WO2023143395A1 - 一种高透皮吸收性的i型重组胶原蛋白及其用途 - Google Patents
一种高透皮吸收性的i型重组胶原蛋白及其用途 Download PDFInfo
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 71
- 108010035532 Collagen Proteins 0.000 title claims abstract description 71
- 229920001436 collagen Polymers 0.000 title claims abstract description 71
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 28
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- 239000007924 injection Substances 0.000 claims abstract description 3
- 239000000463 material Substances 0.000 claims abstract description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 13
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 11
- 102000012422 Collagen Type I Human genes 0.000 claims description 10
- 108010022452 Collagen Type I Proteins 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 7
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 101710135898 Myc proto-oncogene protein Proteins 0.000 claims description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 claims description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 claims description 2
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- 108090000623 proteins and genes Proteins 0.000 description 18
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Definitions
- the invention belongs to the field of biotechnology, and in particular relates to a type I recombinant collagen with high transdermal absorption and its application.
- Collagen is a biological polymer protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. Collagen is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging, etc. It is one of the most critical raw materials in the biotechnology industry. It is used in medical materials, cosmetics , Food industry are widely used. collagen
- Protein also known as collagen, is an important protein component of connective tissue that supports and protects the body. It is also the most abundant structural protein in many vertebrates and invertebrates. It provides mechanical strength to bones, tendons, cartilage and skin. Collagen is one of the most abundant proteins in mammals, accounting for about 20%-30% of the total protein in the body. It mainly exists in skin, bones, tendons, soft tissues, etc. Among them, about 70%-80% of the extracellular matrix of the skin % for collagen. Collagen is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging, etc. It is one of the most critical raw materials in the biotechnology industry. In the field of medical treatment or beauty Has a wide range of applications.
- collagen When collagen is applied to medical dressings, cosmetics, etc., from the perspective of better biological activity, it is hoped that collagen has good transdermal absorption properties.
- collagen is a macromolecular bioactive substance, which itself is not easily absorbed through the skin. Therefore, in previous studies, how to promote the transdermal absorption of collagen has become a research hotspot.
- a mask towel-type collagen dressing can be used, that is, the collagen solution is impregnated in a solid carrier such as non-woven fabric, then sealed in a container, taken out during use, and applied on the face.
- the collagen liquid is impregnated in a liquid-absorbing solid carrier, so that there may be more collagen liquid, and the dryness of the collagen liquid can be delayed, so the contact between the collagen liquid and the skin surface can be prolonged.
- the action time promotes the penetration of collagen molecules with large molecular weight.
- recombinant collagen can be constructed by selecting and repeating short amino acid sequences from natural human collagen.
- the recombinant collagen thus constructed It has advantages such as low immunogenicity, high biological activity, and good stability.
- the transdermal absorption performance of this recombinant collagen may be related to its amino acid sequence, but it has not been clearly confirmed, especially how to design short amino acid sequences as repeating units, so that the constructed recombinant collagen has better For transdermal absorption performance, there is no theory in the prior art that can be used as a guide.
- the inventors conducted in-depth research, and as a result obtained a short amino acid sequence derived from natural human type I collagen and a type I recombinant collagen constructed using the short amino acid sequence as a repeating unit , the type I recombinant collagen has excellent transdermal absorption properties, thereby completing the present invention.
- the present invention includes:
- a polypeptide derived from natural human type I collagen the amino acid sequence of which is shown in SEQ ID No.: 1 (GAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDA).
- a type I recombinant collagen which is composed of a short amino acid sequence from natural human type I collagen as a repeating unit,
- the short amino acid sequence is shown in SEQ ID No.: 1 (GAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDA), and the number of repetitions is more than 3 times.
- the type I recombinant collagen according to item 1 further having a tag for easy purification, said tag being a His tag, a Flag tag or a c-Myc tag.
- Fig. 1 is the SDS-PAGE protein electrophoresis diagram of purified recombinant collagen P-1-P-4.
- Fig. 2 is the SDS-PAGE protein electrophoresis of purified recombinant collagen D-1-D-4.
- the implanted collagen medical devices can promote the adhesion of fibroblasts, adipocytes, dermal cells, etc. Migrate to the implanted device, exhibit cell adhesion, promote cell growth and other characteristics, and achieve rapid repair.
- GER tripeptide is a known tripeptide with adhesion effect, and the collagen containing this tripeptide shows a better effect of promoting adhesion.
- the natural amino acid sequence of type I collagen removes the telopeptide amino acid sequence at both ends, and the remaining 1057 amino acids (162-1219) in the middle are used to screen the fragment containing the GER tripeptide using amino acid analysis software, with 36 amino acids as the basic repeating unit. 18 amino acids were repeated between different short peptides, and a total of 27 peptide fragments were screened out.
