔基体到质膜的载体(Golgi to plasma membrane carriers, GPC)，这些载体及其直径为28 nm的纤维被定位到之前未鉴定的质膜(previously unidentified plasma membrane, PM)突起，这些突起与肌腱轴平行并突出到细胞之间的平行通道中^[39]。如今，这种生物分子可以通过从植物和动物等天然来源中提取，或通过重组蛋白质生产系统(包括酵母、细菌、哺乳动物细胞、昆虫或植物)获得，也可以通过模仿胶原蛋白特征的人工纤维来获得^[40]。

胶原蛋白是参与人体创伤修复的主要蛋白质，因其高生物相容性、可生物降解性以及无毒性和免疫原性而成为有价值的生物材料^[41]，参与调节多种组织的形成，如肌腱、韧带、皮肤和角膜等^[42]。生物纤维胶原蛋白是脊椎动物中最常见的胶原蛋白，主要通过影响组织的分子结构、形状和机械性能(如皮肤的抗拉强度和韧带的牵引力)来发挥其结构功能，尽管数量较少，但对组织的完整性至关重要^[43]。例如，胶原蛋白IX在成人关节软骨中的比例仅为1%^[44]，而胶原蛋白VII对皮肤的完整性极为重要，但其

含量仅约为0.001%^[45]。在纤维化过程中，过量的胶原蛋白会在细胞外基质中沉积，抑制纤维形成的一个新策略是通过阻断端肽介导的胶原蛋白分子相互作用^[46]。胶原蛋白的角色已超出传统的三螺旋和结构支架，它们不仅是“美丽的纤维”，而且胶原蛋白与细胞的相互作用在调节细胞生长、分化和迁移中发挥了重要作用^[47]。

特定的胶原蛋白类型在特定组织中具有独特的生物功能。例如，胶原蛋白VII作为锚定纤维的成分，参与真皮与表皮的黏附^[48]。胶原蛋白X在肥大性软骨中发挥作用，有助于软骨内骨化并建立造血生态位^[49]。胶原蛋白XXII则存在于组织连接处，如骨骼肌与心肌的肌腱交接处^[50]。分析显示，COL22A1与评估肾功能的重要生物标志物血清肌酐水平存在关联^[51]。胶原蛋白XXIV标志着成骨细胞的分化和骨形成^[52]。胶原蛋白XXVII主要存在于成年软骨中，可能在软骨钙化和骨骼形成中起作用^[53]。此外，α5(VI)链在特应性皮炎患者的外表皮中不表达，提示其在维持表皮的完整性和功能中可能起到重要作用^[30]。COL6A5与COL29A1在基因层面与过敏症相关的研究也得到了证实^[54]。

膜胶原蛋白如胶原蛋白XIII可能在骨骼生长和发育过程中对形态和结构具有调整作用，特别是在骨量的调节与骨骼承受机械负荷之间起到桥梁作用，确保骨骼能够根据使用需求保持健康和适应^[55]。胶原蛋白XVII是半桥粒的主要成分^[56]，而胶原蛋白XXIII则与前列腺癌的复发和转移相关^[57]。此外，膜胶原蛋白XIII、XVII和XXV在神经元或相关结构中表达，而胶原蛋白XXVIII主要存在于神经组织中^[58]。胶原蛋白在脊椎动物神经系统发育中也发挥着新的作用，胶原蛋白IV在神经肌肉接头处作为突触前组织者^[59-60]。胶原蛋白XIX由中枢神经元表达，并对海马突触的形成至关重要^[61]。多种胶原蛋白(IV、VI、XVIII和XXV)在阿尔茨海默病(Alzheimer’s disease, AD)患者大脑中的沉积与淀粉样蛋白β肽结合^[62]。此外，遗传证据显示COL25A1基因与阿尔茨海默病风险相关^[63]，而胶原蛋白VI可能在保护神经元免受β-淀粉样蛋白(amyloid-beta)毒性方面发挥作用^[64]。

2 重组胶原蛋白的功能及应用领域

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重组胶原蛋白是通过基因工程技术，将编码胶原蛋白的基因导入适当的宿主细胞中表达和纯化得到的蛋白质。由于其具有与天然胶原蛋白相似的结构和功能，重组胶原蛋白广泛应用于生物医药、组织工程、皮肤护理和伤口修复等领域(图1)。在过去10年中，胶原蛋白生物材料领域出现了许多创新，生产和交联方法不断发展和改进。重组胶原蛋白现在广泛应用于研究和医疗领域，它保留了天然胶原蛋白的多项功能，并且展现出优异的生物相容性、可控的生物活性、低免疫原性、无毒性、可调节的降解性、优化的力学性能以及多样化的加工性能^[65]。

