Method for manufacturing a biosynthetic human structural material

Abstract

The present invention relates to a method for manufacturing a biosynthetic human structural material. The present invention provides a recombinant type IV humanized collagen in which the amino acid sequence contains n (n is an integer of 1 or more) repeating sequences, and the repeating sequence is the sequence of the functional region of human native type IV collagen. The present invention provides the amino acid sequence of a recombinant humanized type IV collagen whose amino acid sequence is derived from native human type IV collagen, does not cause a harmful immune reaction even when administered to the human body, can be accurately expressed in vitro, and has succeeded in biosynthesis in vitro. The recombinant type IV humanized collagen prepared in the present invention has a higher cell adhesion effect than commercially available human-derived collagen. Further, the manufacturing method provided by the present invention is simple, and recombinant type IV humanized collagen can be manufactured at low cost and in high yield.

Description

Technical Field

[0001] Priority and Related Applications This application claims the priority of a Chinese patent application with the application number 202210619454.1 and the invention title "Method for Manufacturing Biosynthetic Human Structural Materials", which was filed with the China National Intellectual Property Administration on June 1, 2022, and incorporates all of its content herein by reference.

[0002] The present invention belongs to the field of biotechnology, and specifically relates to a method for manufacturing biosynthetic human structural materials.

Background Art

[0003] The structural materials of the human body are mainly structural proteins containing collagen. Such proteins have an adhesion function and a support function for cells and tissues, and are the main components of the extracellular matrix.

[0004] Collagen is a protein widely distributed in human connective tissues and is also the most abundant protein in the human body, accounting for 25% - 35% of all proteins. Currently, there are at least 28 subtypes of collagen in the human body, and it has been found that they exist in different tissues and organs. Among them, type IV collagen is mainly present in the hepatic portal vascular region, around the central vein, and is distributed along the hepatic sinusoids. It is an important component forming the framework of the basement membrane, presenting a non-fibrous reticular collagen triple helix structure, playing the role of the extracellular matrix, and having the function of supporting cells. Since collagen is a natural biological resource with biocompatibility not found in other polymer materials, it can be used as an important human structural material and is widely used in the high-end pharmaceutical industry.

[0005] However, currently, the raw materials of collagen are mainly extracted from animal tissues. Unfortunately, since the content of type IV collagen in animals is low, it is impossible to extract a single component of type IV collagen. In addition, the immune response caused by animal origin is also an important reason why the uses of collagen are limited. With the growth of the collagen industry in China, the utilization of biosynthetic pathways to obtain collagen has become increasingly mature. In particular, humanized collagen has already reached the world's leading level. In 2021, the National Medical Products Administration named and classified biosynthetic collagen. Among them, recombinant humanized collagen refers to the full-length or partial fragment of the amino acid sequence encoded by a specific genotype of human collagen synthesized by DNA recombination technology, or a combination containing a functional fragment of human collagen.

[0006] In the living body, type IV collagen has a non-fibrous triple helix structure, and there are six genetically different α-chains: α1(IV)~α6(IV), which can form three different molecular forms in the tissue, namely α1α1α2(IV), α3α4α5(IV), α5α5α6(IV), and they can be selectively expressed in different membranes in specific tissues at different stages. Structurally speaking, the structure of natural type IV collagen in the human body is very complex, so it is extremely difficult to express and prepare a large amount of humanized collagen by conventional means. The synthesis and modification of collagen start from tropocollagen and go through many chemical changes such as hydroxylation, glycosylation, and cross-linking, and are complexly controlled by various biological enzymes. Tropocollagen contains not only collagen chains but also globular heads and tails. Without these heads and tails, the collagen chains cannot be folded into the correct triple helix, lacking the biological activity as collagen. Therefore, the collagen prepared according to the original gene sequence cannot spontaneously organize in vitro to form the correct spatial structure. Due to such difficulties, the research, development, and production of humanized collagen are significantly inhibited.

[0007] Conventionally, the method for producing collagen is a method of treating animal-derived tissues by acid, alkali, or enzymatic degradation methods to extract collagen derivatives. The collagen itself extracted by these methods has already lost its original biological activity and cannot exhibit its original functions when applied in the biomedical field. With the development of modern biotechnology, the use of transgenic technology has been continuously attempted to prepare recombinant human collagen in expression systems of animals, plants, and microorganisms, thereby solving many drawbacks of the conventional extraction process. However, some research institutions have pointed out that receptors that can be expressed in human B cells are not expressed in mice, suggesting that the humanized model can provide expressions that could not be obtained in conventional mice. Therefore, there is an urgent need for a biosynthetic method of humanized type IV collagen that can overcome this defect and can be widely used as a structural material for the human body.

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present invention aims to solve the problem that natural human type IV collagen cannot be correctly expressed in vitro and cannot be prepared in large quantities, and to provide a recombinant type IV humanized collagen, its biosynthetic production method, and its use in the pharmaceutical field, which offer new possibilities for the production and application of humanized collagen.

