Preparation method of recombinant III-type human collagen

Abstract

The invention discloses a method for preparing recombinant III-type human collagen. The method comprises the following steps: constructing genetically engineered bacteria for expressing proline hydroxylase and full-length recombinant III-type human collagen; and culturing the genetically engineered bacteria to obtain the recombinant III-type human collagen, the proline hydroxylase is prepared from P4Ha2, P4Hb and vP4H; the ratio of the copy number of the P4Ha2 to the copy number of the P4Hb to the copy number of the vP4H to the copy number of the full-length recombinant III type human collagen is (1.80 to 2.20) to (1.80 to 2.20) to (0.80 to 1.60) to 1. The method can be used for large-scale production, the purity is high, and the production cost can be reduced; the prepared recombinant III-type human collagen is completely consistent with the full-length sequence of natural III-type human collagen, the hydroxyproline content of the recombinant III-type human collagen is basically consistent with that of the natural III-type human collagen, and the recombinant III-type human collagen has a triple-helix structure, has the biological activity of the natural III-type human collagen, and is free of immunogenicity and virus hidden danger.

Description

Technical Field

[0001] The present application relates to the field of biomedical materials, and in particular to a method for preparing recombinant type III human collagen. Background Art

[0002] Collagen is the most abundant protein in mammals and the main protein of animal connective tissue. Due to its abundance and mechanical strength, collagen has been widely used in the pharmaceutical and cosmetic industries as well as in research. For example, native collagen is often used as an attachment surface for cell culture experiments, and the properties of collagen-based polymer materials have also attracted much attention in tissue regeneration, drug delivery applications, and elucidating the biochemical and biophysical properties of collagen.

[0003] Collagen is first synthesized into procollagen polypeptide chains (protocallagen) in cells, including N-terminal signal peptide, N-terminal propeptide, N-terminal telopeptide, COL3A1 triple helix region, C-terminal telopeptide, and C-terminal propeptide. The three polypeptide chains are twisted together to form procollagen (procollagen), and then moved to the extracellular space. Under the action of procollagen N protease and procollagen C protease, the N-terminal peptide and C-terminal peptide of procollagen are removed respectively to form tropocollagen molecules (tropocollagen, de-telopeptide collagen). At the same time, proline hydroxylase (P4H) catalyzes proline (Pro) into hydroxyproline (HyP). Tropocollagen molecules self-assemble to form collagen microfibrils, which are further polymerized into collagen fibers and collagen bundles.

[0004] Collagen is rich in amino acids and imino acids such as glycine, proline, and hydroxyproline. It is either a homotrimer formed by three identical α chains (such as [α1]3) or a heterotrimer formed by three different α chains (such as [α1]2(α2) or α1α2α3). The trimer is in the shape of a slender rod and constitutes the basic structural unit of collagen fibers. Each α chain contains about 1,000 amino acids and has a left-handed helical configuration. The three left-handed helical chains are intertwined to form a right-handed helical structure. A single α chain has a repeated amino acid sequence (Gly-XY)n, where X and Y are usually proline and hydroxyproline, respectively. Collagen has a triple helical structure that is unique to collagen. The hydroxyl group on hydroxyproline is crucial for the formation of intrachain and interchain hydrogen bonds, maintaining the stability of the collagen triple helical structure, and forming stable collagen fibers to play a biological role in the body.

[0005] Since collagen and collagen-like proteins are beneficial to the human body, they are in great demand. Animal collagen, which is usually extracted from animal bones and skins, has a full-length triple helix structure and complete biological activity, but has extremely high requirements for the source, low purity, high immunogenicity, the presence of animal viruses, certain safety risks, and high production costs. Therefore, it is urgent to provide a method for preparing recombinant type III human collagen with a highly hydroxylated, full-length triple helix structure. Summary of the invention

[0006] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a method for preparing recombinant type III human collagen, wherein the recombinant type III human collagen prepared by the method has a high proline hydroxylation level and a complete triple helical structure.

[0007] This application is completed based on the following findings of the inventors:

[0008] Most recombinant collagens on the market are human-like or humanized collagens, which are mainly fragments and do not have the complete biological activity of collagen. Since the full-length gene sequence of collagen is very long (about 4000bp), the amino acid sequence consists of a large number of repeated sequences (Gly-XY)n, and a large amount of hydroxyproline (HyP) is required to maintain the triple helix structure and functional properties, human proline hydroxylase (P4H) is a tetramer structure (α2β2) and must be in a complete tetramer structure to function. Although many species have been tried to express collagen, none of them can fully achieve the expression of full-length collagen with a complete triple helix structure.

[0009] Most of the methods for preparing recombinant human-like collagen disclosed so far are fragmented repetitive sequences, which do not have the sequence and structure of full-length human collagen, and without the insertion of tool enzymes, the conversion of proline to hydroxyproline cannot be guaranteed. Alternatively, collagen is obtained from cowhide and pig skin as raw materials, which has a full-length sequence and triple helix structure, but its source requirements are extremely high, the production scale is limited, and it also has high immunogenicity and the risk of animal-derived viruses.

[0010] The ideal preparation of recombinant type III human collagen should have the following characteristics: (1) full-length sequence of human collagen, (2) triple helical structure, complete biological activity, and strong stability, (3) scalable preparation with high purity, and (4) non-immunogenicity.

[0011] Based on this, in the first aspect of the present application, the present application proposes a method for preparing recombinant type III human collagen. According to an embodiment of the present application, the method comprises:

[0012] Constructing genetically engineered bacteria that express proline hydroxylase and full-length recombinant human type III collagen;

[0013] Cultivating the genetically engineered bacteria, and digesting the culture product with pepsin to obtain the recombinant type III human collagen;

[0014] Wherein, the amino acid sequence of the recombinant type III human collagen is shown in SEQ ID NO: 1;

[0015] The proline hydroxylases include P4Ha2, P4Hb and vP4H;

[0016] The copy number ratio of P4Ha2, P4Hb, vP4H and full-length recombinant type III human collagen is (1.80-2.20): (1.80-2.20): (0.80-1.60): 1.

[0017] The preparation method of the present application can be mass-produced with high purity and greatly reduces production costs. In addition, the sequence of the recombinant type III human collagen prepared by the above method is completely consistent with the full-length sequence of natural type III human collagen, the hydroxyproline content is basically consistent with that of natural type III human collagen, and it has a triple helix structure and the biological activity of complete natural type III human collagen. Since it is a completely human sequence, it has no immunogenicity and no virus risks.