- the 27 short peptides were synthesized by chemical synthesis, and 27 pure short peptides were prepared with a purity of more than 95%. The peptide synthesis was entrusted to Sichuan Pukang Pharmaceutical Co., Ltd. and confirmed by mass spectrometry and high performance liquid chromatography.
- the effective diffusion area of the diffusion cell is 2.0cm 2
- the volume of the receiving cell is 14ml
- the customized stirring The length of the stirrer is 1.4cm
- the receiving solution is 0.9% NaCl solution.
- the BCA kit method was used to determine the peptide content in the sample solution.
- polypeptide with the sequence number 17 and the amino acid sequence gapgapgsqgapglqgmpgergaaglpgpkgdrgda (SEQ ID No.: 1) had the largest 24h transdermal amount of 2998.75ug.
- the amino acid sequence numbered 12 with better transdermal absorption performance in Table 1 was repeated 4 times (D-1), 12 times (D-2), 24 times (D-3), 48 times (D-4) , after optimizing the codon preference of Escherichia coli, translate it into the corresponding gene sequence, carry out the whole gene synthesis, connect it into the pET24a expression plasmid, and transfer it into BL21 competent cells by heat shock transformation to become the expression strain (4 kinds in total) .
- Example 2 The 8 kinds of recombinant collagens prepared in Example 2 were used to measure the permeation protein by the method of comparison of transdermal properties in Example 1, and the results are shown in Table 2.
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Abstract
本发明涉及一种高透皮吸收性的I型重组胶原蛋白及其用途。本发明的高透皮吸收性的人I型重组胶原蛋白由来自天然人I型胶原蛋白的短氨基酸序列作为重复单元进行多次重复而构成，所述短氨基酸序列如SEQ ID No.:1所示，重复次数为3次以上。本发明的高透皮吸收性的人I型重组胶原蛋白可用作生产各种胶原蛋白产品的原料，所述胶原蛋白产品例如注射剂、面部填充剂、敷料、化妆品、保健食品、组织工程材料、胶原蛋白海绵等。
Description
本发明属于生物技术领域，具体涉及一种高透皮吸收性的I型重组胶原蛋白及其用途。
胶原蛋白是一种生物高分子蛋白，是动物结缔组织中的主要成分，也是哺乳动物体内含量最多、分布最广的功能性蛋白，占蛋白质总量的25％～30％。胶原蛋白与组织的形成、成熟、细胞间信息的传递以及关节润滑、伤口愈合、钙化作用、血液凝固和衰老等有着密切的关系，是生物科技产业最关键的原材料之一，在医学材料、化妆品、食品工业中均有广泛应用。胶原
蛋白又称胶原，是支持和保护机体的结缔组织的重要蛋白质组分，也是很多脊椎和无脊椎动物体内含量最丰富的结构蛋白质，它使骨、腱、软骨和皮肤具有机械强度。胶原蛋白是哺乳动物体内含量最多的蛋白质之一，约占机体总蛋白的20％-30％，主要存在于皮肤、骨骼、肌腱、软组织等中，其中，皮肤细胞外基质中约70％-80％为胶原蛋白。胶原蛋白与组织的形成、成熟、细胞间信息的传递以及关节润滑、伤口愈合、钙化作用、血液凝固和衰老等有着密切的关系，是生物科技产业最关键的原材料之一，在医疗或美容领域有着广泛应用。