Means for Solving the Problems

[0009] The first aspect of the present invention provides a recombinant type IV humanized collagen. Specifically, the present invention screens the sequence of the functional region of human natural type IV collagen and uses it as a repetitive sequence, such that the amino acid sequence of the recombinant type IV humanized collagen contains n (n is an integer of 1 or more) repetitive sequences, that is, the sequence of the functional region of human natural type IV collagen. The recombinant type IV humanized collagen consists of the sequence of the functional region of human natural type IV collagen.

[0010] Furthermore, n may be 1, 2, 3, 4, 5, 6, 7, or 8. When n is an integer of 2 or more, the respective repetitive sequences are directly linked. Thereby, recombinant type-IV humanized collagen having an amino acid sequence containing the sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 can be obtained.

[0011] Furthermore, the amino acid sequence of the recombinant type-IV humanized collagen provided by the present invention may further contain an amino acid sequence cleavable by TEV protease shown in SEQ ID NO.7 in order to facilitate expression and purification in the actual production process. Optionally, the amino acid sequence cleavable by the TEV protease may be directly linked to the repetitive sequence. The amino acid sequence cleavable by TEV protease is preferably located at the N-terminus of the recombinant type-IV humanized collagen.

[0012] The present invention further provides recombinant type-IV humanized collagen having the above amino acid sequence as a core region.

[0013] A second aspect of the present invention provides a polynucleotide encoding the recombinant type-IV humanized collagen according to the first aspect of the present invention. Furthermore, the sequence of the polynucleotide may include the sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, or artificially designed and codon-optimized derivatives thereof.

[0014] A third aspect of the present invention provides a series of recombinant expression vectors containing the polynucleotide according to the second aspect of the present invention. The expression vector selected in the present invention can stably exist and autonomously replicate in various hosts of prokaryotic or eukaryotic cells, such as conventional plasmids (pET series) in the art, shuttle vector PNV 18.1, phage, or viral vectors. The recombinant expression vector is constructed by cloning the nucleotide sequence according to the second aspect of the present invention into the vector by molecular biological operations such as enzymatic digestion and ligation of the vector.

[0015] A fourth aspect of the present invention provides a series of recombinant host cells comprising the recombinant expression vector described in the third aspect of the present invention, which are selected from prokaryotic cells, yeast or eukaryotic cells, and can further be selected from Escherichia coli, Rhodococcus rubber, Bacillus subtilis, and yeast. The recombinant expression vector described in the third aspect of the present invention is transformed into a host cell to obtain such a genetically engineered bacterium.

[0016] A fifth aspect of the present invention provides a method for producing the recombinant type IV humanized collagen described in the first aspect of the present invention, which may include the following steps. S1: A step of fermenting and culturing the recombinant host cell described in the fourth aspect of the present invention to induce protein expression; in this step, the fermenting and culturing may be performed in a shaker at a rotation speed of 200 to 240 rpm and a temperature of 35 to 38 °C, preferably at a rotation speed of 220 rpm and preferably at a temperature of 37 °C. Also, the specific procedure for inducing protein expression may be to lower the temperature to 16 to 30 °C and add IPTG, and the use concentration of any IPTG is 0.3 to 0.7 mM, preferably 0.5 mM. S2: A step of collecting the protein; in this step, the specific procedure for collecting the protein is to centrifuge the mixture obtained in step S1 at 5000 to 7000 rpm for 10 to 15 minutes in an environment at 4 °C, resuspend the precipitate using an aqueous solution containing 20 to 30 mM Tris, 150 to 250 mM sodium chloride, and 15 to 25 mM imidazole, and then homogenize at high pressure or disrupt the cells by ultrasonic waves, followed by centrifuging at 15000 to 18000 rpm for 20 to 40 minutes in an environment at 4 °C. Preferably, the specific procedure for collecting the protein is to centrifuge the mixture obtained in step S1 at 6000 rpm for 12 minutes in an environment at 4 °C, resuspend the precipitate using an aqueous solution containing 25 mM Tris, 200 mM sodium chloride, and 20 mM imidazole, lower the temperature to 15 °C or lower, homogenize at high pressure or disrupt the cells by ultrasonic waves, followed by centrifuging at 17000 rpm for 30 minutes in an environment at 4 °C. S3: Step of purifying the protein; preferably, the step preferably purifies the protein using a Ni affinity chromatography column and / or an ion exchange chromatography column, and optionally includes enzymatically digesting the protein. It is preferable to enzymatically digest the protein using TEV protease, and it is more preferable that the mass ratio of the protein to the TEV protease is (15-25):1.

[0017] Furthermore, before the step S1, the production method may further include a step of screening for functional regions of human native type IV collagen, a step of constructing a recombinant expression vector, and a step of constructing a recombinant host cell. Specifically, the specific procedure of the step of screening for the functional region of human native type IV collagen is as follows: after excluding uncharged amino acid motifs in the helix region of human native type IV collagen, the potential helix functional region with the most inter-chain hydrogen bond structures and capable of most stabilizing the trimer aggregation form is screened by a computer-aided protein structure prediction method, and then the human native type IV collagen functional region with the highest protein expression level, easy to purify, and good stability is screened by a protein expression property prediction method. The amino acid sequences of the functional regions of human native type IV collagen obtained by screening are repeatedly linked, and an amino acid sequence cleavable by TEV protease is added as needed to obtain the amino acid sequence of the recombinant type IV humanized collagen described in the first aspect of the present invention. Then, according to the codon preference of the target, the polynucleotide sequence of the gene encoding the recombinant type IV humanized collagen is artificially designed and optimized to obtain the nucleotide sequence described in the second aspect of the present invention, which is cloned into a vector to construct the recombinant expression vector described in the third aspect of the present invention, and the above vector is transformed into a host cell to obtain the recombinant host cell described in the fourth aspect of the present invention.