[0018] In the second aspect of the present application, the present application proposes a recombinant human type III collagen. According to the embodiments of the present application, the recombinant human type III collagen is prepared by the method described in the first aspect. Thus, the sequence of the recombinant human type III collagen of the present application is completely consistent with the full-length sequence of natural human type III collagen, the hydroxyproline content is basically consistent with that of natural human type III collagen, and it has a triple helix structure and the biological activity of complete natural human type III collagen. Since it is a completely human sequence, it has no immunogenicity and no virus risks.

[0019] In the third aspect of the present application, the present application proposes a composition. According to an embodiment of the present application, the composition comprises recombinant type III human collagen prepared by the method described in the first aspect. Therefore, the composition of the present application has no immunogenicity and no virus risk.

[0020] Beneficial effects:

[0021] 1. The preparation method of the present application adopts Pichia pastoris expression strain as the expression system, and successfully inserts four genes of recombinant type III human collagen (COL3A1), human proline hydroxylase (P4Ha2 and P4Hb), and viral proline hydroxylase (vP4H). Through screening of gene copy number and hydroxyproline content, the expressed recombinant type III human collagen sequence is completely consistent with the full-length sequence of natural type III human collagen, and the hydroxyproline content is basically consistent with that of natural type III human collagen, and has a triple helix structure and the biological activity of complete natural type III human collagen. Since it is a completely human sequence, it has no immunogenicity and no viral risks.

[0022] 2. The preparation method of the present application can be mass-produced with high purity, while greatly reducing production costs.

[0023] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0025] Figure 1 The plasmid map of the human proline hydroxylase P4Hb gene constructed in Example 1 of the present application is shown;

[0026] Figure 2 The plasmid map of the human proline hydroxylase P4Ha2 gene constructed in Example 1 of the present application is shown;

[0027] Figure 3 The WB detection diagram of the expression proteins of human proline hydroxylase P4Hb and P4Ha2 in Example 1 of the present application is shown;

[0028] Figure 4 The plasmid map of the full-length recombinant type III human collagen COL3A1 gene constructed in Example 2 of the present application is shown;

[0029] Figure 5 The electrophoresis diagram of the expression of full-length recombinant type III human collagen COL3A1 in Example 2 of the present application is shown;

[0030] Figure 6 The plasmid map of the viral proline hydroxylase vP4H gene constructed in Examples 4 and 5 of the present application is shown;

[0031] Figure 7 The WB detection diagram of the viral proline hydroxylase vP4H expression protein in Example 5 of the present application is shown;

[0032] Figure 8 The figure shows the result of identifying the N / C-terminal sequence of recombinant type III human collagen using LC / MS technology in Example 7 of the present application;

[0033] Fig. 9 The sequence coverage result diagram of recombinant type III human collagen using LC / MS technology in Example 7 of the present application is shown;

[0034] Fig.10 The electrophoresis (SDS-PAGE) result diagram of the recombinant type III human collagen in Example 7 of the present application is shown;

[0035] Fig.11 The purity (SEC-HPLC) result graph of the recombinant type III human collagen in Example 7 of the present application is shown;

[0036] Fig.12 The circular dichroism (CD) result diagram of the recombinant type III human collagen in Example 7 of the present application is shown;

[0037] Fig.13 The graph shows the self-assembly ability results of the recombinant type III human collagen in Example 7 of the present application. DETAILED DESCRIPTION

[0038] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0039] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0040] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0041] In order to make the present application more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present application belongs.

[0042] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0043] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0044] The present application proposes a method for preparing recombinant type III human collagen, which will be described in detail below.

[0045] Preparation method of recombinant type III human collagen

[0046] In the first aspect of the present application, the present application proposes a method for preparing recombinant type III human collagen. According to an embodiment of the present application, the method comprises:

[0047] Constructing genetically engineered bacteria that express proline hydroxylase and full-length recombinant human type III collagen;

[0048] Cultivating the genetically engineered bacteria, and digesting the culture product with pepsin to obtain the recombinant type III human collagen;

[0049] Wherein, the amino acid sequence of the recombinant type III human collagen is shown in SEQ ID NO: 1;

[0050] The proline hydroxylases include P4Ha2, P4Hb and vP4H;

[0051] The copy number ratio of P4Ha2, P4Hb, vP4H and full-length recombinant type III human collagen is (1.80-2.20): (1.80-2.20): (0.80-1.60): 1.

[0052] The preparation method of the present application can be mass-produced with high purity and greatly reduces production costs. In addition, the sequence of the recombinant type III human collagen prepared by the above method is completely consistent with the full-length sequence of natural type III human collagen, the hydroxyproline content is basically consistent with that of natural type III human collagen, and it has a triple helix structure and the biological activity of complete natural type III human collagen. Since it is a completely human sequence, it has no immunogenicity and no virus risks.

[0053]

[0054] According to an embodiment of the present application, the above method may further include at least one of the following technical features:

[0055] According to an embodiment of the present application, the copy number ratio of P4Ha2, P4Hb, vP4H and full-length recombinant type III human collagen is (1.80-2.20): (1.80-2.20): (0.80-1.60): 1. Thus, the copy number ratio of P4Ha2 to full-length recombinant type III human collagen is 1.80:1, 1.85:1, 1.90:1, 1.95:1, 2.00:1, 2.05:1, 2.10:1, 2.15:1, 2.20:1 or a range value between any two endpoint values ​​therebetween; the copy number ratio of P4Hb to full-length recombinant type III human collagen is 1.80:1, 1.85:1, 1.90:1, 1.95:1, 2.00:1, 2.05:1, 2.10:1, 2.15:1, 2.20:1 or a range value between any two endpoint values ​​therebetween. 1, 2.20:1 or a range between any two endpoint values ​​therebetween; the copy number ratio of vP4H to full-length recombinant human type III collagen is 0.80:1, 0.85:1, 0.90:1, 0.95:1, 1.00:1, 1.05:1, 1.10:1, 1.15:1, 1.20:1, 1.25:1, 1.30:1, 1.35:1, 1.40:1, 1.45:1, 1.50:1, 1.55:1, 1.60:1 or a range between any two endpoint values ​​therebetween.

[0056] According to an embodiment of the present application, the copy number ratio of P4Ha2, P4Hb, vP4H and full-length recombinant type III human collagen is (1.90-2.10): (1.90-2.10): (0.90-1.10): 1.

