Extracellular matrix protein recombinant protein, nucleic acid molecule, recombinant vector, genetically engineered bacterium, preparation method and application

Abstract

The invention provides an extracellular matrix protein recombinant protein, a nucleic acid molecule, a recombinant vector, a genetically engineered bacterium, a preparation method and an application. The provided extracellular matrix protein recombinant protein comprises the following protein fragments: human fibronectin, humanized collagen and elastin-like protein. Wherein the amino acid sequence of the human fibronectin is as shown in SEQ ID NO. 1; the amino acid sequence of the humanized collagen is as shown in SEQ ID NO. 2; the elastin-like protein comprises a pentapeptide repetitive sequence, repetitive units of the pentapeptide repetitive sequence comprise-[valine-proline-glycine-Xaa-glycine] n-, Xaa is isoleucine or lysine, and the number n of the repetitive units is 15-30; in the extracellular matrix protein recombinant protein, any two adjacent ones of the human fibronectin, the humanized collagen and the elastin-like protein are independently connected through a peptide bond or a connecting peptide. The extracellular matrix recombinant fusion protein provided by the invention has good thermal stability and can promote cell adhesion.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and more specifically, to extracellular matrix protein recombinant protein, nucleic acid molecule, recombinant vector, genetic engineering bacteria, and preparation methods and applications thereof. Background Art

[0002] The extracellular matrix (ECM) is a complex network composed of a variety of macromolecular substances, including collagen, elastin, proteoglycans, glycoproteins, etc. It plays an important role in the growth, differentiation, migration of cells and the formation and maintenance of tissues. The components of the extracellular matrix are reorganized and the characteristics of the extracellular matrix components are used to achieve specific biological effects to improve the performance of the extracellular matrix. However, the existing extracellular matrix recombinant fusion proteins have poor thermal stability, are easily degraded at room temperature, and are difficult to preserve. Summary of the invention

[0003] Aiming at the existing problems that the extracellular matrix recombinant fusion protein has poor thermal stability, is easily degraded at room temperature, and is difficult to preserve, a recombinant extracellular matrix protein, a nucleic acid molecule, a recombinant vector, a genetically engineered bacterium, and a preparation method and application are provided.

[0004] In some embodiments, a recombinant extracellular matrix protein is provided, comprising human fibronectin, humanized collagen and elastin-like protein; wherein,

[0005] The amino acid sequence of the human fibronectin is shown in SEQ ID NO.1;

[0006] The amino acid sequence of the humanized collagen is shown in SEQ ID NO.2;

[0007] The elastin-like protein comprises a pentapeptide repeat sequence, wherein the repeating unit of the pentapeptide repeat sequence comprises -[Valine-Proline-Glycine-Xaa-Glycine] n -, Xaa is isoleucine or lysine, and the number of repeating units n is 15 to 30;

[0008] In the recombinant extracellular matrix protein, any two adjacent ones of the human fibronectin, the humanized collagen and the elastin-like protein are independently connected by a peptide bond or a connecting peptide.

[0009] In some embodiments, in the recombinant extracellular matrix protein, the amino acid sequence of the elastin-like protein is as shown in SEQ ID NO.3.

[0010] In some embodiments, the recombinant extracellular matrix protein protein includes, from N-terminus to C-terminus, human fibronectin-first connecting peptide-humanized collagen-second connecting peptide-elastin-His tag sequence; the His tag sequence is shown in SEQ ID NO.4.

[0011] In some embodiments, the amino acid sequence of the extracellular matrix protein recombinant protein is as shown in SEQ ID NO.6.

[0012] In some embodiments, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the recombinant extracellular matrix protein.

[0013] In some embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.7.

[0014] In some embodiments, a recombinant vector is provided, wherein the recombinant vector contains the nucleic acid molecule.

[0015] In some embodiments, a genetically engineered bacterium is provided, which satisfies one or more of the following characteristics:

[0016] The genetically engineered bacteria express the extracellular matrix protein recombinant protein;

[0017] The genetically engineered bacteria carries the nucleic acid molecule;

[0018] The genetically engineered bacteria carries the recombinant vector.