2.1 医药与医疗

随着研究的不断深入，胶原蛋白在再生医学、组织工程以及生物医用材料中的应用前景非常广泛，涵盖了组织修复、药物递送以及蛋白质替代疗法等多个领域^[66-67]。通过采用不同的制备技术，重组胶原蛋白及其片段可以被加工成多种三维结构材料，如多孔海绵、纤维丝和薄膜等，这些材料能够有效促进细胞的附着和生长，从而更好地支持相关的生物医学应用^[68]。皮肤伤口愈合，尤其是对于大面积病变，始终是临床治疗中的一个重大挑战，为了加速愈合过程并提高治疗效果，胶原蛋白在伤口修复领域的应用越来越受到关注。胶原蛋白的天然三聚体结构使其具备良好的凝聚能力，能够促进血小板聚集，从而在创伤部位形成有效的止血和保护屏障^[69]。胶原蛋白作为止血剂或止血海绵，广泛应用于外科手术中，能够显著减少出血并促进伤口愈合^[70]。在皮肤伤口修复的研究中，Wang等^[71]开发了一种创新的多孔支架，该支架结合了胶原蛋白、透明质酸和明胶，旨在改善皮肤伤口的愈合过程；该支架的多孔结构为细胞生长和血管生成提供了理想的微环境，促进了创伤部位的快速愈合。此外，透明质酸和明胶的添加进一步提高了支架的生物相容性和保湿效果，为皮肤再生提供了更加全面的支持^[72]。胶原蛋白不仅在皮肤创伤修复中具有重要作用，Solovieva等^[73]使用冷冻干燥法制造了大量复合海藻酸钠(sodium alginate, AG)-纤维蛋白原(fibrinogen, FG)海绵支架，适用于皮肤生物工程和移植疗法的新型促血管生成材料。在其他医疗领域，如骨科和组织工程中，重组胶原蛋白的应用也日益增多。作为生物可降解材料，胶原蛋白在骨修复和关节治疗中被用作支架材料，帮助促进骨组织再生^[74]。在这些领域，胶原蛋白不仅能够支持细胞生长，还能加速损伤区域的自我修复，从而显著改善患者的恢复速度和治疗效果。

2.2 美容与美发

胶原蛋白具有显著的保湿、美白、抗衰老、防皱及淡斑等多重功效^[75]。胶原蛋白含有丰富的极性基团，使其具备天然的保湿功能^[76-77]。此外，胶原蛋白中还含有酪氨酸残基，这些残基能够有效抑制酪氨酸酶催化皮肤中的酪氨酸转化为多巴，阻止皮肤中黑色素的形成^[78]。除了上述优势，胶原蛋白还具备抗氧化特性，能够帮助清除体内自由基，减缓皮肤衰老过程^[79]。它能够促进皮肤细胞的新陈代谢，增强皮肤的自我修复能力，从而延缓衰老，保持皮肤的弹性和光滑度，使肌肤更加柔嫩细致^[80]。由于其优异的生物活性，胶原蛋白被广泛应用于各种美容、护肤以及美发产品中，成为提升皮肤健康、改善外观、延缓衰老的重要成分。

2.3 食品与保健

随着健康意识的提升和肥胖问题的日益严重，消费者对低脂肪食品的需求不断增加，同时对食品的感官品质要求也日益提高。由于胶原蛋白与多糖的相互作用能够改善胶原蛋白的凝胶特性、乳化特性、起泡性及其稳定性等功能特性，使其广泛应用于保持或改善食品的结构、质地、风味和口感等品质^[81]。重组胶原蛋白作为脂肪替代品，尤其在加工肉制品中具有显著的潜力。Ham等^[82]的研究表明，将胶原蛋白与膳食纤维结合使用，能够有效替代香肠中的猪肉脂肪，在发酵香肠的冷藏过程中，使用该复合物并未显著改变香肠的感官特性，如颜色、风味和口感等(P>0.05)，这表明胶原蛋白复合物不仅可以减少脂肪含量，还能够保持产品的整体可接受性，成为猪肉背脂的理想替代品。胶原蛋白与多糖复合物所形成的纳米颗粒、水凝胶、薄膜及肠衣等材料，能够有效保护活性成分(如香料和生物活性物质)，以及肉类产品和新鲜蔬菜水果等，免受热、剪切力、温度变化、光照、氧气和水分等外部环境因素的负面影响^[83]。Krkić等^[84]研究了胶原蛋白-壳聚糖复合膜在香肠肠衣中的应用，结果表明，与单一的胶原蛋白膜相比，胶原蛋白-壳聚糖复合膜在阻氧性能方面表现出显著的改善。因此，这种复合膜在作为香肠肠衣时，能够更有效地延长产品的保鲜期，并且具有更好的保护作用。

3 重组胶原蛋白生物合成

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重组胶原蛋白凭借其安全性、可控性和生产的灵活性，正逐渐成为天然胶原蛋白的有力替代品，尤其在现代生物技术和医疗应用中展现出巨大的前景^[85]。多年前，一项开创性的实验成功实现了重组人胶原蛋白的表达，从而推动了对其作为动物源胶原蛋白替代品潜在应用的研究^[86]。如今，用于医学和研究的蛋白质不再必须从天然来源(例如动物组织、植物、细菌和海洋生物等)中分离出，科学研究者的关注逐渐从动物源性胶原蛋白转向重组胶原蛋白的表达^[87-88]。目前，重组胶原蛋白的表达体系主要包括原核生物(如大肠杆菌)、酵母、植物、杆状病毒和哺乳动物细胞表达体系^[89]，其中最常被使用的微生物表达系统是大肠杆菌和毕赤酵母。不同胶原蛋白重组表达系统中报道的重组胶原蛋白产量如表2所示。本节内容将阐述胶原蛋白的生物合成机制，对比大肠杆菌和酵母表达体系的优缺点，以及分离纯化工艺的优化。