[0057] According to an embodiment of the present application, the amino acid sequence of P4Ha2 is shown in SEQ ID NO:2.

[0058] MKLWVSALLMAWFGVLSCVQAEFFTSIGHMTDLIYAEKELVQSLKEYILVEEAKLSKIKSWANKMEALTSKSAADAEGYLAHPVNAYKLVKRLNTDWPALEDLVLQDSAAGFIANLSVQRQFFPTDEDEIGAAKALMRLQDTYRLDPGTISRGELPGTKYQAMLSVDDCFGMGRSAYNEGDYYHTVLWMEQVLKQLDAGEEATTTKSQVLDYLSYAVFQLGDLHRALELTRRLLSLDPSHERAGGNLRYFEQLLEEEREKTLTNQTEAELATPEGIYERPVDYLPERDVYESLCRGEGVKLTPRRQKRLFCRYHHGNRAPQLLIAPFKEEDEWDSPHIVRYYDVMSDEEIERIKEIAKPKLARATVRDPKTGVLTVASYRVSKSSWLEEDDDPVVARVNRRMQHITGLTVKTAELLQVANYGVGGQYEPHFDFSRNDERDTFKHLGTGNRVATFLNYMSDVEAGGATVFPDLGAAIWPKKGTAVFWYNLLRSGEGDYRTRHAACPVLVGCKWVSNKWFHERGQEFLRPCGS

[0059] TEVD (SEQ ID NO:2).

[0060] According to an embodiment of the present application, the amino acid sequence of the P4Hb is as shown in SEQ ID NO:3.

[0061] MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNW LKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTH ILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDE KKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDEL(SEQ ID NO:3).

[0062] According to an embodiment of the present application, the amino acid sequence of the vP4H is shown in SEQ ID NO:4.

[0063] MHHHHHHGGSEGFETSDRPGVCDGKYYEKIDGFLSDIECDVLINAAIKKGLIKSEVGGATENDPIK LDPKSRNSEQTWFMPGEHEVIDKIQKKTREFLNSKKHCIDKYNFEDVQVARYKPGQYYYHHYDGDDC DDACPKDQRLATLMVYLKAPEEGGGGETDFPTLKTKIKPKKGT (SEQ ID NO: 4).

[0064] According to an embodiment of the present application, the amino acid sequence of the full-length recombinant type III human collagen is shown in SEQ ID NO:5.

[0065]

[0066] According to an embodiment of the present application, the amount of pepsin added to the culture product is 100-500 U / L.

[0067] According to an embodiment of the present application, the temperature of the enzymatic cleavage treatment is 2 to 10°C.

[0068] According to an embodiment of the present application, the time of the enzymatic digestion treatment is 16 to 30 hours.

[0069] According to an embodiment of the present application, the genetically engineered bacteria is selected from a Pichia pastoris strain. For example, the Pichia pastoris strain includes but is not limited to a Pichia pastoris strain GS115, a Pichia pastoris strain X33, a Pichia pastoris strain GS190, and a Pichia pastoris strain KM71. According to an optional embodiment of the present application, the Pichia pastoris strain is selected from a Pichia pastoris strain GS115.

[0070] According to an embodiment of the present application, the method further comprises:

[0071] The enzyme cleavage product is purified.

[0072] According to an embodiment of the present application, the purification treatment is to perform microfiltration treatment, salting-out treatment, chromatography treatment, and ultrafiltration treatment in sequence.

[0073] According to an embodiment of the present application, the microfiltration treatment is performed using a filter membrane with a pore size of 0.22 μm or more, thereby removing cell impurities.

[0074] According to an embodiment of the present application, the salting-out treatment is performed using a 0.5-3 mol / L sodium chloride solution. According to an embodiment of the present application, the salting-out treatment lasts for 16-30 hours. Thus, a recombinant type III human collagen precipitate can be obtained.

[0075] According to an embodiment of the present application, the chromatography treatment is performed by ion exchange chromatography, thereby removing non-recombinant type III human collagen impurities.

[0076] According to an embodiment of the present application, the ultrafiltration treatment is performed using a cut-off filter membrane with a molecular weight of 50 kD or above, thereby obtaining a retentate to concentrate the recombinant type III human collagen.

[0077] According to an embodiment of the present application, the amino acid sequence of the recombinant type III human collagen is shown in SEQ ID NO: 2. The recombinant type III human collagen of the present application is 100% homologous to the natural type III human collagen sequence, thereby further avoiding adverse reactions such as allergies caused by the heterogeneity of the collagen solution and reducing immune rejection reactions.

[0078] According to an embodiment of the present application, the purity of the recombinant type III human collagen is greater than 95%.

[0079] According to the embodiments of the present application, the hydroxyproline content in the recombinant type III human collagen is greater than 16.5%. The degree of proline hydroxylation of the recombinant type III human collagen in the present application is close to that of natural type III human collagen, thereby further reducing adverse reactions such as allergies caused by the heterologous solution and reducing immune rejection reactions.

[0080] In the present context, the terms "hydroxyproline content" and "degree of proline hydroxylation" are used synonymously.

[0081] According to the embodiments of the present application, the hydroxyproline content in the recombinant type III human collagen is 16.6% to 17.5%. The hydroxyl group on hydroxyproline is crucial for the formation of intra-chain and inter-chain hydrogen bonds and maintaining the stability of the triple helix structure of collagen to play a biological role in the body. When the degree of proline hydroxylation is 16.6%-17.5%, preferably 16.6%-16.9%, it is beneficial for the recombinant type III human collagen to form a triple helix structure and maintain the stability of the triple helix structure.

[0082] According to the embodiments of the present application, the recombinant type III human collagen has a complete triple helix structure. The recombinant type III human collagen in the present application has a complete triple helix structure, which is close to the natural type III human collagen, thereby further avoiding adverse reactions such as allergies caused by the heterogeneity of the solution and reducing immune rejection reactions.

[0083] According to an embodiment of the present application, in the circular dichroism spectrum, the recombinant type III human collagen has a negative absorption peak at a wavelength of 190 to 200 nm, and a positive absorption peak at a wavelength of 210 to 230 nm.

[0084] It should be noted that it is well known to those skilled in the art that the presence of both a negative absorption peak near 197 nm and a positive absorption peak near 221 nm in the circular dichroism spectrum is the gold standard for collagen to have a correct and complete triple helix structure.

