High-stability recombinant I-type collagen and application thereof

Abstract

The invention provides high-stability recombinant I-type collagen and application thereof, and belongs to the technical field of collagen. The invention aims to avoid potential restriction enzyme cutting sites and unstable sequence fragments during sequence design through directional selection of the existing amino acid sequence, so that the amino acid sequence can be efficiently expressed in pichia pastoris, and the recombinant I-type collagen obtained after purification has very high stability; the recombinant I-type collagen gives consideration to factors such as related biological active sites, hydrophilicity and the like, has related biological activity while being relatively high in stability, and has very good practicability.

Description

Technical Field

[0001] The invention belongs to the technical field of collagen, and in particular relates to high-stability recombinant type I collagen and applications thereof. Background Art

[0002] Collagen is an important component of the extracellular matrix (ECM) of animal organisms. It is a spiral fibrous protein twisted from three peptide chains. It forms a fine elastic network in the skin, locking in moisture, supporting the skin, making cells plump and hydrated, and making the skin elastic, moist, delicate, and smooth. In connective tissues such as cartilage, bones, tendons, ligaments, etc., the triple helix structure of collagen provides mechanical properties, constructs a fibrous scaffold, and provides cells with high tensile strength and stability. Collagen is now widely used in many fields such as chemical industry, medicine, food, and cosmetics because of its good biocompatibility, biological activity, and degradability.

[0003] At present, collagen is mainly obtained through animal tissue extraction or recombinant expression technology. Among them, the technology of extracting collagen from animal tissue is relatively mature, and this type of collagen has a long history of application. However, in the traditional collagen production process, acid and alkali and other raw materials are generally used to extract animal tissues, which is difficult to extract. The properties of the obtained collagen are uneven, the batch differences are large, and there is a safety hazard of viral infection. The research and application of recombinant collagen has a history of more than 30 years. The existing literature and patents mainly focus on the expression of human collagen single chains in different hosts and the expression of truncated collagen gene single chains. There are also a small number of focuses on the co-expression of collagen and related post-expression modification enzymes to obtain triple helical collagen.

[0004] At present, the recombinant expression of type I collagen focuses more on its effective secretion and biological activity, while ignoring the stability of recombinant collagen. This makes most of the type I recombinant collagen on the market not very stable and prone to degradation, which increases the immunogenicity of the raw materials, making subsequent purification more difficult and reducing the yield; unstable recombinant collagen, even if it passes the purification process to obtain a high-purity freeze-dried sponge, will produce a lot of degradation after rethawing, which increases the risk of subsequent use of the finished product and greatly limits its subsequent application. Therefore, it is necessary to develop highly stable recombinant type I collagen. Summary of the invention

[0005] In view of some deficiencies in the prior art, the present invention provides a highly stable recombinant type I collagen and its application; the present invention aims to avoid potential enzyme cleavage sites and unstable sequence fragments during sequence design through directional selection of existing amino acid sequences, so that the recombinant type I collagen obtained after purification has high stability; the recombinant type I collagen takes into account relevant biological active sites, hydrophilicity and other factors, has relevant biological activity while having high stability, and has good practicality.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0007] The present invention first provides a high-stability recombinant type I collagen, which includes a repeatedly connected fragment A, wherein the fragment A includes a plurality of sequence fragments with high stability, easy to be efficiently expressed by Pichia pastoris, and not easily cleaved by enzymes, and the sequence fragments are one or more of 302-322AA, 413-445AA, 485-532AA, 554-577AA, 593-613AA, 668-679AA, 740-763AA, 776-799AA, 899-922AA, 962-976AA, 1010-1039AA, and 1070-1102AA in human type I collagen CO1A1.

[0008] Preferably, the fragment A comprises any one of the following sequence fragment combinations:

[0009] a) 554-577AA, 1070-1102AA;

[0010] b)668-679AA, 776-799AA, 1082-1102AA;

[0011] c)593-613AA, 776-799AA, 962-976AA;

[0012] d)593-613AA, 668-679AA, 776-799AA, 962-976AA, 1082-1102AA;

[0013] e)413-445AA, 899-922AA, 1082-1102AA;

[0014] f)302-322AA, 485-532AA, 740-763AA, 1010-1039AA;

[0015] g)302-322AA, 485-532AA, 776-799AA;

[0016] h)668-679AA, 740-763AA, 1010-1039AA, 1082-1102AA.

[0017] Preferably, the high-stability recombinant type I collagen has a size of 30-50 kDa.

[0018] Preferably, LEKR is added to the N-terminus of the high-stability recombinant type I collagen.

[0019] Preferably, the high-stability recombinant type I collagen includes an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, or an amino acid sequence that is more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identical to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

[0020] The present invention also provides nucleotides encoding the above-mentioned highly stable recombinant type I collagen.