3.1 生物合成机制

胶原蛋白的生物合成过程见图2。整个过程始于其编码基因的转录和翻译，信号序列引导生长中的pre-pro-α链到内质网后，信号肽裂解，生成的新生胶原蛋白α链被称为pro-α链^[87,90]。在内质网中，这些pro-α链经历了一系列的酶促修饰。脯氨酸和赖氨酸残基的羟基化反应分别由脯氨酰3-羟化酶、脯氨酰-4-羟化酶以及赖氨酰羟化酶催化^[102]。这3种酶的催化作用依赖于若干辅助因子，包括亚铁离子、2-酮戊二酸、分子氧和抗坏血酸^[102]。目前，3-羟基脯氨酸的功能尚不清楚^[103]，4-羟基脯氨酸的存在对于分子内氢键至关重要，因此有助于三螺旋结构域的热稳定性，以及单体和胶原原纤维的完整性。

首先，脯氨酸-4-羟化酶(prolyl 4-hydroxylase, P4H)和赖氨酸羟化酶(lysyl hydroxylase, LH)分别对Gly-Xaa-Yaa序列中Yaa位点的脯氨酸和赖氨酸残基进行羟基化；若新生的胶原蛋白α链在折叠过程中发生异常，可能导致不良的三螺旋结构形成，这种错误的结构不仅在细胞内造成应激反应，还可能影响内质网的功能^[104-107]。除了P4H和LH的修饰外，前胶原蛋白α链还需经过脯氨酰-3-羟化酶(prolyl 3-hydroxylase, P3H)进行进一步地修饰^[108]。此外，部分赖氨酸残基还会进一步进行羟基化^[109]。随后C端前肽在膜结合伴侣分子，如凝集素样分子伴侣、钙联蛋白及内质网氧化还原酶(protein disulfide isomerase, PDI)的协同作用下，促进了二硫键的形成，从而推动了胶原蛋白α链的三聚体化，最终组装成三螺旋，形成原胶原蛋白^[110]。在信号肽酶去除信号肽后，原胶原分子经历多个步骤的翻译后修饰，这些修饰对于胶原蛋白的三螺旋结构的稳定性以及其在细胞外的成熟交联至关重要^[86]。在这个过程中，伴侣分子HSP47与三螺旋前胶原蛋白结合，能够防止前胶原蛋白的局部展开和不当聚集^[90]。经过HSP47的修饰后，形成的原胶原蛋白被内质网出口蛋白TANGO1识别并包装在高尔基体内^[111]。在高尔基体内，HSP47从前胶原蛋白中解离，进入分泌囊泡并向细胞外基质转移^[112]。在形成纤维的原胶原蛋白分泌到细胞外空间后，原胶原蛋白的3条pro-α链的N端和C端被前胶原肽酶切割去除前肽，形成成熟的胶原蛋白分子^[113]。

3.2 重组胶原蛋白在大肠杆菌中的表达

大肠杆菌表达体系因其清晰的遗传背景、低廉的发酵成本、短的生产周期和高效率，已被广泛用于大规模生产外源蛋白。在大肠杆菌(Escherichia coli)等原核表达系统中，胶原蛋白的表达面临一系列挑战。首先，原核系统由于缺乏复杂的内质网和高尔基体等细胞器，不能有效地进行蛋白质的翻译后修饰(如糖基化、二硫键的形成等)^[114]。这导致了胶原蛋白等复杂蛋白在大肠杆菌中的表达往往会出现折叠错误，形成不溶性的包涵体，或者即使未形成包涵体，表达的蛋白也常常缺乏功能性，无法发挥预期的生物学作用^[115]。具体来说，胶原蛋白的高分子量和特有的三级结构要求其正确折叠以维持其功能，而原核系统中的折叠机制和修饰过程通常无法满足这一需求^[93]。然而，Yu等^[116]研究表明，大肠杆菌表达重组胶原蛋白时通常缺乏羟基化，却仍能产生相对稳定的三螺旋胶原蛋白结构。为解决这一问题，一种可行的策略是引入p4h基因及相关胶原蛋白基因来促进胶原蛋白的羟基化过程，从而提高其结构稳定性。Liu等^[117]的研究证实炭疽芽孢杆菌(Bacillus anthracis)的脯氨酰-4-羟化酶(P4H)的羟基化率最高(63.6%)，并对其在5 L发酵罐中进行分批补料发酵，获得了产量为0.8 g/L的羟基化胶原蛋白。刘斌^[91]的研究在大肠杆菌中高效表达了重组类人胶原蛋白，平均得率达到500 mg/L。

不可否认的是，缺乏适当的翻译后修饰(如二硫键的正确配对)和氨基酸序列的精确加工，也会导致胶原蛋白在大肠杆菌中失去生物活性，无法发挥其在医药、食品或美容领域的功能。因此，为了提高重组胶原蛋白在大肠杆菌中的表达质量和功能性，研究者们努力开发更先进的大肠杆菌表达系统，这些系统能够完成蛋白酶的加工、二硫键的正确形成，并具备更完善的翻译后修饰能力，类似于真核细胞的修饰机制，由此提高胶原蛋白的表达产量、纯度，并确保其结构和功能的完整性。此外，溶解氧(dissolved oxygen, DO)是大肠杆菌高密度发酵过程中的一个关键因素，若溶氧水平不适当，会对菌体的代谢过程产生负面影响，从而抑制目标产物的积累^[118]。在重组胶原蛋白的发酵生产中，有通气量、搅拌速度、温度和压力4个主要因素会影响溶氧浓度。其中，通气量被认为是对溶氧水平影响最为显著的因素^[119]。