[0018] The sixth aspect of the present invention provides the use of the recombinant type IV humanized collagen described in the first aspect of the present invention in the manufacture of products including medical devices, cosmetics, health foods, or pharmaceuticals.

[0019] Specifically, the present invention is as follows.

[0020] [1] Recombinant type IV humanized collagen whose amino acid sequence contains n (n is an integer of 1 or more) repeating sequences, wherein the repeating sequence is the sequence of the functional region of human native type IV collagen, preferably, n is 1, 2, 3, 4, 5, 6, 7, or 8, and when n is an integer of 2 or more, the repeating sequences are directly linked to each other, more preferably, the amino acid sequence of the recombinant type IV humanized collagen is recombinant type IV humanized collagen containing any one of the following (i) to (iii). (i) The amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 (ii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or modified in the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and which retains the cell adhesion effect of human native type IV collagen (iii) An amino acid sequence that retains the cell adhesion effect of human native type IV collagen and is encoded by the following nucleotide sequence, wherein the nucleotide sequence hybridizes with a polynucleotide sequence encoding the sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 under stringent conditions, and the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions, or ultra-high stringent conditions

[0021] [2] A recombinant type IV humanized collagen in which the amino acid sequence further contains an amino acid sequence cleavable by the TEV protease shown in SEQ ID NO.7, Preferably, the amino acid sequence cleavable by the TEV protease and the repeating sequence are directly linked, The recombinant type IV humanized collagen according to [1], wherein preferably, the amino acid sequence cleavable by the TEV protease is located at the N-terminus of the recombinant type IV humanized collagen.

[0022] [3] A polynucleotide encoding the recombinant type IV humanized collagen according to [2], Preferably, a polynucleotide containing the sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

[0023] [4] A recombinant expression vector containing the polynucleotide according to [3], Preferably, the recombinant expression vector includes a pET series vector, a shuttle vector, a phage or a virus vector, More preferably, the recombinant expression vector is pET-28a-Trx-His, a recombinant expression vector.

[0024] [5] A recombinant host cell containing the recombinant expression vector according to [4], Preferably, the recombinant host cell is a prokaryotic cell, yeast or eukaryotic cell, More preferably, the host cell is Escherichia coli BL21(DE3), a recombinant host cell.

[0025] [6] A method for producing the recombinant type IV humanized collagen according to [1] or [2], S1: A step of fermenting and culturing the recombinant host cell according to claim 5 to induce protein expression; S2: A step of collecting the protein; S3: A step of purifying the protein, and includes, It is preferable to purify the protein using a Ni affinity chromatography column and / or an ion exchange chromatography column. Optionally including enzymatic digestion of the protein, it is preferable to enzymatically digest the protein using TEV protease, and it is more preferable that the mass ratio of the protein to the TEV protease is (15 - 25):1. A manufacturing method.

[0026] [7] In the S1 step, the fermentation culture is carried out in a shaker with a rotation speed of 200 - 240 rpm and a temperature of 35 - 38 °C. The manufacturing method according to [6].

[0027] [8] In the S1 step, the specific procedure for inducing protein expression is to lower the temperature to 16 - 30 °C, add IPTG, and preferably, the usage concentration of the IPTG is 0.3 - 0.7 mM. The manufacturing method according to [6] or [7].

[0028] [9] In the S2 step, the specific procedure for collecting the protein is to centrifuge the mixture obtained in the S1 step at 5000 - 7000 rpm for 10 - 15 minutes in an environment at 4 °C, resuspend the precipitate using an aqueous solution containing 20 - 30 mM Tris, 150 - 250 mM sodium chloride, and 15 - 25 mM imidazole, homogenize under high pressure, or disrupt the cells by ultrasonic waves, and then centrifuge at 15000 - 18000 rpm for 20 - 40 minutes in an environment at 4 °C. The manufacturing method according to any one of [6] - [8].

[0029]

[10] Use in the manufacture of products including medical devices, cosmetics, health foods, or pharmaceuticals containing the recombinant type IV humanized collagen according to [1] or [2].

Advantages of the Invention

[0030] By implementing the above-described embodiments, the present invention provides an amino acid sequence of recombinant humanized type IV collagen that is derived from natural human type IV collagen, does not cause harmful immune reactions even when administered to the human body, can be accurately expressed in vitro, and has been successfully biosynthesized in vitro. The recombinant type IV humanized collagen prepared in the present invention has a higher cell adhesion effect than commercially available human-derived collagen. In addition, the production method provided by the present invention is simple, and recombinant type IV humanized collagen can be produced at low cost and in high yield.