当将胶原蛋白应用于医用敷料、化妆品等用途时，从更好地发挥生物活性的角度考虑，希望胶原蛋白具有良好的透皮吸收性能。然而，胶原蛋白是大分子生物活性物质，其本身不易透皮吸收。因此，在以往的研究中，如何促进胶原蛋白的透皮吸收成为研究热点。例如，为了促进分子量大的胶原蛋白分子透皮吸收，可以利用面膜巾型胶原蛋白敷料，即，将胶原蛋白液含浸在无纺布等固体载体，然后密封于容器中，使用时取出，贴敷于面部。上述面膜巾型胶原蛋白敷料中胶原蛋白液被含浸在具有吸液性的固体载体中，可以存在较多的胶原蛋白液、且可以延缓胶原蛋白液干涸，因此能够延长胶原蛋白液与皮肤表面的作用时间，促进分子量大的胶原蛋白分子的渗透。
近年来随着基因工程技术的广泛应用，研发人员创造出各种类型的重组胶原蛋白，例如可以通过选择来自天然人胶原蛋白的短氨基酸序列进行重复来构建重组胶原蛋白，这样构建的重组胶原蛋白具有例如免疫原性低、生物活性高、稳定性好等优点。理论上，这种重组胶原蛋白的透皮吸收性能可能与其氨基酸序列相关，但并未得到明确证实，特别是对于如何设计作为重复单元的短氨基酸序列，才能使得构建的重组胶原蛋白具有更好的透皮吸收性能，现有技术中没有任何的理论可以作为指导。
发明内容
为了解决现有技术中存在的上述技术问题，发明人进行了深入研究，结果获得了来源于天然人I型胶原蛋白的短氨基酸序列及以该短氨基酸序列作为重复单元构建的I型重组胶原蛋白，所述I型重组胶原蛋白具有优异的透皮吸收性能，从而完成了本发明。
即，本发明包括：
1.来自天然人I型胶原蛋白的多肽，其氨基酸序列如SEQ ID No.:1(GAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDA)所示。
2.一种I型重组胶原蛋白，其由来自天然人I型胶原蛋白的短氨基酸序列作为重复单元进行多次重复而构成，
其中，所述短氨基酸序列如SEQ ID No.:1(GAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDA)所示，重复次数为3次以上。
3.根据项1所述的I型重组胶原蛋白，其中，所述重复次数为5～150次，优选10～100次。
4.根据项1所述的I型重组胶原蛋白，其还带有使其易于纯化的标签，所述标签为His标签、Flag标签或c-Myc标签。
5.根据项1～4中任一项所述的I型重组胶原蛋白在制备胶原蛋白产品中的用途。
6.根据项5所述的用途，其中，所述胶原蛋白产品选自注射剂、面部填充剂、敷料、化妆品、保健食品、组织工程材料、胶原蛋白海绵等，
图1为纯化后的重组胶原蛋白P-1～P-4的SDS-PAGE蛋白电泳图。
图2为纯化后的重组胶原蛋白D-1～D-4的SDS-PAGE蛋白电泳图。
以下将通过具体的实施例对本发明进行详细地描述。需要特别指出的是，这些描述仅仅是示例性的描述，并不构成对本发明范围的限制。
我们首先通过对各种来源于天然人I型胶原蛋白的短氨基酸序列(这样的短氨基酸序列与天然人I型胶原蛋白100％同源，可避免外源物质的免疫原性等问题)进行筛选，获得透皮吸收性能优异的短氨基酸序列。然后，构建以该短氨基酸序列作为重复单元的各种分子量的I型重组胶原蛋白，并验证其透皮吸收性能。从而获得了透皮吸收性能优异的I型重组胶原蛋白。
还需要说明的是，胶原蛋白具有良好的促粘附效果是其能够广泛应用于植入类医疗器械的重要原因，置入的胶原类医疗器械，能够促进成纤维细胞、脂肪细胞、真皮细胞等向置入的器械迁移，表现出细胞粘附，促细胞生长等特性，实现快速的修复作用。GER三肽是已知的具有粘附作用的三肽，含有该三肽的胶原蛋白表现出更好的促粘效果。为了保证获得的透皮吸收性能优异的I型重组胶原蛋白的生理活性，在筛选高透皮吸收性能的短氨基酸序列时，我们优先选择了含有GER三肽的天然短氨基酸序列，再通过该短氨基酸序列重复构建新的I型重组胶原蛋白。
实施例1：高透皮吸收性多肽的获得
1)胶原肽的制备
I型胶原的天然氨基酸序列去除两端的端肽氨基酸序列，剩余的中间1057个氨基酸(162-1219)，利用氨基酸分析软件对含有GER三肽的片段进行筛选，以36个氨基酸作为基本重复单元，不同短肽之间重复18个氨基酸，共筛得多肽片段27个，通过化学合成的方式进行27个短肽的合成，制备27个短肽纯品，纯度大于95％以上。多肽合成委托四川普康药业有限公司完成，并通过质谱和高效液相色谱进行了确认。
2)透皮性能的比较
1.离体鼠皮的制备
实验用昆明小鼠20～22g 10只，雌雄各半，处死后，除去腹部毛发，然后剥离去毛发部位的皮肤，除去脂肪和筋络，用蒸馏水反复冲洗，鼠皮洗净后用生理盐水冲洗，再用10％甘油处理，置于-20℃储藏备用(7天内用完)。
2.实验装置
单室扩散池：扩散池有效扩散面积2.0cm2，接受池体积14ml，定制搅
拌子长度1.4cm，接受液为0.9％的NaCl溶液。
3.样品液制备