常海燕等^[120]通过调整搅拌转速及空气流量，将发酵罐的DO分别控制为10%、20%、30%和40%，实验结果表明，当DO为20%时，细菌密度和类人胶原蛋白II浓度最高，分别达到83.9 g/L和13.4 g/L，且代谢副产物乙酸浓度较低。范代娣等^[121]在其研究中对人类已知序列的胶原蛋白基因进行特定的重复与修饰，并将其重组至大肠杆菌中，他们对重组胶原蛋白的水溶性、免疫排异性、稳定性、成胶性及吸收性等方面进行了优化改进；通过自动调节溶氧水平，并将搅拌转速保持在600-1 400 r/min范围内，确定了最佳的培养条件，其中基质中葡萄糖浓度为1%时有利于细胞的生长，通过温度诱导的方式，成功实现了胶原蛋白的高效、稳定表达，最终获得的表达量达到了总蛋白的29.4%。滕飞等^[92]基于I型人源化胶原蛋白hCOL1A1设计了一种重组表达载体，并将其转化到大肠杆菌BL21(DE3)中进行表达，其中将小分子泛素样修饰蛋白(small ubiquitin-like modifier, SUMO)和6×His标签融合到hCOL1A1的N末端；实验确定了25 ℃培养温度和0.4 mmol/L异丙基-β-d-硫代半乳糖苷(isopropyl β-d-1-thiogalactopyranoside, IPTG)浓度为最佳诱导条件，在5 L发酵罐中获得了1.43 g/L的蛋白产量。Xie等^[93]设计了一个功能性人I型胶原蛋白片段rhLCOLI，并在大肠杆菌BL21(DE3) PlysS中使用热诱导质粒pBV-rhLCOL-I进行表达，研究结果显示，在42 ℃条件下，rhLCOL-I的表达量可达到总蛋白的36.3%，且以可溶性形式存在；在7 L发酵培养中，纯化的rhLCOLI产量为1.88 g/L。

3.3 重组胶原蛋白在酵母中的表达

酵母作为真核生物，具备对分泌的重组蛋白进行多种后期修饰的能力^[122]。与动物细胞表达系统相比，酵母表达的重组蛋白不含病原微生物、病毒包涵体或热源，从而在生产过程中具有更高的安全性，且其发酵成本通常较低^[123]。目前，已经开发了多种以酵母为宿主的表达系统，如毕赤酵母(Pichia pastoris)、汉逊酵母和啤酒酵母等^[124]。在这些系统中，毕赤酵母因其广泛的应用优势而成为最常用的酵母种类。与其他酵母相比，毕赤酵母作为甲基营养型酵母，能够在重组胶原蛋白的翻译和后期加工中展现出更多的优势特征，例如有效形成二硫键、进行糖基化以及促使蛋白水解过程的高效进行^[125]。这些特性使得毕赤酵母在生产重组蛋白方面具有显著的优势，有助于提升最终产品的功能性和稳定性。

将人类I型、II型和III型胶原蛋白的编码基因导入到携带脯氨酸羟化酶基因的毕赤酵母工程菌中，所表达的重组胶原蛋白能够实现充分的脯氨酸羟基化；此外，通过持续供氧，最终实现了产量在0.2-0.6 g/L之间的水平^[94-95]。侯增淼等^[96]通过设计编码亲水性Gly-X-Y胶原肽段的核苷酸序列，基于人类I型胶原蛋白的Gly-X-Y重复序列，构建了毕赤酵母工程菌，该系统成功表达了类人胶原蛋白，并实现了4.5 g/L的表达量。Fang等^[97]成功建立了人类III型胶原蛋白的真核表达系统，并成功在毕赤酵母GS115中分泌表达分子量高达96.3 kDa的外源蛋白rhCOL3A1，蛋白初始产量约为0.7 mg/mL。由于存在强诱导性醇氧化酶1 (aldehyde oxidase 1, AOX1)启动子，600多种重组蛋白在P. pastoris中可实现高效表达，产量达到每升培养物毫克至克的范围内。毕赤酵母表达重组蛋白的关键之一是加入甲醇诱导氧化酶启动子^[126]。梁鑫等^[98]在15 L发酵罐中进行毕赤酵母发酵重组人III型胶原蛋白的生产实验，在甲醇诱导阶段，他们通过控制甲醇的流加量，设定0.05%、0.10%、0.20%和0.40%等4种不同的甲醇浓度，经过多次发酵实验的比较分析，发现当甲醇浓度保持在0.20%时，胶原蛋白的表达量显著提高，从3 g/L增加到8 g/L。石艺平^[99]的研究利用优化设计的III型胶原蛋白和脯氨酸羟化酶基因，在毕赤酵母中实现了共表达，探索了胶原蛋白的生产工艺及性质，并且通过摇瓶发酵条件和发酵罐高密度发酵条件优化，提高至0.2 g/L蛋白表达量。