[0021] The present invention also provides a recombinant expression vector, which contains nucleotides encoding the above-mentioned high-stability recombinant type I collagen.

[0022] The present invention also provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium comprises the nucleotide encoding the above-mentioned high-stability recombinant type I collagen or the above-mentioned recombinant expression vector.

[0023] Preferably, the host bacteria of the recombinant engineered bacteria are eukaryotic bacteria or prokaryotic bacteria.

[0024] Preferably, the host bacterium is Pichia pastoris.

[0025] Preferably, the recombinant engineered bacteria are deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration with the deposit numbers CGMCC No.31952, CGMCC No.31953 and CGMCC No.31954.

[0026] The present invention also provides a composition, which comprises the above-mentioned high-stability recombinant type I collagen.

[0027] The present invention also provides a product, which comprises the above-mentioned high-stability recombinant type I collagen or composition.

[0028] The present invention also provides the use of the above-mentioned high-stability recombinant type I collagen, composition or product in the preparation of medical equipment, biomaterials, tissue engineering products and cosmetics.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The recombinant type I collagen of the present invention has high stability, the main band accounts for more than 90% in the fermentation stage, the purification difficulty is low, and it is not easy to degrade during the purification process, which greatly improves the yield of the full-length protein. The purity of the pilot sponge is above 95%;

[0031] (2) The sample obtained after purification of the recombinant type I collagen of the present invention performed better than the original protein in the stability experiment, and no significant degradation occurred under all conditions of the stability experiment, which reduced the storage difficulty and extended the storage time;

[0032] (3) The recombinant protein involved in the present invention has similar physical and chemical properties and biological functions as the original sequence. When used as a biological material, it has higher stability and high batch consistency, which facilitates quality control and is beneficial to product stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the SDS-PAGE image of the supernatant of recombinant type I collagen expression in a shake flask (induced for 48 hours).

[0034] Figure 2 This is the SDS-PAGE image of the supernatant of the 5L tank fermentation of recombinant type I collagen.

[0035] Figure 3 This is a small test sponge gel diagram of 102-105 recombinant type Ⅰ collagen.

[0036] Figure 4 This is the gel image of recombinant type I collagen 102~105 at different pH values, where a is 102, b is 103, c is 104, and d is 105.

[0037] Figure 5 This is the repeated freeze-thaw gel image of recombinant type I collagen 102~104.

[0038] Figure 6 This is the circular dichroism spectrum detection result of recombinant type I collagen 102~105. In the figure, a is 102, b is 103, c is 104, and d is 105.

[0039] Figure 7These are the results of the cytotoxicity experiment of recombinant type I collagen 102~105. In the figure, a is 102, b is 103, c is 104, and d is 105.

[0040] Figure 8 This is a bar graph of the results of the cell adhesion experiment of recombinant type I collagen 102-105, where a is 0.5 mg / mL and b is 1.0 mg / mL.

[0041] Fig. 9 It is a statistical chart of the migration area ratio of recombinant type I collagen 102~105.

[0042] Fig.10 This is a diagram of the magnified SDS-PAGE test results of three batches, including the gel running images of Marker, two samples of SF2406002 batch, two samples of SF2406003, and two samples of SF2406004.

[0043] Fig.11 This is the mass spectrometry identification result of recombinant type Ⅰ collagen 103 sponge.

[0044] Fig.12 This is the mass spectrometry identification result of recombinant type Ⅰ collagen 105 sponge.

[0045] Fig.13 These are the results of mass spectrometry identification of three batches of pilot-scale sponges of recombinant type I collagen 102. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. The technical scheme of the embodiment of the present invention is clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0047] Unless otherwise defined, technical or scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0048] Example 1: Design of highly stable recombinant type I collagen

[0049] Using human type I collagen CO1A1 as the parent sequence (https: / / www.uniprot.org / uniprotkb / P02452), we selected sequence fragments that were highly stable, easy to express in Pichia pastoris, and not easily cleaved by enzymes. Any two or more of them were selectively concatenated and repeated a certain number of times so that the size of the repeated sequence met the optimal size for Pichia pastoris to express exogenous proteins. LEKR was added at the front end to ensure the correctness of the N-terminus.

[0050] Among them, the sequence fragments with high stability, easy to be efficiently expressed in Pichia pastoris, and not easily cleaved by enzymes are 302-322AA, 413-445AA, 485-532AA, 554-577AA, 593-613AA, 668-679AA, 740-763AA, 776-799AA, 899-922AA, 962-976AA, 1010-1039AA, and 1070-1102AA of the parent sequence.