[0085] According to an embodiment of the present application, the recombinant type III human collagen has a negative absorption peak at a wavelength of 192 to 198 nm.

[0086] According to an embodiment of the present application, the recombinant type III human collagen has a positive absorption peak at a wavelength of 215 to 225 nm.

[0087] In an optional embodiment of the present application, the recombinant type III human collagen consistent with natural type III human collagen in the present application must have the following characteristics: (1) Structural composition, 100% homologous to the natural type III human collagen sequence, including complete consistency with the full-length amino acid sequence composition and collagen length of natural type III human collagen; (2) The degree of post-translational modification, that is, the degree of proline hydroxylation is close to or consistent with that of natural type III human collagen, which is an important basis for the formation of the triple helix structure of collagen; (3) It has a complete triple helix structure, which is a necessary basis and condition for natural collagen to exert its biological effects in vivo; (4) It has biological activity compared with animal-derived collagen.

[0088] Recombinant human type III collagen and compositions

[0089] In the second aspect of the present application, the present application proposes a recombinant human type III collagen. According to the embodiments of the present application, the recombinant human type III collagen is prepared by the method described in the first aspect. Thus, the sequence of the recombinant human type III collagen of the present application is completely consistent with the full-length sequence of natural human type III collagen, the hydroxyproline content is basically consistent with that of natural human type III collagen, and it has a triple helix structure and the biological activity of complete natural human type III collagen. Since it is a completely human sequence, it has no immunogenicity and no virus risks.

[0090] In the third aspect of the present application, the present application proposes a composition. According to an embodiment of the present application, the composition comprises recombinant type III human collagen prepared by the method described in the first aspect. Therefore, the composition of the present application has no immunogenicity and no virus risk.

[0091] According to an embodiment of the present application, the composition further includes an auxiliary material.

[0092] According to an embodiment of the present application, the composition is selected from a recombinant type III human collagen hydrogel, a recombinant type III human collagen repair solution, a recombinant type III human collagen solution, or a recombinant type III human collagen dressing.

[0093] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0094] In this embodiment, the Pichia pastoris strain GS115 expression system was used, and the synthesis of the full-length recombinant type III human collagen gene sequence was entrusted to Beijing Anbiqi Biotechnology Co., Ltd. The vector construction, strain screening, and recombinant type III human collagen preparation were all completed by our company. The specific steps are as follows:

[0095] Example 1: Construction of Pichia pastoris strain containing human proline hydroxylase

[0096] The P4H hydroxylase gene (the amino acid sequence of P4Ha2 is shown in SEQ ID NO: 2, and the nucleotide sequence encoding P4Ha2 is shown in SEQ ID NO: 6; the amino acid sequence of P4Hb is shown in SEQ ID NO: 3, and the nucleotide sequence encoding P4Hb is shown in SEQ ID NO: 7) was synthesized.

[0097]

[0098]

[0099] Construction of GS115-P4Hb strain expressing human proline hydroxylase P4Hb protein:

[0100] AOX1 promoter gene and α-factor secretion signal gene were synthesized upstream of P4Hb gene, and inserted into pGAPZA between Bgl II and Not I by double restriction enzyme digestion to construct pPIC-P4Hb plasmid. The schematic diagram is shown in Figure 1 . Using plasmid pPIC-P4Hb as a template, the fragment was amplified with primers AOX1F11: AAACGCTGTCTTGGAACCTAATATGACAAAAG (SEQ ID NO: 8) and AOX1R11: AAACTGTCAGTTTTGGGCCATTTGGGGAACATG (SEQ ID NO: 9), and the fragment was recovered by agarose gel electrophoresis and electrotransformed into Pichia pastoris GS115. YPD plates containing 50 μg / ml bleomycin (Zeo) were cultured at 28°C for 2-3 days to screen positive colonies. Single clones on the YPD screening plate were picked and cultured in BMGY medium. Samples were taken after induced expression for 72 hours, and protein immunoblotting (WB) detection was performed using anti-P4Hb antibody. The spectrum is shown as follows Figure 3 , indicating that the constructed strain GS115-P4Hb successfully expressed the P4Hb protein.

[0101] Construction of GS115-P4Hb-P4Ha2 strain expressing human proline hydroxylase P4Hb protein and P4Ha protein:

[0102] Add the Strep tag sequence to the C-terminus of the P4Ha2 gene (with its natural signal peptide at the N-terminus) and insert it into the multiple cloning site of the Glyco13 plasmid to obtain the pPIC-P4Ha2 plasmid. Figure 2 The plasmid pPIC-P4Ha2 was linearized by PmeI digestion, and the fragment was recovered by agarose gel electrophoresis. The fragment was electroporated into Pichia pastoris GS115-P4Hb, and cultured on a YPD plate containing 100 μg / ml nourseothricin at 28°C for 2-3 days to screen positive colonies. The single clone on the YPD screening plate was selected and cultured in BMGY medium. After induced expression for 72 hours, samples were taken and protein immunoblotting (WB) was performed using anti-Strep-tag antibodies. The pattern is shown in the figure. Figure 3 , indicating that the constructed strain GS115-P4Hb-P4Ha2 successfully expressed the P4Ha2 protein.

[0103] Example 2: Construction of a Pichia pastoris strain containing human proline hydroxylase and full-length recombinant human type III collagen

[0104] The amino acid sequence of full-length recombinant type III human collagen comes from UniProtKB-P02461, which is natural type III human collagen with a total length of 1466 amino acids. After removing the N-terminal telopeptide and C-terminal telopeptide that are prone to cause immunogenicity, the sequence length is 1061 amino acids (i.e., recombinant type III human collagen). The theoretical degree of hydroxylation is 16.9%. The amino acid sequence of full-length recombinant type III human collagen is shown in SEQ ID NO:5, the amino acid sequence encoding the recombinant type III human collagen with the N-terminal telopeptide and C-terminal telopeptide removed is shown in SEQ ID NO:1 (i.e., the sequence length is 1061 amino acids), and the nucleotide sequence encoding the amino acid sequence of full-length recombinant type III human collagen is shown in SEQ ID NO:10.

[0105]

[0106]

[0107] Construction of pPIC3.5K-Col3A1 plasmid expressing full-length recombinant type III human collagen COL3A1: The gene sequence of full-length recombinant type III human collagen (nucleotide sequence as shown in SEQ ID NO: 10) was inserted between the BamH I and Not I sites of the pPIC3.5 plasmid to obtain the plasmid pPIC3.5K-Col3A1, as shown in the map Figure 4 .