3.4 重组胶原蛋白在其他系统中的表达

在重组胶原蛋白的生产中，除了常规的细胞培养系统外，研究人员还探索了多种其他表达系统，并取得了一定的进展。Stein等^[100]提出了一种简单、安全且可扩展的方法，通过转基因烟草植物生产和纯化I型胶原蛋白，获得了高达20 g/L的产量，该胶原蛋白具有与人类组织来源胶原蛋白相似的生物功能，为人类重组胶原蛋白在再生医学中的应用提供了新的可能性。Nokelainen等^[101]使用改良的昆虫表达载体，在悬浮培养系统中成功获得了高达50 mg/L的II型胶原蛋白表达量。哺乳动物细胞表达系统因其成熟的转录和后转录修饰机制，成为重组胶原蛋白表达的理想选择。然而，由于该系统的表达产量较为有限，因此针对通过哺乳动物细胞表达的重组胶原蛋白性质的研究相对较少。Wang等^[127]成功利用仓鼠卵巢细胞表达全长人类I型胶原蛋白α1链。

4 重组胶原蛋白分离纯化工艺

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研究胶原蛋白的关键是获得高纯度、生物学活性且稳定的目标蛋白，这样才能进行性质研究并推动其大规模生产^[128]。由于胶原蛋白在组织或细胞中通常与其他蛋白和细胞器混合存在，并且不同细胞类型中的蛋白质在结构和功能上存在差异，导致重组胶原蛋白的分离和纯化变得复杂。因此，高效的蛋白纯化技术是胶原蛋白研究的基础。

蛋白质下游分离和纯化工艺的理论基础主要依赖于蛋白质的理化特性，这些特性包括溶解度、密度、电离行为、亲水性、分子配位吸附特性、分子大小与形状以及其生物学功能等。其中，胶原蛋白制备常用的技术包括超滤、层析和磁性吸附(表3)。

Saallah等^[129]采用胃蛋白酶溶解法从海参(Holothuria scabra)中提取胶原蛋白，然后用透析和超滤膜分离，结果表明超滤法的胶原蛋白产量(11.39%)高于透析法(5.15%)。层析是生物技术行业蛋白质纯化的主要方式，重组胶原蛋白纯化常采用的层析方式主要包括亲和层析、离子层析和凝胶过滤层析。刘斌^[91]的研究结果表明，一步Sephadex G100凝胶过滤层析是针对重组人源胶原蛋白的最佳纯化方法，通过该方法得到的重组胶原蛋白纯度为95.5%，回收率为93.6%。采用磁珠吸附方法纯化大肠杆菌发酵产物中的重组胶原蛋白，得到的重组蛋白在稳定性和生物活性方面优于天然人源III型胶原蛋白^[130]。

5 总结与展望

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胶原蛋白是人体和动物体内最丰富的结构蛋白，广泛应用于生物医学、材料科学等领域。尽管在胶原蛋白的重组表达技术上取得了显著进展，但大规模生产稳定且具有天然特性的胶原蛋白仍面临多项技术难题。

胶原蛋白分子的独特三螺旋结构和稳定性依赖于一些天然酶的作用，特别是脯氨酸-4-羟化酶(P4H)和脯氨酰-3-羟化酶(P3H)。然而，大规模生产胶原蛋白时，最初的一个重大挑战是缺乏这些天然酶，导致重组胶原蛋白无法正确折叠或形成所需的三螺旋结构。为了克服这一问题，科学家尝试将胶原蛋白编码基因与P4H亚基编码基因共表达，取得了一定的成功。这一方法能够促进胶原蛋白分子正确折叠，进而形成热稳定的三螺旋结构^[10,131]。尽管P4H的共表达在一定程度上解决了三螺旋结构的稳定性问题，但仍有必要考虑其他关键酶的作用，特别是脯氨酰-3-羟化酶(P3H)，该酶对于胶原蛋白功能的完整性至关重要。进一步研究表明，若没有这些辅助酶的作用，重组胶原蛋白的性能可能会与天然胶原蛋白存在差异，从而影响其在生物医学和工业应用中的表现。另一个需要解决的技术瓶颈是胶原蛋白的糖基化。胶原蛋白在天然状态下常伴有复杂的糖基化修饰，尤其是羟赖氨酸残基是其糖基化的主要位点。为了提高重组胶原蛋白与天然胶原蛋白的相似性，未来的表达系统需要能够进行糖基化处理。赖氨酰羟化酶的共表达可能是解决这一问题的关键。通过合理设计表达系统，可以实现重组胶原蛋白的糖基化，进而提升其生物活性和临床应用潜力。传统上，胶原蛋白的重组表达多依赖于哺乳动物细胞系统。然而，细菌衍生的胶原蛋白片段(如短的胶原样三螺旋肽)也成为一个研究热点。尽管细菌胶原蛋白缺乏羟脯氨酸残基，但它仍能在高温下保持稳定性^[132]。这些细菌来源的胶原蛋白具有较好的生物相容性，且在大规模生产上具有潜力。然而，如何确保其安全性、有效性及与细胞的相互作用仍然是需要进一步研究的课题^[133]。