Brief Description of the Drawings

[0031]

Figure 1

Figure 2

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Figure 6

Modes for Carrying Out the Invention

[0032] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited thereto.

[0033] In the present invention, the term "may" includes both the meaning of performing a certain process and the meaning of not performing it.

[0034] In the present invention, "any" or "optionally" means that the situations or circumstances described below may or may not occur, and this description includes the meanings in both cases where this situation occurs and does not occur.

[0035] In the present invention, the terms "comprising", "having", "including", or "containing" are inclusive and open-ended and do not exclude additional unrecited elements or method steps. On the other hand, "comprising", "having", "including", or "containing" can also mean a closed form that excludes additional unrecited elements or method steps.

[0036] In the present invention, the numerical ranges represented by "numerical value A to numerical value B", "numerical value A or more", and "numerical value A or less" mean ranges including the limit values A and B.

[0037] In the present invention, any numerical value includes the standard deviation of the error of the apparatus or method used to measure that value. The numerical ranges and parameters used to define the present invention are all approximate values, but reproduce as accurately as possible the numerical values according to specific examples. However, essentially, any numerical value necessarily includes the standard deviation by the above measurement apparatus or method.

[0038] In the present invention, the terms "polypeptide" and "protein" mean a series of at least two amino acid residues linked to each other via a covalent bond (for example, a peptide bond), and may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptide may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. This term also includes amino acid polymers modified by any other optional operations such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component.

[0039] In the present invention, the term "amino acid" may include all of natural amino acids, unnatural amino acids, amino acid analogs, and their D and L stereoisomers.

[0040] In the present invention, deletion of an amino acid can mean deleting one, two, or more than two amino acids from an amino acid sequence, provided that the modified sequence retains all or part of the activity of the original amino acid sequence.

[0041] In the present invention, addition of an amino acid can mean adding one, two, or more than two amino acids at any position at the C-terminus, N-terminus, or between the C-terminus and the N-terminus of an amino acid sequence, provided that the modified sequence retains all or part of the activity of the original amino acid sequence.

[0042] In the present invention, the substitution of an amino acid can mean that the amino acid is replaced by another amino acid at any position in the amino acid sequence, provided that the modified sequence retains all or part of the activity of the original amino acid sequence. The substitution of an amino acid may be a conservative amino acid substitution, which means that some amino acids are replaced by amino acids having similar properties to form a peptide (conservative mutant peptide) as compared with the original amino acid sequence. For example, these conservative mutant peptides can be generated by the following amino acid substitutions: substitution of Ala with Val, Leu or Ile; substitution of Arg with Lys, Gln, Asn or His; substitution of Asn with Gln, His, Lys or Arg; substitution of Asp with Glu or Asn; substitution of Cys with Ser or Ala; substitution of Gln with Asn or Glu; substitution of Glu with Asp or Gln; substitution of Gly with Ala; substitution of His with Asn, Lys, Gln or Arg; substitution of Ile with Leu, Met, Ala, Val or Phe; substitution of Leu with Ile, Met, Ala, Val or Phe; substitution of Lys with Asn, Gln or Arg; substitution of Met with Ile, Leu or Phe; substitution of Phe with Leu, Val, Ile, Ala or Tyr; substitution of Pro with Ala; substitution of Ser with Thr; substitution of Thr with Ser or Val; substitution of Trp with Phe or Tyr; substitution of Tyr with Trp, Phe, Thr or Ser; and substitution of Val with Phe, Ala, Met, Ile or Leu. The amino acid substitution may also be a non-conservative amino acid substitution.

[0043] In the present invention, amino acid modification can include modification of functional groups, intramolecular covalent bonds (for example, formation of a ring between side chains), methylation, acylation, ubiquitination, phosphorylation, aminohexanation, biotinylation and other modifications to the native sequence.

[0044] In the present invention, "hybridize" means the ability of a polynucleotide or oligonucleotide to bind to a substantially complementary sequence under stringent conditions and not to cause non-specific binding with non-complementary partners under these conditions. For this reason, the above sequences are preferably 90-100% complementary. The property of complementary sequences that can specifically bind to each other is utilized, for example, in Northern or Southern blot techniques or primer binding in PCR or RT-PCR. According to the present invention, hybridization is carried out under medium stringent conditions, medium-high stringent conditions, high stringent conditions, or ultra-high stringent conditions. Such hybridization conditions are described in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, specific hybridization conditions are as follows. (1) Low stringent hybridization conditions are those with 6× sodium chloride / sodium citrate (SSC) at about 45°C and then at least 50°C, followed by washing twice with 0.2× SSC, 0.1% SDS (under low stringent conditions, the washing temperature can be raised to 55°C); (2) Medium stringent hybridization conditions are those with 6× SSC at about 45°C and then at 60°C, followed by washing once or multiple times with 0.2× SSC, 0.1% SDS; (3) High stringent hybridization conditions are those with 6× SSC at about 45°C and then at 65°C, followed by washing once or multiple times with 0.2× SSC, 0.1% SDS, and these are preferred conditions; (4) Ultra-high stringent hybridization conditions are those with 0.5 M sodium phosphate, 7% SDS at 65°C and then at 65°C, followed by washing once or multiple times with 0.2× SSC, 1% SDS.