[0019] In some embodiments, the host bacteria of the genetically engineered bacteria are selected from Escherichia coli. Optionally, the host bacteria of the genetically engineered bacteria are Escherichia coli BL21 (DE3).

[0020] In some embodiments, a method for preparing a recombinant extracellular matrix protein is provided, comprising the following steps:

[0021] In the presence of an inducer, the genetically engineered bacteria are cultured to express the extracellular matrix protein recombinant protein.

[0022] In some embodiments, the use of the recombinant extracellular matrix protein in the preparation of an active additive for use in tissue engineering, pharmacy, or beauty and skin care, or in the preparation of a preparation for use in tissue engineering, pharmacy, or beauty and skin care is provided.

[0023] In some embodiments, an active additive or preparation for use in the fields of tissue engineering, pharmacy, or cosmetic skin care is provided, wherein the active additive or preparation comprises the recombinant extracellular matrix protein.

[0024] The extracellular matrix recombinant fusion protein provided has good thermal stability, is not easy to degrade in 25°C and 37°C environments, and can promote cell adhesion. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0026] Figure 1 is the map of the plasmid pET-28a(+)-rhECMp constructed in Example 1;

[0027] Figure 2 This is a graph showing the results of colony PCR detection in Example 1, wherein the bands corresponding to lanes 1-8 are the results of colony PCR of 8 different monoclonal strains;

[0028] Figure 3 This is a diagram showing the result of restriction enzyme digestion identification of the plasmid pET-28a(+)-rhECMp extracted in Example 1, wherein the bands corresponding to lanes 1-6 are parallel control results of 6 groups of plasmid pET-28a(+)-rhECMp restriction enzyme digestion;

[0029] Figure 4 The figure is the result of SDS-PAGE electrophoresis identification of rhECMp in Example 2, wherein lane M is a marker, lane 1 is the bacterial solution before induction of expression, lane 2 is the supernatant after ultrasonic disruption, lane 3 is the supernatant filtrate after ultrasonic disruption, lane 4 is the protein obtained after purification, and lane 5 is the flow-through liquid collected during the purification process;

[0030] Figure 5 The figure is the result of western blot detection of rhECMp in Example 2, wherein the bands corresponding to lanes 1-7 are the parallel control results of 7 recombinant protein rhECMp samples;

[0031] Figure 6Figure 3 is a graph showing the results of the stability test in Example 3, wherein A is a graph showing the results of the thermal stability test of rhECMp in Example 3, wherein lane M is a marker, lane 1 is placed at 37°C (relative humidity 75%) for 0 days, lane 2 is placed at 37°C (relative humidity 75%) for 1 day, lane 3 is placed at 37°C (relative humidity 75%) for 5 days, lane 4 is placed at 37°C (relative humidity 75%) for 10 days, lane 5 is placed at 37°C (relative humidity 75%) for 15 days, and lane 6 is placed at 37°C (relative humidity 75%) for 30 days; B is the thermal stability test result of humanized collagen, wherein M is a marker, lane 1 is placed at 37°C (relative humidity 75%) for 15 days, and lane 2 is placed at 37°C (relative humidity 75%) for 30 days;

[0032] Figure 7 These are the results of the rhECMp cell adhesion promotion test in Example 4, wherein A1 is a blank control first visual field image, A2 is a blank control second visual field image, B1 is an elastin-like polypeptide first visual field image, B2 is an elastin-like polypeptide second visual field image, C1 is a humanized collagen first visual field image, C2 is a humanized collagen second visual field image, D1 is a rhECMp first visual field image, and D2 is a rhECMp second visual field image. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present invention, the present invention is described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0036] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0037] In this application, the "suitable" mentioned in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0038] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0039] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0040] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0041] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0042] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0043] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0044] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0045] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0046] In one embodiment, a recombinant extracellular matrix protein is provided, comprising the following protein fragments: human fibronectin, humanized collagen and elastin.