尽管胶原蛋白重组表达技术面临诸多挑战，但随着基因工程、合成生物学、细胞培养技术和生物打印技术的不断发展，未来在这一领域的研究仍有广阔的前景^{[117,134-135]}。为了进一步提升胶原蛋白的重组表达效率和质量，酶工程和优化表达系统将成为研究的重点。通过改造现有的酶或者开发新的酶，可能会有效解决胶原蛋白分子折叠不完全和糖基化不充分的问题。例如，进一步研究如何在表达系统中共同引入P4H、P3H等多种关键酶，可能会使重组胶原蛋白更加接近天然状态，且功能更加完善。尽管细菌衍生的胶原蛋白已经在稳定性和生物相容性方面展示了巨大的潜力，但如何进一步优化其生产工艺、确保其与细胞的良好相互作用以及扩展其在临床应用中的可行性仍是未来研究的关键方向。利用基因工程手段优化细菌胶原蛋白的质量和功能，可能使其成为一种低成本、高产量的生物材料，适用于大规模应用。

糖基化是胶原蛋白的重要特性之一，未来的研究可以关注如何在重组胶原蛋白的生产过程中实现高效、精确的糖基化。通过共表达赖氨酰羟化酶等关键酶，或者利用合成生物学技术设计特定的糖基化途径，可能为重组胶原蛋白的糖基化修饰提供新的思路。此外，基于胶原蛋白的糖基化模式和细胞相互作用机制，还可以开发新的细胞特异性胶原蛋白变体，以满足不同生物医学应用中的需求。在生物医学应用方面，重组胶原蛋白的支架材料和细胞培养仍是关键研究领域。近年来，3D打印技术和电纺丝法的进展使得科学家能够更精确地制造胶原蛋白支架，并能够在其中嵌入细胞以模拟组织的生长和发育。这为未来的组织工程提供了巨大的发展空间。通过将重组胶原蛋白与干细胞、3D生物打印等技术结合，可能为临床上更复杂的组织修复和再生提供新的解决方案^[136-138]。

作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。

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2025年第65卷第5期

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doi: 10.13343/j.cnki.wsxb.20240758

接收时间：2024-11-28
首发时间：2026-02-05
出版时间：2025-05-04

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收稿日期：2024-11-28
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福建师范大学生命科学学院，工业微生物发酵技术国家地方联合工程研究中心，工业微生物教育部工程中心，福建福州

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Type	Distribution	Subunit composition	Function
I	Skin, bones, tendons, cornea, organs, and blood vessels	α1[I]₂α2[I]	Mutations can lead to osteoporosis, tooth deformities, bluish sclera, thinning skin, weak tendons, and hearing loss. It binds to bone morphogenetic protein-2 and transforming growth factor P, promoting cartilage development
II	Cartilage	α1[II]₃	Binds to bone morphogenetic protein-2 and transforming factor P, which promotes the development of cartilage
III	Reticular fibers, blood vessels, skin, uterus, and intestines	α1[III]₃	Mutations in this gene cause Ehler-Danlos syndrome
IV	Basement membrane and capillaries	α1[IV]₂α2[IV] α3[IV]α4[IV] α5[IV]α5[IV]₂α6[IV]	Support structure of cells and tissues, inhibit angiogenesis and tumor growth
V	Cells, bones, skin, placenta, cornea, and hair	α1[V]₃, α1[V]₂α2[V], α1[V]α2[V]α3[V]	Neural development and regeneration, mutations in which cause Ehler-Danlos syndrome
VI	Skin, bones, blood vessels, cartilage, and cornea	α1[VI]α2[VI]α3[VI], α1[VI]α2[VI]α4[VI]	Support structure of cells and tissues, muscle function
VII	Mucous membranes, bladder, skin, amniotic fluid, and umbilical cord	α1[VII]₂α2[VII]	Mutations in which cause epidermolysis bullosa
VIII	Heart, skin, kidneys, brain, bones, blood vessels, and cartilage	α1[VIII]₃, α2[VIII]₃, α1[VIII]₂α2[VIII]	Serves as a support structure for cells and tissues
IX	Cornea, cartilage	α1[IX]α2[IX]α3[IX]	Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen
X	Cartilage	α1[X]₃	Acts as a support structure for cells and tissues, promoting cartilage development
XI	Cartilage and intervertebral disc	α1[XI]α2[XI]α3[XI]	Promote cartilage development
XII	Cartilage, skin, tendons	α1[XII]₃	Maintain the integrity and stability of the extracellular matrix and tissues, and regulate the formation of collagen
XIII	Skeletal muscle, eyes, heart, endothelial cells, and skin	α1[XIII]₃	Transmembrane collagen associated with neuromuscular junction development
XIV	Blood vessels, nerves, eyes, bones, tendons, cartilage, and skin	α1[XIV]₃	Maintain the integrity and stability of the extracellular matrix and tissues, and regulate the formation of collagen
XV	Capillaries, heart, ovaries, skin, testicles, kidneys, and placenta	α1[XV]₃	Inhibits angiogenesis and tumor growth
XVI	Skin, heart, smooth muscle, and kidneys	α1[XVI]₃	Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen
XVII	Skin	α1[XVII]₃	Mutations in this gene cause epidermolysis bullosa, inhibit angiogenesis and tumor growth, signaling molecule receptors, and maintain kidney morphology
XVIII	Kidneys, liver, and lungs	α1[XVIII]₃	Mutations in this gene cause epidermolysis bullosa, inhibit angiogenesis and tumor growth, signaling molecule receptors, and maintain kidney morphology
XIX	Skin, liver, kidneys, spleen, placenta, and prostate	α1[XIX]₃	Regulates the collagen formation process
XX	Corneal epithelium	α1[XX]₃	Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen
XXI	Stomach, heart, kidneys, placenta, skeletal muscle, and blood vessels	α1[XXI]₃	Extracellular matrix component of the blood vessel wall, secreted by smooth muscle cells
XXII	Organizational connections	α1[XXII]₃	Structurally and functionally independent aggregates of cartilage matrix that are integrated with the extracellular matrix of cartilage fibers
XXIII	Metastatic carcinoma cells	α1[XXIII]₃	Essential for tissue proliferation, key structure of the extracellular matrix
XXIV	Bones and cornea	α1[XXIV]₃	Participates in the formation of bones, bone mineralization and regulation of bone homeostasis
XXV	Eyes, heart, brain, and testicles	α1[XXV]₃	Plays a role in neuromuscular development and cancer metastasis and has been implicated in Alzheimer’s disease
XXVI	Testicles and ovaries	α1[XXVI]₃	Associated with thyroid cancer
XXVII	Cartilage, dermis, cornea, retina, and heart arteries	α1[XXVII]₃	Involved in notochord morphogenesis, vertebral mineralization, and post-embryonic axial growth
XXVIII	Renal tubular epithelial cells	α1[XXVIII]₃	Associated with kidney disease
XXIX	Skin, lungs, stomach, and intestines	α1[XXIX]₃	Plays an important role in epidermal integrity and function