[0045] In the present invention, suitable vectors are those known in the field of vector construction, including the selection of promoters and other regulatory elements such as enhancer elements. The vectors according to the present invention contain sequences suitable for introduction into cells. For example, the vector may be an expression vector in which the coding sequence of the protein is controlled by its own cis-acting regulatory elements and is designed to facilitate gene integration or gene replacement in host cells. Those skilled in the art will understand that in the present invention, a "vector" includes DNA molecules such as plasmids, phages, viruses, or other vectors containing one or more heterologous or recombinant nucleotide sequences. Suitable phage and viral vectors include, but are not limited to, λ-phage, EMBL phage, simian virus, bovine papillomavirus, Epstein-Barr virus, adenovirus, herpes virus, mouse sarcoma virus, mouse mammary tumor virus, lentivirus, etc.

[0046] In the present invention, the host cell may be a eukaryotic cell such as a fungus and yeast, or a prokaryotic cell such as a bacterium of the family Enterobacteriaceae.

[0047] In the present invention, the "functional region of human native type IV collagen" refers to a sequence fragment in human native type IV collagen α1 that has functions such as cell adhesion activity possessed by the amino acid sequence of collagen.

[0048] The application fields of the recombinant type IV humanized collagen provided by the present invention include the manufacture of high-end medical devices such as biological dressings, human biomimetic materials, plastic surgery materials, organoid culture, cardiovascular stents, coatings, tissue injection fillers, ophthalmic materials, gynecological biological materials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, 3D printed artificial organ biological materials, etc.; high-end cosmetic raw materials, high-end health foods, and additives for high-end pharmaceuticals, etc. Examples

[0049] The present invention will be further described by the following examples, but any example or combination thereof should not be construed as limiting the scope or embodiments of the present invention. The scope of the present invention is limited by the appended claims. Those skilled in the art can clearly understand the scope defined by the claims based on this specification and common general knowledge in the art. Without departing from the spirit and scope of the present invention, those skilled in the art can make any changes or modifications to the technical solution of the present invention, and such changes and modifications are also included in the scope of the present invention.

[0050] When specific conditions are not described in the examples, they are carried out according to general conditions or the conditions recommended by the manufacturer. All reagents and equipment not described by the manufacturer are ordinary products that can be purchased commercially. To better explain the present invention, numerous specific details are described in the following specific embodiments. Those skilled in the art will understand that the present invention can be implemented without such details. In another embodiment, in order to clarify the gist of the present invention, well-known methods, means, devices, and processes to those skilled in the art are not described in detail.

[0051] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, all units used in this specification are international standard units, and it should be understood that the numerical values and numerical ranges appearing in the present invention include inevitable systematic errors.

[0052] Example 1: Construction and Expression of Recombinant Humanized Type IV Collagen 1. Implementation of Large-Scale Protein Functional Region Screening 1) Screening of the array: The Gly-X-Y repeat sequences in the helical region of native type IV collagen contain many charged amino acids, and these charges bind to cells through interactions. Therefore, regions that do not contain these important charge motifs were excluded. 2) Using a computer-aided protein structure prediction method, potential helical functional regions with the most intermolecular hydrogen bond structures and the ability to most stabilize the trimeric aggregation form were screened. 3) Using a method for predicting protein expression characteristics, human type IV collagen functional regions with the highest protein expression level, easy purification, and good stability were screened. 4) To ensure that the molecular weight of recombinant type IV humanized collagen falls within a certain range and is easy to purify and stabilize, amino acid fragments in the regions obtained by screening were directly ligated n times in succession and optimized. Finally, the target fragments of recombinant type IV humanized collagen were obtained as follows. (1) Amino acid sequence of recombinant type IV humanized collagen C007: GQKGDQGEKGQIGPIGEKGSRGDPGTPGVPGKDGQAGQPGQPGPKGDPGISGTPGAPGLPGPKGSVGGMGLPGTPGEKGVPGIPGPQGSPGLPGDKGAKGEKGQAGPPGIGIPGLRGEKGDQGIAGFPGSPGEKGEKGSIGIPGMPGSPGLKGSPGSVGYPGSPGLPGEKGDKGLPGLDGIPGVKGEAGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHATE (SEQ ID NO.1); (2) Amino acid sequence of recombinant type IV humanized collagen C4P7C6: GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHA (SEQ ID NO.2); (3) Amino acid sequence of recombinant type IV humanized collagen C4P7E5: GLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFM (SEQ ID NO.3).

[0053] When the recombinant type IV humanized collagen of the present invention is expressed, an amino acid sequence ENLYFQ (SEQ ID NO.7) cleavable by TEV protease can be added to its N-terminus. Preferably, the amino acid sequence cleavable by the above TEV protease and the sequence of the functional region of the above human native type IV collagen are directly linked.

[0054] 2. The synthesized polynucleotide fragments were each inserted into the pET-28a-Trx-His expression vector (the vector map is shown in Figure 6.) to obtain the corresponding recombinant expression plasmids. Here, the polynucleotide fragments of each recombinant type IV humanized collagen are as follows.