[0047] In one embodiment, in the recombinant extracellular matrix protein, the amino acid sequence of human fibronectin is as shown in SEQ ID NO.1: VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT.

[0048] In one embodiment, in the recombinant extracellular matrix protein, the amino acid sequence of the humanized collagen is as shown in SEQ ID NO.2: GAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQP.

[0049] In one embodiment, in the recombinant extracellular matrix protein, the elastin-like protein comprises a pentapeptide repeat sequence, and the repeating unit of the pentapeptide repeat sequence comprises -[Valine-Proline-Glycine-Xaa-Glycine] n -, Xaa is isoleucine or lysine, and the number of repeating units n is 15 to 30. For example, the number of repeating units of the pentapeptide repeat sequence can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or a range consisting of any two of the foregoing values.

[0050] In one embodiment, in the recombinant extracellular matrix protein, the amino acid sequence of elastin-like protein is as shown in SEQ ID NO.3: VPGIGVPGIGVPGKGVPGIGVPGIGVPGIGVPGIGVPGKGVPGIGVPGIGAVTGRGDSPASSVPGIGVPGIGVPGKGVPGIGVPGIGVPGIGVPGIGVPGKGVPGIGVPGIGVPG.

[0051] In one embodiment, in the recombinant extracellular matrix protein, any two adjacent human fibronectin, humanized collagen and elastin are independently connected by a peptide bond or a connecting peptide.

[0052] In one embodiment, in the recombinant extracellular matrix protein, human fibronectin, humanized collagen and elastin are sequentially connected via a connecting peptide.

[0053] Fibronectin (FN) is a high molecular weight (about 440 kDa) glycoprotein of the extracellular matrix that binds to integrin transmembrane receptor proteins. Fibronectin can bind to extracellular matrix components such as collagen, fibrin and heparin sulfate proteoglycans (such as syndecan). Fibronectin has many biological functions, such as being essential for wound healing and embryonic development. Cellular fibronectin aggregates in the extracellular matrix, an insoluble network that separates and supports the organs and tissues of an organism. Fibronectin and fibrin are deposited at the site of injury to form blood clots, stop bleeding and protect subcutaneous tissue. Secondly, FN plays an important role in cell adhesion, extension, growth, migration and differentiation. Fibronectin has a wide range of applications and a huge market in medicine, cosmetics and scientific research.

[0054] As the core component of the extracellular matrix, collagen has multiple biological functions, such as maintaining various tissue structures, tissue development and remodeling, affecting cell attachment and proliferation, etc. Among them, type III collagen is the most important and most abundant protein in the human body. Recombinant humanized collagen is a biological polymer substance with the advantages of strong water solubility, high biological activity, low immunogenicity and high biocompatibility. It is widely used in many fields such as tissue engineering, biomaterials and drug delivery systems.

[0055] Elastin is the main component of elastic fibers, which are mainly found in ligaments and vascular walls, coexisting with collagen fibers, giving tissues elasticity and tensile strength. In addition, as the main component of the extracellular matrix, elastin plays a certain role in cell adhesion and support. Human skin aging is related to the degradation of true elastic fibers by elastase. The pentapeptide repeat sequence Val-Pro-Gly-Xaa-Gly (VPGXG) n is the basic sequence unit of elastin-like polypeptides, in which Xaa is called a guest residue, and the Xaa in the pentapeptide repeat sequence of elastin in the extracellular matrix protein recombinant protein provided is isoleucine or lysine. Elastin-like proteins are artificially synthesized proteins that imitate the amino acid sequence characteristics of natural elastin, have good cell compatibility, and can be naturally degraded into amino acids in the body. In addition, elastin is derived from the extracellular matrix, and elastin-like proteins with a similar structure to elastin can provide similar extracellular matrix environments to cell tissues.

[0056] In one embodiment, the recombinant extracellular matrix protein protein includes, from N-terminus to C-terminus, humanized fibronectin-first connecting peptide-humanized collagen-second connecting peptide-elastin-His tag sequence. The first connecting peptide and the second connecting peptide may be the same or different.