取出储藏的鼠皮，解冻，生理盐水洗净，将其夹在接受室与供给室之间，贴药面朝向供给室，皮肤面朝向接受室，调整水浴系统温度为37.5℃，搅拌速度100rpm/min，在接受室中加入预温37℃的0.9％NaCl溶液，排尽气泡，为降低干扰，再不给药的情况下，先将鼠皮内层表面与接受液接触，更换全部接受液，再将合成的27个胶原肽分别用0.9％NaCl配成5mg/ml的溶液，分别注入供给室紧贴于鼠皮皮肤上，24h后用注射器吸取部分接受液作为样品液，以进行胶原蛋白肽透皮量的测定。
4.目标肽的检测
采用BCA试剂盒法测定样品液中的多肽含量。
5.透皮吸收结果
表1，不同胶原肽的24h透过量的比较
结果显示序号为17，氨基酸序列为gapgapgsqgapglqgmpgergaaglpgpkgdrgda(SEQ ID No.:1)的多肽的24h透皮量最大，为2998.75ug。
实施例2利用大肠杆菌表达系统制备各种I型重组胶原蛋白
1)不同重复次数的SEQ ID No.:1的I型重组胶原蛋白的制备
将SEQ ID No.:1的氨基酸序列进行重复，分别重复3次(P-1)、重复10次(P-2)、重复20次(P-3)、重复40次(P-4)，进行大肠杆菌的密码子偏好优化后，翻译成对应的基因序列，进行全基因合成后连接入pET24a表达质粒，通过热击转化的方式转入BL21感受态细胞即为表达菌株(共4种)。
分别挑取上述4种表达菌株单菌落，转接LB液体摇瓶，37℃震荡培养过夜即为种子液，以1％的接种量转接入100ml LB液体培养基中，37℃，
200rpm培养至OD值在2～3左右时，添加终浓度为1.5mM的IPTG，降温至28℃进行诱导培养，诱导14h，离心收集菌体，以pH6.0的PB缓冲液配制10％(菌体湿重/PB体积)的菌悬液，1000bar条件下高压匀浆3min，离心收集上清即为粗蛋白表达液，通过离子交换色谱进行分离纯化，分别收集11.9KD(P-1)、39.6KD(P-2)、79.2KD(P-3)、158.4KD(P-4)的蛋白即为不同重复次数的I型重组胶原蛋白。各种胶原蛋白的纯化后的SDS-PAGE蛋白电泳图见图1，泳道A、B、C、D分别为P-1、P-2、P-3、P-4。
2)相同重复次数的含GER三肽其它I型胶原氨基酸序列对应的重组I型胶原蛋白的表达
将表1中透皮吸收性能较好的编号为12的氨基酸序列分别重复4次(D-1)、12次(D-2)、24次(D-3)、48次(D-4)，进行大肠杆菌的密码子偏好优化后，翻译成对应的基因序列，进行全基因合成后连接入pET24a表达质粒，通过热击转化的方式转入BL21感受态细胞即为表达菌株(共4种)。
分别挑取上述4种表达菌株单菌落，转接LB液体摇瓶，37℃震荡培养过夜即为种子液，以1％的接种量转接入100ml LB液体培养基中，37℃，200rpm培养至OD值在2～3左右时，添加终浓度为1.5mM的IPTG，降温至28℃进行诱导培养，诱导14h，离心收集菌体，以pH6.0的PB缓冲液配制10％(菌体湿重/PB体积)的菌悬液，1000bar条件下高压匀浆3min，离心收集上清即为粗蛋白表达液，通过离子交换色谱进行分离纯化，分别收集12.7KD(D-1)、37.9KD(D-2)、78.28KD(D-3)、164.95KD(D-4)的蛋白即为不同重复次数的重组胶原蛋白。各种重组胶原蛋白的纯化后的SDS-PAGE蛋白电泳图见图2，泳道E、F、G、H分别为D-1、D-2、D-3、D-4。
实施例3各种I型重组胶原蛋白的透皮吸收性能的对比
将实施例2中制备的8种重组胶原蛋白，分别采用实施例1中透皮性能比较的方法进行透过蛋白的测定，结果见表2。
表2不同氨基酸序列的重组胶原蛋白的24h透过量的比较
如表2所示随着分子量的增大，蛋白的透皮吸收效率逐渐降低，然而通过本专利筛选的SEQ ID No.:1重复所形成的重组胶原，在分子量相近，重复次数相近的重组胶原中均能表现出良好的透皮吸收效果。
Claims (6)
- 来自天然人I型胶原蛋白的多肽，其氨基酸序列如SEQ ID No.:1(GAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDA)所示。
- 一种I型重组胶原蛋白，其由来自天然人I型胶原蛋白的短氨基酸序列作为重复单元进行多次重复而构成，其中，所述短氨基酸序列氨基酸序列如SEQ ID No.:1(GAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDA)所示，重复次数为3次以上。
- 根据权利要求1所述的I型重组胶原蛋白，其中，所述重复次数为5～150次，优选10～100次。
- 根据权利要求1所述的I型重组胶原蛋白，其还带有使其易于纯化的标签，所述标签为His标签、Flag标签或c-Myc标签。
- 根据权利要求1～4中任一项所述的I型重组胶原蛋白在制备胶原蛋白产品中的用途。
- 根据权利要求5所述的用途，其中，所述胶原蛋白产品选自胶原蛋白注射剂、面部填充剂、敷料、化妆品、保健食品、组织工程材料和胶原蛋白海绵。
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