[0055] (1) Polynucleotide sequence of recombinant type IV humanized collagen C007 with an amino acid sequence that can be cleaved by TEV protease added to the N-terminus: GAAAACCTGTATTTCCAGGGTCAGAAAGGAGATCAAGGAGAAAAAGGACAGATAGGACCAATAGGAGAAAAAGGGAGCCGGGGAGATCCGGGAACACCGGGAGTGCCGGGAAAAGATGGACAGGCAGGCCAGCCGGGACAGCCGGGTCCTAAAGGAGATCCGGGTATAAGCGGAACACCGGGTGCACCGGGATTACCTGGTCCGAAAGGGAGCGTAGGGGGAATGGGACTGCCGGGAACACCTGGTGAAAAAGGCGTACCTGGTATTCCGGGTCCTCAGGGGTCCCCGGGACTGCCTGGTGATAAAGGTGCAAAAGGGGAAAAAGGGCAAGCAGGTCCGCCGGGTATTGGTATACCGGGTCTGCGTGGGGAAAAAGGTGATCAGGGTATTGCCGGTTTTCCGGGTAGCCCGGGTGAGAAAGGTGAAAAAGGAAGTATAGGAATACCGGGTATGCCTGGTAGTCCGGGTCTGAAAGGTAGCCCGGGAAGCGTGGGGTATCCGGGTAGCCCTGGTCTGCCTGGGGAAAAAGGCGATAAAGGTCTGCCGGGTCTGGACGGTATTCCTGGTGTTAAAGGGGAAGCAGGTCTGCCGGGGACCCCGGGTCCAACAGGTCCTGCAGGGCAGAAAGGTGAACCAGGTAGCGATGGTATTCCGGGGTCTGCGGGGGAAAAAGGAGAACCGGGTCTGCCGGGAAGAGGTTTTCCGGGCTTTCCGGGTGCAAAAGGAGATAAGGGTAGCAAAGGCGAAGTGGGTTTTCCGGGACTGGCAGGTAGTCCGGGAATCCCTGGTAGCAAAGGTGAACAGGGTTTTATGGGGCCGCCGGGTCCGCAGGGTCAGCCTGGTTTACCGGGTAGCCCAGGTCATGCGACAGAG (SEQ ID NO.4);

[0056]

[0057]

[0058] 3. The successfully constructed expression plasmid was transformed into competent cells BL21(DE3) of Escherichia coli. The specific procedure is as follows. (1) Take out the competent cells BL21(DE3) of Escherichia coli from the ultra-low temperature refrigerator, place them on ice, take 2 μL of the plasmid to be transformed in a semi-thawed state, add it to the Escherichia coli competent cells BL21(DE3), and gently mix 2 - 3 times. (2) After placing the mixture in an ice bath on ice for 30 minutes, perform heat shock in a 42°C water bath for 45 - 90 seconds. After taking it out, place it in an ice bath on ice for 2 minutes. (3) Transfer it to a biosafety cabinet, add 700 μL of liquid LB medium, and then culture it at 37°C and 220 rpm for 60 minutes. (4) Take 200 μL of the bacterial solution and evenly spread it on an LB plate containing kanamycin sulfate. (5) Culture the plate in an incubator at 37°C for 15 - 17 hours until colonies of uniform size grow.

[0059] 4. Pick up 5 - 6 single colonies from the transformed LB plate, transfer them to an Erlenmeyer flask containing the antibiotic stock solution (100 mg / L of kanamycin sulfate), and culture them on a constant temperature shaker at 220 rpm and 37°C until they become foggy. Next, lower the temperature of the Erlenmeyer flask after culture to 16 - 30°C, add IPTG (final concentration 0.5 mM), induce expression for a certain period, then dispense the bacterial solution into a centrifuge bottle, centrifuge at 6000 rpm and 4°C for 12 minutes to collect the bacterial cells, record the weight of the bacteria, collect samples, and perform electrophoresis measurement (the sample name is recorded as "bacterial solution").

[0060] 5. The recovered bacterial cells were resuspended in an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole), the temperature of the bacterial suspension was lowered to 15 °C or below, and it was homogenized twice or the cells were disrupted by ultrasonic waves. After completion, the bacterial suspension was collected and subjected to electrophoresis measurement (the sample names were described as "Homogeneous 1", "Homogeneous 2", or "Ultrasonic" respectively). The bacterial suspension after cell disruption was dispensed into centrifuge bottles and centrifuged at 17,000 rpm at 4 °C for 30 minutes. The supernatant was collected, and electrophoresis measurements were performed on the supernatant and the precipitate (the sample names were described as "Supernatant" and "Precipitate" respectively).