[0057] In one embodiment, in the extracellular matrix protein recombinant protein, the His tag sequence is as shown in SEQ ID NO.4: HHHHHH.

[0058] In one embodiment, the first connecting peptide is the same as the second connecting peptide. Furthermore, the amino acid sequences of the first connecting peptide and the second connecting peptide are both as shown in SEQ ID NO.5: GGGGS.

[0059] In one embodiment, the amino acid sequence of the extracellular matrix protein recombinant protein is as shown in SEQ ID NO.6: VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGGGGSGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGGGGSVPGIGVPGIGVPGKGVPGIGVPGIGVPGIGVPGIGVPGIGVPGKGVPGIGVPGIGAVTGRGDSPASSVPGIGVPGIGVPGKGVPGIGVPGIGVPGIGVPGIGVPGKGVPGIGVPGIGHHHHHH.

[0060] In one embodiment, a nucleic acid molecule is provided, which encodes the aforementioned extracellular matrix protein recombinant protein.

[0061] In one embodiment, the nucleotide sequence of the provided nucleic acid molecule is as shown in SEQ ID NO.7: ATGGTCTCAGATGTACCCAGGGACCTAGAAGTTGTCGCAGCGACCCCGACCAGCTTGCTGATTTCCTGGGATGCGCCTGCGGTTACCGTTCGTTATTACCGTATTACCTATGGTGAGACAGGCGGAAACAGCCCGGTTCAAGAATTCACCGTGCCAGGTTCGAAAAGCACCGCAACGATTTCTGGCCTGAAACCGGGCGTCGATTATACCATCACCGTTTACGCAGTTACGGGTAGAGGCGACAGCCCAGCGTCTTCCAAACCGATTAGTATTAACTACCGCACCGGCGGTGGTGGCTCCGGTGCCGCGGGCGAGCGCGGTGCGCCGGGTTTTCGTGGTCCGGCCGGTCCGAATGGTATCCCGGGTGAGAAAGGCCCGGCTGGTGAACGTGGTGCTCCGGGTCCGGCGGGTCCGCGCGGTGCCGCGGGTGAGCCGGGCCGCGACGGCGTGCCGGGCGGTCCGGGCATGCGTGGTATGCCGGGTAGCCCGGGCGGTCCGGGTTCAGATGGTAAGCCTGGGCCTCCGGGTTCGCAGGGTGAAAGCGGTCGTCCGGGTCCGCCTGGCCCAAGCGGTCCGCGTGGTCAGCCGGGCGGGGGCGGTTCTGTTCCAGGCATCGGCGTCCCGGGTATTGGCGTCCCGGGGAAGGGCGTGCCGGGTATCGGCGTTCCGGGCATTGGCGTGCCGGGTATCGGCGTGCCGGGTATCGGCGTTCCGGGCAAGGGCGTTCCGGGTATCGGGGTGCCGGGAATTGGCGCTGTAACTGGTCGTGGCGACAGCCCGGCGAGCAGCGTTCCGGGTATTGGCGTTCCGGGCATCGGTGTGCCGGGTAAGGGTGTGCCGGGTATCGGCGTGCCGGGCATCGGTGTTCCAGGTATCGGTGTTCCGGGTATCGGCGTGCCGGGTAAGGGCGTGCCGGGCATTGGCGTGCCGGGTATTGGCCACCATCATCACCACCAC。

[0062] In one embodiment, a recombinant vector is provided, which contains the aforementioned nucleic acid molecule.

[0063] In this application, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. The vector can be a plasmid.

[0064] In some embodiments, a "vector" is a nucleic acid molecule carrying a gene of interest, which can deliver the gene of interest in a desired manner, for example, can deliver the gene of interest into a host cell.

[0065] In this application, unless otherwise specified, "plasmid" or "plasmid vector" refers to an artificially constructed plasmid. Without limitation, a plasmid vector can be a circular DNA molecule that can replicate autonomously independent of the cell nucleus.

[0066] In some embodiments, the recombinant vector further comprises a terminator.