[0108] Construction of GS115-COL3A1-P4Hb-P4Ha2 strain expressing full-length recombinant type III human collagen COL3A1, human proline hydroxylase P4Hb protein and P4Ha protein: plasmid pPIC3.5K-Col3A1 was used as a template, primers AOXF33: TTTGGTTCGTTGAAATGCTAACGGCCAGTTGGT (SEQ ID NO: 11) and AOXR33: CTTAGTTCATCTTGGATGAGATCACGCTTTTGTC (SEQ ID NO: 12) were used to amplify the fragment, and the linearized pPIC3.5k-Col3A1 fragment was purified and recovered by ethanol precipitation. Pichia pastoris GS115-P4Hb-P4Ha2 was transformed by electroporation, and cultured on YPD plates containing 600 μg / ml geneticin G418 at 28°C for 2-3 days, and positive colonies were screened. The single clones on the YPD screening plate were selected and cultured in BMGY medium. Samples were taken after 72 hours of induction and tested by electrophoresis SDS-PAGE. The pattern is shown in the figure. Figure 5 It can be seen that there is protein expression above the molecular weight of 245kD on the electrophoresis graph, and the molecular weight is significantly greater than the single-chain 130kD, indicating that after the full-length recombinant type III human collagen is expressed in Pichia pastoris, a triple helical structure is formed, indicating that the constructed strain GS115-COL3A1-P4Hb-P4Ha2 successfully expresses the full-length recombinant type III human collagen.

[0109] Example 3: Screening of strains producing highly hydroxylated, full-length triple-helical recombinant human type III collagen

[0110] For the Pichia pastoris strain GS115-COL3A1-P4Hb-P4Ha2 containing human proline hydroxylase and full-length recombinant human type III collagen co-expressed constructed in Example 2, relative quantitative PCR was used to detect the plasmid copy number to confirm the strain with the highest degree of hydroxylation corresponding to the optimal insertion ratio of the three genes.

[0111] 1. Yeast genomic DNA extraction: Use a yeast genomic DNA extraction kit and follow the instructions to extract yeast whole genome DNA. Use the Nanodrop one device to measure the DNA concentration of the extracted DNA.

[0112] 2. Gene copy number detection: Relative quantitative PCR ΔΔCt method was used. For the primers used in each gene PCR, two sets of primers were provided (see Table 1, including upstream primers and downstream primers). Each set of primers was selected for detection. For each gene, a 25 μl PCR reaction solution was prepared as follows: TBGreen Premix Ex Taq (2X) (Tli RNaseH Plus), ROX plus (2×) 12.5 μl, 1 μl of each upstream and downstream primer (10 μmol / L) of each gene, 9.5 μl of sterile water, and 1 μl of genomic DNA extracted from each strain (prepared to 10 ng / ml) were added to a 96-well PCR reaction plate in sequence. Duplicate wells were set up. After addition, the wells were sealed with a sealing film and centrifuged instantly to centrifuge the liquid in each tube to the bottom of the well. The wells were placed in a fluorescent quantitative PCR instrument, and the ΔΔCt method was selected for operation. The PCR conditions were set to preheat at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, and then the melting curve program (Melt curve), after the overall reaction was completed, GAP gene was selected as the internal reference gene, and one strain (serial number 1) was selected as the control sample for analysis, and the mean Ct value of each group of genes was calculated (Ct value: the number of cycles experienced when the fluorescence signal in each reaction tube reached the set threshold value); the first ΔCt value was calculated, that is, the Ct value of the target gene to be tested in each group minus the Ct value of the internal reference gene, and then each ΔCt value was subtracted from the ΔCt value of the control group just calculated to obtain the ΔΔCt value, and then the relative expression (2^ -ΔΔCt The gene copy number results of each sample are shown in Table 2.

[0113] Table 1: Quantitative PCR primer information for each gene

[0114] Primer name Primer sequences P4Ha2-F1 TTGCCAACTTGTCCGTTCAG P4Ha2-R1 TCAACGGACAACATGGCTTG P4Ha2-F2 GGCCTTGATGAGATTGCAGG P4Ha2-R2 AGTCACCCTCGTTGTAAGCA P4Hb-F1 CCGAAGAATTGACTGCCGAG P4Hb-R1 CTTAACTGGCTGCTTGTCCC P4Hb-F2 TGTCCAGCCGTCAGATTGAT P4Hb-R2 GGTGTGGCTTGATCTTTCCC COL3A1-F1 ACCCGGTTTTAGAGGTCCTG COL3A1-R1 GCATACCTCTCATTCCGGGA COL3A1-F2 TCCTGGATCTAATGGTGCCC COL3A1-R2 GGACCCATTTCGCCTTTACC GAP-F1 TCTTCCACTGGTGCTGCTAA GAP-R1 ACACCCTTGAGCTTACCCTC GAP-F2 GGCGAACACCTTTCCCAATT GAP-R2 GTCCAATACGTCCGAAACCG

[0115] Note: GAP is a Pichia pastoris housekeeping gene, P4Ha2 and P4Hb are two human proline hydroxylase genes, and COL3A is a full-length recombinant type III human collagen gene.

[0116] Table 2: Detection results of gene copy number and hydroxyproline content of each strain

[0117]

[0118]

[0119] 3. Hydroxyproline content detection

[0120] Hydroxyproline is a specific amino acid contained in collagen, and its content is positively correlated with the stability of the triple helix structure formed by collagen. The sample to be tested (i.e., the full-length recombinant type III human collagen prepared in step 2 of this embodiment) is hydrolyzed into hydroxyproline at 105°C and 6 mol / L hydrochloric acid. Hydroxyproline is oxidized by chloramine T and reacts with p-dimethylaminobenzaldehyde to form a red compound, which is measured colorimetrically at a wavelength of 560 nm. The detection method is as follows:

[0121] 3.1 Solution preparation:

[0122] Hydrochloric acid solution, c(HCl) = 6 mol / L: Mix equal volumes of high-purity hydrochloric acid and water;

[0123] pH = 6.0 buffer: weigh 57g sodium acetate trihydrate, 37.5g trisodium citrate, 5.5g citric acid monohydrate, 385ml isopropanol, add 500ml water, adjust pH to 6.0 with citric acid monohydrate, dilute to 1000ml with water; Chloramine T solution: weigh 3.5g chloramine T, dilute to 50mL with water;

[0124] Oxidant solution: Mix chloramine T solution and pH 6.0 buffer in a ratio of 1:4; 60% perchloric acid solution: Measure 43 ml of perchloric acid and dilute to 50 ml with water;

[0125] p-Dimethylaminobenzaldehyde solution: weigh 10g p-dimethylaminobenzaldehyde and dissolve it in 15ml 60% perchloric acid solution;

[0126] Color developer: Measure 15 ml of p-dimethylaminobenzaldehyde solution and dissolve it in 65 ml of isopropanol; Sodium hydroxide solution, c(NaOH)=6 mol / L: Weigh 24 g of sodium hydroxide and dilute it to 100 ml with water.