[0061] 6. Recombinant humanized type IV collagen was purified and enzymatically digested. The specific procedure is as follows. (1) Crude purification: a. Equilibration of the column packing material: The column packing material was equilibrated at a flow rate of 10 mL / min using an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole). b. Sample injection: Until the liquid stopped flowing, the supernatant after centrifugation was added to the column packing material at a flow rate of 5 mL / min, and the flow-through was collected for electrophoresis measurement (the sample name was described as "Flow-through"). c. Washing of impurities: Until the liquid flow stopped, 100 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) was added at a flow rate of 10 mL / min, and the flow-through of the impurity washing was collected for electrophoresis measurement (the sample name was described as "Impurity wash"). d. Recovery of the target protein: 20 mL of elution solution (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole) was added at a flow rate of 10 mL / min, the flow-through solution was collected, the protein concentration was measured by ultraviolet-visible spectrophotometry, and the protein concentration was calculated from the formula (C (mg / mL) = A280 × dilution factor × extinction coefficient), and electrophoresis measurement was performed (the sample name was described as "Elution"). e. The column packing material was washed at a flow rate of 10 mL / min using a 1 M imidazole working solution, and electrophoresis measurement was performed on the flow-through (the sample name was described as "1 M wash"). (2) Enzymatic digestion: TEV enzyme was added such that the ratio of the total mass of the protein to the total mass of the TEV enzyme was 20:1, and enzymatic digestion was carried out at 16 °C for 2 hours. Samples before and after digestion were collected for electrophoresis measurement (the sample names were described as "before digestion" and "after digestion", respectively). The protein solution after enzymatic digestion was placed in a dialysis bag, dialyzed at 4 °C for 2 hours, then transferred to a new dialysis solution (20 mM sodium chloride, 20 mM Tris), and dialyzed at 4 °C overnight. A sample of the protein solution was collected for electrophoresis measurement (the sample name was described as "after solution exchange"). (3) Purification: a. Equilibration of the column packing material: The column packing material was equilibrated with Solution A (20 mM Tris, 20 mM sodium chloride) at a flow rate of 10 mL / min. b. Sample injection: The sample was injected at a flow rate of 5 mL / min, and the flow-through was collected for electrophoresis measurement (the sample name was described as "FL"). c. Gradient elution: 0 - 15% aqueous Solution B was set for 2 minutes each, then held for 3 CV, 15 - 30% aqueous Solution B was set for 2 minutes each, then held for 3 CV, 30 - 50% aqueous Solution B was set for 2 minutes each, then held for 3 CV. Here, Solution B contains 1 M sodium chloride and 20 mM Tris, and the above ratios are volume fractions. The peaks were collected for electrophoresis measurement (the sample name was described as "B elution"). d. The column packing material was washed. e. The content of the target protein was measured, the protein yield was calculated, and the protein was stored in an environment at 4 °C. (4) Water exchange: The recovered protein solution was added to a 10 kDa ultrafiltration concentration tube, and concentration and water exchange based on centrifugation at 3500 rpm for 15 minutes were repeated 3 times for electrophoresis measurement (the sample name was described as "water exchange").

[0062] 7. Test results: The electrophoresis detection results in the preparation process of each recombinant protein are shown in Figures 1 - 3. Figure 4 is the comparison result of the stability in the expression and purification of recombinant type IV humanized collagen C4P7C6 and C007.

[0063] Here, a part of Figures 1 and 4 is the electrophoresis detection result of recombinant type IV humanized collagen C007, and it was found that the purification and enzymatic digestion effects were good. Parts of FIGS. 2 and 4 are the results of electrophoretic detection of recombinant type IV humanized collagen C4P7C6, and it was found that the purification and enzymatic digestion effects were good. FIG. 3 is the result of electrophoretic detection of recombinant type IV humanized collagen C4P7E5, and it was found that the purification and enzymatic digestion effects were good.

[0064] Example 2: Measurement of Biological Activity of Recombinant Type IV Humanized Collagen For the method of measuring the activity of collagen, refer to the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004). The specific method is as follows. (1) By the ultraviolet absorption method, the concentrations of commercial human collagen (Sigma, C7774) (as a positive control) and target protein samples containing recombinant type IV humanized collagen C4P7C6, C4P7E5, and C007 with good purification and enzymatic digestion effects provided by the present invention were measured. Specifically, the ultraviolet absorptions of the samples at 215 nm and 225 nm were measured respectively, and the protein concentration was calculated by the empirical formula C (μg / mL) = 144×(A215 - A225). In addition, it is necessary to detect when A215 < 1.5. The principle of this method is to measure the characteristic absorption of peptide bonds under far ultraviolet light, which is not affected by the chromophore content, has few interfering substances, is easy to operate, and is suitable for the detection of human collagen and its analogs that cannot be colored with Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. HumanaPress. 43-45.). After measuring the protein concentration, the protein concentrations of all the samples to be measured were adjusted to 0.5 mg / ml with PBS. (2) 100 μL of various types of protein solutions and blank PBS control solution were added to a 96-well plate and left standing at room temperature for 60 minutes. (3) 10 5 cultured 3T3 cells in good condition were placed in each well and incubated at 37 °C for 60 minutes. (4) Each well was washed 4 times with PBS. (5) Using an LDH detection kit (Roche, 04744926001), the absorbance of OD 492nm was detected. The cell adhesion rate can be calculated from the numerical values of the blank control. The calculation formula is: cell adhesion rate = (test well - blank well) × 100% / (positive well - blank well). The cell adhesion rate can reflect the activity of collagen. The higher the activity of the protein, the better the external environment it can provide for the cells to adhere to the wall in a short time.