[0067] In one embodiment, the recombinant vector is a pET28a plasmid containing the nucleotide sequence shown in SEQ ID NO.7.

[0068] In one embodiment, a genetically engineered bacterium is provided, which expresses the aforementioned extracellular matrix protein recombinant protein.

[0069] In one embodiment, a genetically engineered bacterium is provided, which carries the aforementioned nucleic acid molecule.

[0070] In one embodiment, a genetically engineered bacterium is provided, which carries the aforementioned recombinant vector.

[0071] In some embodiments, the aforementioned nucleic acid molecule carried in the recombinant vector is integrated into the genome of the genetically engineered bacteria.

[0072] In some embodiments, the recombinant vector is contained in the genetically engineered bacteria in the form of a free plasmid.

[0073] In one embodiment, the host bacteria of the provided genetically engineered bacteria are selected from Escherichia coli.

[0074] In one embodiment, the host bacteria of the provided genetically engineered bacteria is Escherichia coli BL21 (DE3).

[0075] In one embodiment, a method for preparing a recombinant extracellular matrix protein is provided, comprising the following steps: culturing genetically engineered bacteria to express the recombinant extracellular matrix protein in the presence of an inducer.

[0076] In one embodiment, a method for preparing a recombinant extracellular matrix protein protein comprises the following steps: constructing a recombinant vector containing a nucleotide sequence encoding a recombinant extracellular matrix protein protein, transforming a host bacterium to obtain a genetically engineered bacterium, inducing the genetically engineered bacterium to express the recombinant extracellular matrix protein protein, collecting and disrupting the bacterial cells, collecting the supernatant, and purifying the recombinant extracellular matrix protein protein in the supernatant by affinity chromatography or ion exchange chromatography.

[0077] In one embodiment, in the step of inducing genetically engineered bacteria to express extracellular matrix protein recombinant protein, the inducer is isopropyl-β-D-thiogalactoside (IPTG) with a concentration of 0.1 mM to 10 mM.

[0078] In one embodiment, in the step of inducing the genetically engineered bacteria to express the extracellular matrix protein recombinant protein, the induction is performed at 35°C to 40°C.

[0079] In one embodiment, the method for disrupting the bacteria is high-pressure homogenization, osmotic pressure shock, freeze-thaw or ultrasonic disruption, but is not limited thereto.

[0080] In one embodiment, the extracellular matrix protein recombinant protein in the supernatant is purified by Ni-NTA affinity chromatography.

[0081] In one embodiment, the steps of purifying the extracellular matrix protein recombinant protein in the supernatant by Ni-NTA affinity chromatography are as follows: the supernatant is loaded onto a purification column, and the Ni-NTA affinity column is equilibrated with a binding buffer (20mM NaH2PO3, 50mMNaCl, 10mM imidazole, pH 8.0). Then, the protein supernatant is added and incubated for 0.5h~1h to allow the target recombinant protein to fully bind to the column material. Then, the impurity protein is rinsed with a washing buffer containing imidazole (1M imidazole, 20mMNaH2PO3, 500mM NaCl, pH 8.0).

[0082] In one embodiment, there is provided the use of a recombinant extracellular matrix protein in preparing an active additive for use in tissue engineering, pharmacy, or beauty and skin care, or in preparing a preparation for use in tissue engineering, pharmacy, or beauty and skin care.

[0083] In one embodiment, an active additive or preparation for use in the fields of tissue engineering, pharmacy, or cosmetic skin care is provided, wherein the additive or preparation comprises a recombinant extracellular matrix protein.

[0084] In one embodiment, the preparation used in the field of tissue engineering, pharmacy or cosmetic skin care further comprises an acceptable carrier.

[0085] In this application, unless otherwise specified, "acceptable" means suitable for administration to subjects within the scope of reasonable judgment and commensurate with a reasonable benefit / risk ratio, and can be used to describe ligands, materials, compositions, dosage forms, etc. As an example, "pharmaceutically acceptable" means suitable for administration to subjects within the scope of reasonable medical judgment and commensurate with a reasonable benefit / risk ratio.