Type

Distribution

Subunit composition

Function

Skin, bones, tendons, cornea, organs, and blood vessels

α1[I]₂α2[I]

Mutations can lead to osteoporosis, tooth deformities, bluish sclera, thinning skin, weak tendons, and hearing loss. It binds to bone morphogenetic protein-2 and transforming growth factor P, promoting cartilage development

Cartilage

α1[II]₃

Binds to bone morphogenetic protein-2 and transforming factor P, which promotes the development of cartilage

III

Reticular fibers, blood vessels, skin, uterus, and intestines

α1[III]₃

Mutations in this gene cause Ehler-Danlos syndrome

Basement membrane and capillaries

α1[IV]₂α2[IV] α3[IV]α4[IV] α5[IV]α5[IV]₂α6[IV]

Support structure of cells and tissues, inhibit angiogenesis and tumor growth

Cells, bones, skin, placenta, cornea, and hair

α1[V]₃, α1[V]₂α2[V], α1[V]α2[V]α3[V]

Neural development and regeneration, mutations in which cause Ehler-Danlos syndrome

Skin, bones, blood vessels, cartilage, and cornea

α1[VI]α2[VI]α3[VI], α1[VI]α2[VI]α4[VI]

Support structure of cells and tissues, muscle function

VII

Mucous membranes, bladder, skin, amniotic fluid, and umbilical cord

α1[VII]₂α2[VII]

Mutations in which cause epidermolysis bullosa

VIII

Heart, skin, kidneys, brain, bones, blood vessels, and cartilage

α1[VIII]₃, α2[VIII]₃, α1[VIII]₂α2[VIII]

Serves as a support structure for cells and tissues

Cornea, cartilage

α1[IX]α2[IX]α3[IX]

Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen

Cartilage

α1[X]₃

Acts as a support structure for cells and tissues, promoting cartilage development

Cartilage and intervertebral disc

α1[XI]α2[XI]α3[XI]

Promote cartilage development

XII

Cartilage, skin, tendons

α1[XII]₃

Maintain the integrity and stability of the extracellular matrix and tissues, and regulate the formation of collagen

XIII

Skeletal muscle, eyes, heart, endothelial cells, and skin

α1[XIII]₃

Transmembrane collagen associated with neuromuscular junction development

XIV

Blood vessels, nerves, eyes, bones, tendons, cartilage, and skin

α1[XIV]₃

Maintain the integrity and stability of the extracellular matrix and tissues, and regulate the formation of collagen

Capillaries, heart, ovaries, skin, testicles, kidneys, and placenta

α1[XV]₃

Inhibits angiogenesis and tumor growth

XVI

Skin, heart, smooth muscle, and kidneys

α1[XVI]₃

Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen

XVII

Skin

α1[XVII]₃

Mutations in this gene cause epidermolysis bullosa, inhibit angiogenesis and tumor growth, signaling molecule receptors, and maintain kidney morphology

XVIII

Kidneys, liver, and lungs

α1[XVIII]₃

Mutations in this gene cause epidermolysis bullosa, inhibit angiogenesis and tumor growth, signaling molecule receptors, and maintain kidney morphology

XIX

Skin, liver, kidneys, spleen, placenta, and prostate

α1[XIX]₃

Regulates the collagen formation process

Corneal epithelium

α1[XX]₃

Maintain the integrity and stability of the extracellular matrix and regulate the formation process of collagen

XXI

Stomach, heart, kidneys, placenta, skeletal muscle, and blood vessels

α1[XXI]₃

Extracellular matrix component of the blood vessel wall, secreted by smooth muscle cells