[0065] The results are shown in Figure 5. It was found that the recombinant type IV humanized collagen of the present invention has better biological adhesion activity compared to commercially available human collagen, that is, recombinant type IV humanized collagen C4P7C6 > C007 > C4P7E5 > human collagen.

[0066] Example 3: Detection of recombinant type IV humanized collagen by mass spectrometry

[0067]

Table 1

[0068] After the protein sample was treated with DTT reduction and iodoacetamide alkylation, trypsin was added and digested overnight. The peptide fragments obtained after digestion were further desalted with a C18 ZipTip and then mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA) on a plate. Finally, analysis was performed using a matrix-assisted laser desorption ionization-time of flight mass spectrometer MALDI-TOF / TOF UlraflextremeTM, Brucker, Germany (for peptide mass fingerprinting technology, see Protein J. 2016;35:212-7).

[0069] The database search was processed from the MS / MS Ion Search web page of the local mascot site. The protein identification results were obtained based on the primary mass spectrometry of the peptide fragments generated after digestion. Search parameters: Trypsin digestion, two missed cleavage sites were set. The alkylation of cysteine was a defined modification, and the oxidation of methionine was an optional modification. The database used for identification was NCBprot.

[0070]

Table 2

[0071]

Table 3

[0072]

Table 4

Claims

1. A recombinant type IV humanized collagen having an amino acid sequence containing n (n is an integer of 1 or more) repeating sequences, wherein the repeating sequence is the sequence of the functional region of human native type IV collagen, preferably, n is 1, 2, 3, 4, 5, 6, 7 or 8, and when n is an integer of 2 or more, the repeating sequences are directly linked to each other, more preferably, the amino acid sequence of the recombinant type IV humanized collagen is characterized by containing any one of the following (i) to (iii): Recombinant type IV humanized collagen. (i) The amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 (ii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or modified in the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, and which retains the cell adhesion effect of human native type IV collagen (iii) An amino acid sequence that retains the cell adhesion effect of human native type IV collagen and is encoded by the following nucleotide sequence, wherein the nucleotide sequence hybridizes with a polynucleotide sequence encoding the sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 under stringent conditions, and the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions, or ultra-high stringent conditions

2. A recombinant type IV humanized collagen having an amino acid sequence further containing an amino acid sequence cleavable by TEV protease shown in SEQ ID NO. 7, preferably, the amino acid sequence cleavable by the TEV protease and the repeating sequence are directly linked to each other, preferably, the amino acid sequence cleavable by the TEV protease is located at the N-terminus of the recombinant type IV humanized collagen. The recombinant type IV humanized collagen according to Claim 1.

3. A polynucleotide encoding the recombinant type IV humanized collagen according to Claim 2, preferably, a polynucleotide containing the sequences shown in SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO.

6.

4. A recombinant expression vector comprising the polynucleotide according to claim 3, preferably, the recombinant expression vector comprises a pET series vector, a shuttle vector, a phage or a viral vector, more preferably, the recombinant expression vector is pET-28a-Trx-His, characterized in that it is a recombinant expression vector.

5. A recombinant host cell comprising the recombinant expression vector according to claim 4, preferably, the recombinant host cell is a prokaryotic cell, yeast or eukaryotic cell, more preferably, the recombinant host cell is Escherichia coli BL21(DE3), characterized in that it is a recombinant host cell.

6. A method for producing the recombinant type IV humanized collagen according to claim 1 or 2, S1: A step of fermentatively culturing the recombinant host cell according to claim 5 and inducing protein expression; S2: A step of collecting the protein; S3: A step of purifying the protein, and it is preferable to purify the protein using a Ni affinity chromatography column and / or an ion exchange chromatography column, optionally including enzymatically digesting the protein, preferably enzymatically digesting the protein using TEV protease, and more preferably the mass ratio of the protein to the TEV protease is (15-25):

1.

7. In the step S1, the fermentative culture is characterized in that it is carried out in a shaker at a rotational speed of 200-240 rpm and a temperature of 35-38 °C, according to the production method according to claim 6.

8. In the step S1, the specific procedure for inducing protein expression is to lower the temperature to 16-30 °C and add IPTG, preferably, the concentration of IPTG used is 0.3-0.7 mM, according to the production method according to claim 6 or 7.

9. In the S2 step, the specific procedure for collecting the protein is as follows: centrifuge the mixture obtained in the S1 step at 5000-7000 rpm for 10-15 minutes in an environment of 4°C, resuspend the precipitate using an aqueous solution containing 20-30 mM Tris, 150-250 mM sodium chloride, and 15-25 mM imidazole, homogenize it under high pressure, or disrupt the cells by ultrasonic waves, and then centrifuge at 15000-18000 rpm for 20-40 minutes in an environment of 4°C. The manufacturing method according to any one of claims 6 to 8.

10. Use in the manufacture of a product comprising a medical device, a cosmetic, a health food or a pharmaceutical product of the recombinant type IV humanized collagen according to claim 1 or 2.

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