[0086] In some embodiments, "acceptable carrier" refers to an acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material.

[0087] The recombinant extracellular matrix protein is obtained by fusing different functional domains of human fibronectin, humanized collagen peptide and elastin-like polypeptide. The recombinant extracellular matrix protein can be expressed soluble in Escherichia coli and purified by affinity chromatography or ion exchange chromatography. The elastin-like protein in the recombinant extracellular matrix protein can form hydrogen bonds with water molecules and can also form hydrogen bonds with other protein molecules to increase the thermal stability of the recombinant extracellular matrix protein.

[0088] The extracellular matrix protein recombinant protein provided has good thermal stability and is easy to purify. It can promote cell adhesion and wound repair, capillary angiogenesis and tissue collagen regeneration. It can be used as a tissue engineering matrix material, an active additive for cell culture, and can be directly added to medicines and skin care products. As an active raw material for tissue repair, it can also be used as a cosmetic raw material, and has great application prospects.

[0089] The following specific examples further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0090] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0091] Example 1 Construction and expression of recombinant extracellular matrix protein (rhECMp) expression vector

[0092] (1) Construction of a recombinant expression vector of rhECMp: Design the amino acid sequence of rhECMp, which includes, from N-terminus to C-terminus, human fibronectin-connecting peptide-humanized collagen-connecting peptide-elastin-His tag sequence; the human fibronectin sequence is shown in SEQ ID NO.1, the humanized collagen sequence is shown in SEQ ID NO.2, the elastin sequence is shown in SEQ ID NO.3, the His tag sequence is shown in SEQ ID NO.4, the connecting peptide sequence is shown in SEQ ID NO.5, and the complete amino acid sequence of the fusion protein is shown in SEQ ID NO.6. Then design the nucleotide sequence encoding the above rhECMp, and the complete nucleotide sequence encoding rhECMp is shown in SEQ ID NO.7. Entrust Guangzhou Ruibo Xingke Biotechnology Co., Ltd. to synthesize the nucleotide sequence shown in SEQ ID NO.7, and pre-insert it between BamH I and Xol I of the pET-28a(+) vector to obtain the recombinant plasmid pET-28a(+)-rhECMp. The map can be found at Figure 1 . The heat shock method was used to transfer the competent cells to DH5α, and the colonies were cultured on kanamycin-resistant plates at 37°C. Single clones were picked for PCR identification (primer rhECMp F: GGATCCATGGTCTCAGATGTACCCAGGGAC, SEQ ID NO.8; primer rhECMp R: CTCGAGTTAGTGGTGGTGATGATGGTGGCCAATAC, SEQ ID NO.9); PCR amplification conditions were as follows: denaturation at 95°C for 10 min before entering the cycle, and the first step of the cycle parameters was denaturation at 95°C for 60 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 60 seconds, for 30 cycles. The amplified products were detected by gel electrophoresis, and the test results can be found in Figure 2 .Depend on Figure 2 The results can be identified as positive clones.

[0093] The single clone was picked and incubated in 3 mL of ordinary LB culture medium containing kanamycin at a final concentration of 50 μg / mL at 37°C overnight. The expression plasmid pET-28a(+)-rhECMp was extracted using a plasmid extraction kit and the enzyme digestion was positive. The results can be found in Figure 3 .

[0094] (2) Induced expression of rhECMp: pET-28a(+)-rhECMp was heat-shock transformed into competent cells BL21(DE3)plysS and screened and cultured in LB(Amp) medium. Positive clones were picked, and plasmids were extracted and then verified by enzyme digestion. 3 mL of ordinary LB culture medium containing kanamycin at a final concentration of 50 μg / mL was added to a 15 mL test tube and shaken overnight at 37°C. Then, 10 mL of ordinary LB culture medium containing kanamycin at a final concentration of 50 μg / mL was added to a 50 mL test tube and 0.5 mL of bacterial solution was shaken at 37°C for 2 h to 3 h. When the OD600 value reached 0.4 to 0.6, 1 mmol / L IPTG was added and shaken at 37°C for 4 h. 1 mL of the expressed bacteria was collected respectively, centrifuged at 8000 rpm for 5 min, 1 mL of Tris-HCl was added, ultrasonically disrupted, and the supernatant was centrifuged.