[0127] 3.2 Preparation of L-hydroxyproline reference solution:

[0128] L-Hydroxyproline reference stock solution: Take L-Hydroxyproline reference, weigh accurately, dissolve in water and quantitatively dilute to a solution containing 20ug per 1ml;

[0129] L-Hydroxyproline reference substance series solution: Accurately measure 2.5ml, 3.75ml, 5.0ml, 7.5ml, and 10.0ml of L-Hydroxyproline reference substance stock solution into 10ml volumetric flasks respectively, make up to volume with water, and prepare L-Hydroxyproline reference substance series solution.

[0130] 3.3 Preparation of test samples: Take the full-length recombinant type III human collagen samples of each strain obtained according to step 2 of this example, place them in a hydrolysis tube, accurately weigh, add an appropriate amount of 6 mol / L HCl, and seal the tube with nitrogen; hydrolyze at 105°C for 22h-24h, cool, transfer the hydrolyzate to a volumetric flask, add water to wash the hydrolysis tube, combine the washings and transfer them to a volumetric flask, add 2 drops of phenolphthalein indicator, add 6 mol / L NaOH until the solution turns pink, make up to volume with water, shake well, and prepare the test solution.

[0131] 3.4 Detection: Accurately measure 0.5 ml of blank (water), hydroxyproline reference solution series and test solution, add 1 ml of isopropanol and 0.5 ml of oxidant solution respectively, mix and place at room temperature for 4 minutes, then add 6.5 ml of color developer respectively, mix, place each tube in a 60°C water bath and heat for 15 minutes, and cool. Use blank as control, measure absorbance at 560 nm, perform linear regression on absorbance with hydroxyproline reference solution series concentration, obtain linear regression equation, and calculate hydroxyproline content in test solution. The results of hydroxyproline content of each sample are shown in Table 2.

[0132] From the results of gene copy number and hydroxyproline content, it can be seen that when the copy number of P4Ha2 gene and P4Hb gene is controlled at 1:1, and the copy number of P4Ha2 gene and P4Hb gene is twice the copy number of COL3A1 gene, a relatively high hydroxylation degree of recombinant type III human collagen can be obtained. That is, the target strain of recombinant type III human collagen with high hydroxylation can be obtained by the gene copy number ratio, but the hydroxylation degree cannot reach 16.5% or above.

[0133] Example 4: Construction of yeast strain for co-expression of virus-derived proline hydroxylase gene and recombinant human type III collagen gene

[0134] The pPIC3.5K-Col3A1 plasmid expressing recombinant type III human collagen COL3A1 constructed in Example 2 was transformed into the GS115 strain according to the transformation operation in Example 2 to obtain the GS115-COL3A1 strain.

[0135] Construction of pPIC6A-vP4H plasmid: Synthesize the vP4H gene sequence (amino acid sequence as shown in SEQ ID NO:4, nucleotide sequence as shown in SEQ ID NO:13), N-terminal contains His Tag tag for protein expression screening, insert into the EcoRI and Xho I sites of pPIC6A plasmid, obtain plasmid pPIC6A-vP4H, plasmid map as shown Figure 6 .

[0136] ATGCATCACCACCATCATCATGGAGGGTCAGAAGGGTTTGAGACCTCCGACAGACCAGGTGTTTGCGATGGTAAGTACTATGAGAAAATCGACGGATTCCTCTCGGATATTGAGTGTGACGTACTAATCAATGCAGCTATAAAGAAAGGCTTGATAAAGTCAGAGGTTGGTGGCGCTACCGAAAACGATCCTATAAAACTGGACCCAAAGTCACGGAATAGCGAACAAACGTGGTTCATGCCCGGAGAGCACGAAGTCATAGATAAGATACAGAAGAAAACTAGGGAATTTCTCAATAGTAAAAAGCACTGTATCGATAAATATAACTTCGAGGATGTGCAGGTCGCCAGATATAAGCCTGGTCAATACTACTATCATCACTACGATGGAGACGATTGCGACGACGCGTGCCCCAAAGACCAACGACTTGCCACATTGATGGTATACTTAAAAGCCCCGGAGGAAGGTGGCGGCGGGGAAACGGACTTCCCAACATTAAAAACCAAGATCAAACCGAAGAAAGGAACTTCTATTTTTTTTTGGGTGGCTGATCCTGTGACACGGAAACTTTATAAGGAGACTCTGCATGCTGGGTTACCTGTTAAGTCCGGAGAAAAGATAATTGCGAACCAGTGGATTCGCGCAGTAAAGTAA(SEQ ID NO:13)。

[0137] Construction of GS115-COL3A1-vP4H strain: plasmid pPIC6A-vP4H was used as a template, and the primers GAPFc1: ACGTTGCGGGTAAAACGGAGGTCGTGTACCCGACCTAGC (SEQ ID NO: 14) and GAPRc1: AATGCTGGGAAGAGCATTGCTGCAAGGGGGCCGTAGAA (SEQ ID NO: 15) were used to amplify the fragment, and the linearized pPIC6A-vP4H fragment was recovered. Pichia pastoris GS115-COL3A1 was electrotransformed, and the YPD plate containing 300 μg / ml blasticidin was used for constant temperature culture at 28°C for 2-3 days to screen the positive GS115-COL3A1-vP4H strain. The single clones on the YPD screening plate were picked and cultured in BMGY medium. Samples were taken after 72 hours of induction expression, and protein immunoblotting (WB) detection was performed using anti-His-Tag antibody. The gene copy number and hydroxyproline content of the full-length recombinant type III human collagen expressed by the GS115-COL3A1-vP4H strain were detected according to the method in Example 3.