XXII

Organizational connections

α1[XXII]₃

Structurally and functionally independent aggregates of cartilage matrix that are integrated with the extracellular matrix of cartilage fibers

XXIII

Metastatic carcinoma cells

α1[XXIII]₃

Essential for tissue proliferation, key structure of the extracellular matrix

XXIV

Bones and cornea

α1[XXIV]₃

Participates in the formation of bones, bone mineralization and regulation of bone homeostasis

XXV

Eyes, heart, brain, and testicles

α1[XXV]₃

Plays a role in neuromuscular development and cancer metastasis and has been implicated in Alzheimer’s disease

XXVI

Testicles and ovaries

α1[XXVI]₃

Associated with thyroid cancer

XXVII

Cartilage, dermis, cornea, retina, and heart arteries

α1[XXVII]₃

Involved in notochord morphogenesis, vertebral mineralization, and post-embryonic axial growth

XXVIII

Renal tubular epithelial cells

α1[XXVIII]₃

Associated with kidney disease

XXIX

Skin, lungs, stomach, and intestines

α1[XXIX]₃

Plays an important role in epidermal integrity and function

表达系统 Expression system	表达重组胶原蛋白类型 Recombinant collagen types	重组胶原蛋白表达水平 Recombinant collagen expression level (g/L)	参考文献 References
细菌(大肠杆菌) Bacteria (E. coli)	羟基化胶原蛋白Hydroxylated collagen	0.80	[90]
人类I型胶原蛋白Human type I collagen	0.50	[91]
人类I型胶原蛋白Human type I collagen	1.43	[92]
人类I型胶原蛋白Human type I collagen	1.88	[93]
真核细胞(酵母) Eukaryotic cells (yeast)	I、II、III型胶原蛋白Type I, II, III collagen	0.20-0.60	[94]
I型胶原蛋白Type I collagen	0.50	[95]
重组类人胶原蛋白 Recombinant human collagen	4.50	[96]
III型胶原蛋白Type III collagen	0.70	[97]
	人类III型胶原蛋白Human type III collagen	8.00	[98]
	III型胶原蛋白Type III collagen	0.20	[99]
植物(烟草) Plant (Tobacco)	I型胶原蛋白Type I collagen	20.00	[100]
昆虫Insect	II型胶原蛋白Type II collagen	0.05	[101]

表达系统

Expression system

表达重组胶原蛋白类型

Recombinant collagen types

重组胶原蛋白表达水平

Recombinant collagen expression level (g/L)

参考文献

References

细菌(大肠杆菌)

Bacteria (E. coli)

羟基化胶原蛋白Hydroxylated collagen

0.80

[90]

人类I型胶原蛋白Human type I collagen

0.50

[91]

人类I型胶原蛋白Human type I collagen

1.43

[92]

人类I型胶原蛋白Human type I collagen

1.88

[93]

真核细胞(酵母)

Eukaryotic cells (yeast)

I、II、III型胶原蛋白Type I, II, III collagen

0.20-0.60

[94]

I型胶原蛋白Type I collagen

0.50

[95]

重组类人胶原蛋白

Recombinant human collagen

4.50

[96]

III型胶原蛋白Type III collagen

0.70

[97]

人类III型胶原蛋白Human type III collagen

8.00

[98]

III型胶原蛋白Type III collagen

0.20

[99]

植物(烟草)

Plant (Tobacco)

I型胶原蛋白Type I collagen

20.00

[100]

昆虫Insect

II型胶原蛋白Type II collagen

0.05

[101]

Technology	Principle	Advantage	Shortcoming	Application
Ultrafiltration	Use a semi-permeable membrane to filter collagen based on molecular weight, separating it from impurities	Simple operation, low cost, and efficient removal of macromolecular impurities	Less efficient at separating small molecules or dissolved salts; membranes require regular replacement	Protein purification, macromolecular impurity removal, and collagen concentration
Chromatography	Separate collagen based on properties such as molecular size, hydrophilicity, or charge	Good separation effect; enables targeted separation of different collagen types	Complex, time-consuming process with high equipment costs	Different types of collagen separation and purification
Magnetic adsorption	Collagen is adsorbed onto magnetic particles and separated using a magnetic field	Fast, reusable, with high collagen adsorption efficiency	Magnetic particles may impact collagen, requiring careful control during operation	Separation and purification of collagen, loaded drug delivery system, etc.

Technology

Principle

Advantage

Shortcoming

Application

Ultrafiltration

Use a semi-permeable membrane to filter collagen based on molecular weight, separating it from impurities

Simple operation, low cost, and efficient removal of macromolecular impurities

Less efficient at separating small molecules or dissolved salts; membranes require regular replacement

Protein purification, macromolecular impurity removal, and collagen concentration

Chromatography

Separate collagen based on properties such as molecular size, hydrophilicity, or charge

Good separation effect; enables targeted separation of different collagen types

Complex, time-consuming process with high equipment costs

Different types of collagen separation and purification

Magnetic adsorption

Collagen is adsorbed onto magnetic particles and separated using a magnetic field

Fast, reusable, with high collagen adsorption efficiency

Magnetic particles may impact collagen, requiring careful control during operation

Separation and purification of collagen, loaded drug delivery system, etc.