[0095] Example 2 Purification of rhECMp

[0096] (1) The bacterial ultrasonic lysate obtained in Example 1 was centrifuged at 8000 rpm for 5 min at 4°C to obtain the supernatant;

[0097] (2) Load all the supernatant onto the purification column and equilibrate the Ni-NTA (Hangzhou New Dragon, catalog number: NRPB58S-Y15) affinity column with 5 column volumes of binding buffer (50mM Tris-HCl, 300mM NaCl, 20mM imidazole, pH 8.0). Then add the protein supernatant and incubate at 4°C for 0.5h~1h to allow the target recombinant protein to fully bind to the column material. Then rinse the impurities with washing buffer (300mM imidazole, 50mM Tris-HCl, 300mM NaCl, pH8.0). Perform SDS-PAGE electrophoresis on the supernatant after bacterial disruption and the purified protein. The identification results can be found in Figure 4 At the same time, the purified protein was tested by western blot. The identification results can be found in Figure 5 . Figure 4 and Figure 5 The results showed that rhECMp recombinant protein was successfully expressed.

[0098] Example 3 rhECMp thermal stability test

[0099] Take an equal amount of the liquid rhECMp obtained in Example 2, seal it with a cap and place it in an environment of 37°C (relative humidity 75%). Take samples at 0 days, 1 day, 5 days, 10 days, 15 days, and 30 days. Detect the results by 10% SDS-PAGE electrophoresis. Analyze the depth of the electrophoresis bands by software to evaluate the remaining percentage of protein at each time point. The experimental results are shown in Figure 6 A in the figure. At the same time, the same amount of recombinant humanized collagen as the fusion protein rhECMp was taken, sealed with a cap, and placed in an environment of 37°C (relative humidity 75%). Samples were taken at 15 days and 30 days, and the results were detected by 10% SDS-PAGE electrophoresis. The depth of the electrophoresis bands was analyzed by software to evaluate the remaining percentage of protein at each time point. The experimental results are shown in Figure 6 B in.

[0100] Depend on Figure 6 It can be seen that the obtained rhECMp has not been degraded after being placed in an environment of 37°C (relative humidity 75%) for 30 days. However, after the recombinant humanized collagen was placed in an environment of 37°C (relative humidity 75%) for 15 days and 30 days, the sample was basically completely degraded. This experiment compared the stability of rhECMp under different conditions, proving that the rhECMp of the present invention has good thermal stability, and its thermal stability is significantly better than that of recombinant humanized collagen.

[0101] Example 4 rhECMp cell adhesion promotion test

[0102] Take the rhECMp purified in Example 2, humanized collagen and elastin-like polypeptide, wherein the amino acid sequence of humanized collagen is shown in SEQ ID NO.2, and the amino acid sequence of elastin-like polypeptide is shown in SEQ ID NO.3. The preparation method of humanized collagen and elastin-like polypeptide is the same as the preparation method of rhECMp.

[0103] (1) Coating preparation

[0104] 100 μL of the purified rhECMp obtained in Example 2, humanized collagen, elastin-like peptide and D-PBS blank control were added to the 96-well plate. Four wells were prepared for each sample coating and incubated in a 37°C, 5% CO2 incubator for 1 h to 4 h. Excess coating solution was removed from the wells, 100 μL of 1% BSA-PBS solution was added, and incubated in a 37°C, 5% CO2 incubator for 1 h. After removing the liquid in the wells, wash three times with D-PBS, discard the washing solution, seal with sealing film and place at 4°C for use.

[0105] (2) Cell preparation

[0106] NIH / 3T3 cells were cultured in a 37°C, 5% CO2 cell culture incubator, and the cell density and status were observed under an inverted microscope every day. When the cells grew to 80% to 90% of the culture flask, cell passage or cell inoculation was performed. The cells were diluted to 5×10 using complete culture medium premixed with Hoechst 33342 fluorescent dye (10%). 4 / mL. 100 μL of cells were added to the wells, covered with aluminum foil, and incubated for 1 h at 37 °C, 5% CO2. Three replicates were measured, and the fourth well was used to adjust the microscope parameters and its measurement value was not used.