[0138] The results showed that the prepared recombinant type III human collagen had the triple helix structure characteristics of the full-length sequence. When the gene copy number ratio of COL3A1 gene:vP4H gene was close to 1:1, the hydroxyproline content could reach 15.9%, which was basically consistent with the characteristics of the recombinant type III human collagen expressed by the GS115-COL3A1-P4Hb-P4Ha2 strain, but the degree of hydroxylation could not reach 16.5% or above.

[0139] Example 5: Further insertion of viral proline hydroxylase gene

[0140] Based on the construction and screening of the highly hydroxylated, triple-helix structured full-length recombinant type III human collagen strain GS115-COL3A1-P4Hb-P4Ha2 (the gene copy number ratio of P4Ha2 gene: P4Hb gene: COL3A1 gene is 1.98:1.93:1, i.e. strain number 8) in Example 3, the vP4H gene was further inserted into the strain to increase the hydroxyproline level of the recombinant type III human collagen.

[0141] Construction of pPIC6A-vP4H plasmid: Synthesize the vP4H gene sequence (amino acid sequence as shown in SEQ ID NO:4, nucleotide sequence as shown in SEQ ID NO:13), N-terminal contains His Tag tag for protein expression screening, insert into the EcoRI and Xho I sites of pPIC6A plasmid, obtain plasmid pPIC6A-vP4H, plasmid map as shown Figure 6 .

[0142] Construction of GS115-COL3A1-P4Hb-P4Ha2-vP4H strain: plasmid pPIC6A-vP4H was used as template, primers GAPFc1: ACGTTGCGGGTAAAACGGAGGTCGTGTACCCGACCTAGC (SEQ ID NO: 14) and GAPRc1: AATGCTGGGAAGAGCATTGCTGCAAGGGGGCCGTAGAA (SEQ ID NO: 15) were used to amplify the fragment, and the linearized pPIC6A-vP4H fragment was recovered. Pichia pastoris GS115-COL3A1-P4Hb-P4Ha2 was electrotransformed, and cultured at 28°C for 2-3 days on a YPD plate containing 300 μg / ml blasticidin, and the positive GS115-COL3A1-P4Hb-P4Ha2-vP4H strain was screened. The monoclones on the YPD screening plate were selected and cultured in BMGY medium. Samples were taken after 72 hours of induction and Western blotting (WB) was performed using anti-His-Tag antibody. The pattern is shown in Figure 2. Figure 7 , indicating that the constructed strain GS115-COL3A1-P4Hb-P4Ha2-vP4H successfully expressed the vP4H protein.

[0143] At the same time, the gene copy number and hydroxyproline content of the recombinant type III human collagen expressed by the strain GS115-COL3A1-P4Hb-P4Ha2-vP4H were detected according to the method of Example 3. The detection results are shown in Table 3.

[0144] Table 4: Detection results of gene copy number and hydroxyproline content of GS115-COL3A1-P4Hb-P4Ha2-vP4H strain

[0145]

[0146] The results showed that the degree of proline hydroxylation of recombinant human type III collagen was further improved, and recombinant human type III collagen could obtain the highest hydroxyproline content of 16.8%. However, with the further increase of the vP4H gene copy number ratio, the hydroxyproline content tended to decrease, which may be due to the overexpression of hydroxyproline protein, which increased the energy consumption of the strain and was not conducive to the further post-translational modification of recombinant human type III collagen.

[0147] Example 6: Preparation of recombinant human type III collagen

[0148] The highly hydroxylated, triple-helical full-length recombinant type III human collagen GS115-COL3A1-P4Hb-P4Ha2-vP4H strain No. 3 selected in Example 5 was cultured in a 500 ml shake flask using BMGY medium for 72 h and confirmed by electrophoresis (the protein expression on the map is the same as Figure 7 ), take the fermentation liquid and centrifuge it at 5000rpm for 15min to obtain the bacterial precipitate containing recombinant type III human collagen. After being broken by a homogenizer, centrifuge it at 12000rpm for 30min, discard the supernatant and obtain the precipitate. The obtained precipitate is further treated with pepsin at a final concentration of 400U / L at 4°C for 20 hours to remove the terminal peptide, and then microfiltration is performed with a 0.44μm filter membrane to obtain the permeate, and then salting out with a 1mol / L sodium chloride solution for 16 to 30 hours, ion exchange chromatography is used to remove non-recombinant type III human collagen impurities, and ultrafiltration and concentration with a 100kD filter membrane is performed to obtain a high-purity recombinant type III human collagen stock solution.

[0149] Example 7: Detection of the structure and properties of recombinant human type III collagen

[0150] In order to confirm the consistency between the recombinant human type III collagen prepared in Example 6 and the natural human type III collagen, the structure and properties of the recombinant human type III collagen were tested.

[0151] 1. Sequence determination

[0152] The recombinant type III human collagen was sequenced and the N / C-terminal sequence results were identified by LC / MS (e.g. Figure 8 ) and Trypsin, Glu-C double digestion sequence coverage results (such as Fig. 9 ), the results showed that the sequence composition of the recombinant type III human collagen sample was completely consistent with the amino acid sequence of natural human collagen after removing the N-terminal telopeptide and the C-terminal telopeptide (amino acid sequence such as SEQ ID NO: 2).

[0153] 2. Molecular weight determination

[0154] The molecular weight was determined by non-reducing and reducing polyacrylamide gel electrophoresis detection methods. Prepare 4% concentrated gel / 7.5% separation gel, take 50μg of recombinant type III human collagen sample, add it to a 1.5ml centrifuge tube, add 0.01mol / L hydrochloric acid to a total volume of 75ul, add 25ul loading buffer (4x) (add 10μl 10× reducing agent to the reduced sample), and mix well. Pipette 6-10μl of the prepared sample into the gel hole, connect it to the electrophoresis power supply for electrophoresis, the first step: 80V, 20min; the second step: 160V or 180V, 60min. After electrophoresis, stain and decolorize, use a gel imager to take pictures, and the test results are shown in Fig.10 It can be seen that the reduced electrophoresis band is around 130kD, and the non-reduced electrophoresis band is around 390kD, which is the same as the triple helix molecular weight of recombinant type III human collagen. The non-reduced electrophoresis molecular weight shows that the expressed recombinant type III human collagen has a triple helix structure.