[0107] (3) Detection

[0108] Use an inverted microscope to capture fluorescent tile images of each of the 3 wells. Fill each well with D-PBS to form a "reverse meniscus", blow out bubbles and cover with parafilm. Centrifuge (inverted) at 22 °C for 5 min at optimal relative centrifugal force. After centrifugation, discard the parafilm and remove the supernatant from the wells. After washing once with D-PBS, add 100 μL D-PBS. Take an image for each of the 3 wells. For staining results of cell nuclei, see Figure 7 Based on the distribution of cell nuclei, the number of cells can be observed and compared. Figure 7 It can be seen that compared with humanized collagen and elastin-like peptides, the same amount of rhECMp fusion protein can adhere to more cells. It can be concluded that the ability of rhECMp fusion protein to promote cell adhesion is significantly better than that of humanized collagen peptides and elastin-like peptides.

[0109] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0110] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A recombinant extracellular matrix protein, characterized in that The invention comprises the following protein fragments: human fibronectin, humanized collagen and elastin-like protein; wherein, The amino acid sequence of the human fibronectin is shown in SEQ ID NO.1; The amino acid sequence of the humanized collagen is shown in SEQ ID NO.2; The elastin-like protein comprises a pentapeptide repeat sequence, wherein the repeating unit of the pentapeptide repeat sequence comprises -[Valine-Proline-Glycine-Xaa-Glycine] n -, Xaa is isoleucine or lysine, and the number of repeating units n is 15 to 30; In the recombinant extracellular matrix protein, any two adjacent ones of the human fibronectin, the humanized collagen and the elastin-like protein are independently connected by a peptide bond or a connecting peptide.

2. The recombinant extracellular matrix protein according to claim 1, characterized in that The amino acid sequence of the elastin-like protein is shown in SEQ ID NO.

3.

3. The recombinant extracellular matrix protein according to claim 1 or 2, characterized in that From the N-terminus to the C-terminus, it includes: human fibronectin-first connecting peptide-humanized collagen-second connecting peptide-elastin-like protein-His tag sequence; the His tag sequence is shown in SEQ ID NO.4; Optionally, the amino acid sequence of the extracellular matrix protein recombinant protein is as shown in SEQ ID NO.

6.

4. A nucleic acid molecule, characterized in that Encoding the recombinant extracellular matrix protein according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

7.

6. A recombinant vector, characterized in that: Containing the nucleic acid molecule according to claim 4 or 5.

7. A genetically engineered bacterium, characterized in that: Meet one or more of the following characteristics: The genetically engineered bacteria express the extracellular matrix protein recombinant protein according to any one of claims 1 to 3; The genetically engineered bacteria carries the nucleic acid molecule of claim 4 or 5; The genetically engineered bacteria carries the recombinant vector described in claim 6.

8. The genetically engineered bacterium according to claim 7, characterized in that: The host bacteria of the genetically engineered bacteria is selected from Escherichia coli. Optionally, the host bacteria of the genetically engineered bacteria is Escherichia coli BL21 (DE3).

9. A method for preparing a recombinant extracellular matrix protein, characterized in that: The steps include: In the presence of an inducer, the genetically engineered bacteria according to claim 7 or 8 are cultured to express the recombinant extracellular matrix protein.

10. Use of the recombinant extracellular matrix protein according to any one of claims 1 to 3 in the preparation of an active additive for use in the fields of tissue engineering, pharmacy or beauty and skin care, or in the preparation of a preparation for use in the fields of tissue engineering, pharmacy or beauty and skin care.

11. An active additive or preparation for use in tissue engineering, pharmacy or beauty and skin care, characterized in that: The active additive or preparation comprises the recombinant extracellular matrix protein according to any one of claims 1 to 3.

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