[0155] 3. Purity Determination

[0156] Size exclusion chromatography (SEC-HPLC) was used for detection, and the detection conditions were as follows:

[0157]

[0158]

[0159] Test results see Fig.11 The main peak RT8.550min is the location of the recombinant type III human collagen triple helix structure, and the front peak RT8.302min is the location of the dimer based on the recombinant type III human collagen triple helix structure. Both are recombinant type III human collagen components, and the purity is 100%.

[0160] 4. Triple helix structure detection

[0161] Circular dichroism spectroscopy was used to analyze the structure of recombinant human type III collagen by utilizing the circular dichroism of proteins and the difference in absorption of left and right circularly polarized light by asymmetric molecules.

[0162] Take a sample of recombinant type III human collagen, dilute it with 20mM PB to 0.1mg / ml, and directly scan it in the far-ultraviolet band. The scanning conditions are as follows:

[0163] bandwidth 1.0nm Far UV wavelength range 190-260nm Recording interval duration 0.5s Detection duration 3min Cuvette path length 0.5mm Detection temperature Room temperature

[0164] Test results see Fig.12It can be seen that in the far-ultraviolet spectrum of the circular dichroism spectrum, there is a maximum absorption near 223nm and a minimum absorption near 197nm. The spectrum characteristics are consistent with the far-ultraviolet spectrum characteristics of collagen with a triple helix structure.

[0165] 5. Self-assembly ability test

[0166] Collagen with a triple helix structure can self-assemble to form collagen fibers under appropriate conditions. During this process, the turbidity of the collagen solution will change. The change in turbidity during the self-assembly process is detected at different times using an ELISA instrument at a wavelength of 313nm, which can reflect the self-assembly performance of collagen. The self-assembly curve of the collagen solution presents a typical S-shape, including three stages: static phase, rapid growth phase and plateau phase.

[0167] Preparation of 10mmol / L PBS buffer (pH10.0): weigh 0.27g of potassium dihydrogen phosphate, 1.14g of disodium hydrogen phosphate, 0.20g of potassium chloride, and 8.00g of sodium chloride, add 800ml of water to dissolve, adjust the pH value to 10.0±0.05 with 1mol / L sodium hydroxide solution, and add water to 1000ml.

[0168] Sample dilution was performed on ice. Take 200 μl of recombinant type III human collagen sample, add 400 μl of 10 mmol / L PBS buffer (pH 10.0) solution, mix well, and make the final pH between 7.0 and 7.4. Pipette 200 μL of the prepared test solution and add it to a 96-well plate. Set the detection wavelength to 313 nm, control the temperature at 37 ° C, and detect the OD313 nm value on an enzyme reader. Detect the OD value every 30 seconds, and the measurement time is 1.5 hours. See the detection spectrum for details. Fig.13 It can be seen that a typical S-shaped curve is presented, indicating that recombinant type III human collagen has the ability to further self-assemble on the basis of the triple helix.

[0169] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0170] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for preparing recombinant human type III collagen, characterized in that: include: Constructing genetically engineered bacteria that express proline hydroxylase and full-length recombinant human type III collagen; Cultivating the genetically engineered bacteria, and enzymatically digesting the culture product with pepsin to obtain the recombinant type III human collagen; Wherein, the amino acid sequence of the recombinant type III human collagen is shown in SEQ ID NO: 1; The proline hydroxylases include P4Ha2, P4Hb and vP4H; The copy number ratio of P4Ha2, P4Hb, vP4H and full-length recombinant type III human collagen is (1.80-2.20): (1.80-2.20): (0.80-1.60):

1.

2. The method according to claim 1, characterized in that The copy number ratio of P4Ha2, P4Hb, vP4H and full-length recombinant type III human collagen is (1.90-2.10): (1.90-2.10): (0.90-1.10):

1.

3. The method according to claim 1, characterized in that The amount of pepsin added to the culture product is 100-500 U / L; And / or, the temperature of the enzyme digestion treatment is 2 to 10°C; And / or, the enzyme digestion treatment time is 16 to 30 hours; And / or, the amino acid sequence of P4Ha2 is as shown in SEQ ID NO: 2; And / or, the amino acid sequence of P4Hb is as shown in SEQ ID NO:3; And / or, the amino acid sequence of vP4H is shown in SEQ ID NO:4; And / or, the amino acid sequence of the full-length recombinant human type III collagen is as shown in SEQ ID NO: 5; And / or, the genetically engineered bacteria is selected from Pichia pastoris strains.

4. The method according to claim 1, characterized in that: Further including: The enzyme cleavage product is purified.

5. The method according to claim 4, characterized in that The purification treatment is to carry out microfiltration treatment, salting-out treatment, chromatography treatment and ultrafiltration treatment in sequence.

6. The method according to claim 5, characterized in that The microfiltration treatment is carried out using a filter membrane with a pore size of 0.22 μm or more; And / or, the salting-out treatment is carried out using a 0.5-3 mol / L sodium chloride solution; And / or, the salting-out treatment time is 16 to 30 hours; And / or, the chromatography treatment is performed using ion exchange chromatography; And / or, the ultrafiltration treatment is performed using a cut-off filter membrane with a molecular weight of 50 kD or above.

7. The method according to claim 1, characterized in that The purity of the recombinant type III human collagen is greater than 95%; And / or, the hydroxyproline content in the recombinant type III human collagen is greater than 16.5%, preferably 16.6% to 17.5%; And / or, the recombinant human type III collagen has a complete triple helical structure; And / or, in the circular dichroism spectrum, the recombinant type III human collagen has a negative absorption peak at a wavelength of 190 to 200 nm and a positive absorption peak at a wavelength of 210 to 230 nm; And / or, the recombinant type III human collagen has a negative absorption peak at a wavelength of 195 to 200 nm; And / or, the recombinant type III human collagen has a positive absorption peak at a wavelength of 215-225 nm.

8. A recombinant type III human collagen, characterized in that: The recombinant type III human collagen is prepared by the method according to any one of claims 1 to 7.

9. A composition, characterized in that The invention relates to recombinant human type III collagen prepared by the method according to any one of claims 1 to 7.

10. The composition according to claim 9, characterized in that The composition further comprises an auxiliary material; And / or, the composition is selected from recombinant type III human collagen hydrogel, recombinant type III human collagen repair solution, recombinant type III human collagen solution, or recombinant type III human collagen dressing.

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