[0051] The sequence fragments with high stability, easy to be efficiently expressed in Pichia pastoris, and not easily cleaved by enzymes are as follows:

[0052] 302-322AA: GPRGLPGERGRPGAPGPAGAR (SEQ ID NO. 17);

[0053] 413-445AA:GARGPSGPQGPGGPPPGPKGNSGEPGAPGSKGDT (SEQ ID NO. 18);

[0054] 485-532AA: ERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEA (SEQ ID NO. 19);

[0055] 554-577AA:PPGPAGQDGRPGPPGPPGARGQA (SEQ ID NO. 20);

[0056] 593-613AA: KAGERGVPGPPGAVGPAGKD (SEQ ID NO. 21);

[0057] 668-679AA:GAPGPSGARGER(SEQ ID NO.22);

[0058] 740-763AA: GPKGDRGDAGPKGADGSPGKDGVR (SEQ ID NO. 23);

[0059] 776-799AA: GAPGDKGESGPSGPAGPTGARGAP (SEQ ID NO. 24);

[0060] 899-922AA:GPAGKEGGKGPRGETGPAGRPGEV(SEQ ID NO.25);

[0061] 962-976AA: GLPGQRGERGFPGLP (SEQ ID NO. 26);

[0062] 1010-1039AA: GESGREGAPGAEGSPGRDGSPGAKGDRGET (SEQ ID NO. 27);

[0063] 1070-1102AA: GPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQ (SEQ ID NO.28). 554-577AA and 1070-1102AA were selected and connected in series, and then repeated 6 times and LEKR was added in the front part to obtain recombinant type I collagen 101, the amino acid sequence of which is shown in SEQ ID NO.1.

[0064] SEQ ID NO.1:

[0065] LEKRGPPGPAGQDGRPGPPGPPGARGQAGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGPPGPAGQDGRPGPPGPPGARGQAGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGPPGPAGQDGRPGPPGPPGARGQAGGDKGETGEQGPPGPAGQDGRPGPPGPPGARGQAGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQ

[0066] 668-679AA, 776-799AA, and 1082-1102AA were selected and connected in series, and then repeated 6 times and LEKR was added in the front section to obtain recombinant type I collagen 102. The amino acid sequence of the recombinant type I collagen 102 is shown in SEQ ID NO.2.

[0067] SEQ ID NO.2:

[0068] LEKRGAPGPSGARGERGAPGDKGESPSGPAGPTGARGAPGARGPAGPQGPRGDKGETGEQGAPGPSGARGERGAPGDKGESGPSGPAGPTGARGAPGARGPAGPQGPRGDKGETGEQGAPGPSGARGERGAPGDKGESGPSKGETGEQGAPGPSGARGERGAPGDKGESGPSGPAGPTGARGAPGARGPAGPQGPRGDKGETGEQ

[0069] 593-613AA, 776-799AA, and 962-976AA were selected and connected in series, and then repeated 6 times and LEKR was added in the front section to obtain recombinant type I collagen 103. The amino acid sequence of the recombinant type I collagen 103 is shown in SEQ ID NO.3.

[0070] SEQ ID NO.3:

[0071] LEKRGKAGERGVPGPPGAVGPAGKDGAPGDKGESGPSGPAGPTGARGAPGLPGQRGERGFPGLPGPSGKAGERGVPGPPGAVGPAGKDGAPGDKGESGPSGPAGPTGARGAPGLPGQRGERGFPGL PGPSGKAGERGVPGPPGAVKDGAPGDKGESGPSGPAGPTGARGAPGLPGQRGERGFPGLPGPSGKAGERGVPGPPGAVGPAGKDGAPGDKGESGPSGPAGPTGARGAPGLPGQRGERGFPGLPGPS

[0072] 593-613AA, 668-679AA, 776-799AA, 962-976AA, and 1082-1102AA were selected and connected in series, and then repeated 5 times and LEKR was added in the front section to obtain recombinant type I collagen 104. The amino acid sequence of the recombinant type I collagen 104 is shown in SEQ ID NO.4.

[0073] SEQ ID NO.4:

[0074] LEKRGAPGPSGARGERGARGPAGPQGPRGDKGETGEQGKAGERGVPGPPGAVGPAGKDGLPGQRGERGFPGLPGPSGAPGDKGESGPSGPAGPTGARGAPGAPGPSGARGERGARGPAGPQGPRGDKGETGEQGKAGERGVPGA VGPAGKDGLPGQRGERGFPGLPGPSGAPGDKGESGPSGPAGPTGARGAPGAPGPSGARGERGARGPAGPQGPRGDKGETGEQGKAGERGVPGPPGAVGPAGKDGLPGQRGERGFPGLPGPSGAPGDKGESGPSGPAGPTGARGAP

[0075] 413-445AA, 899-922AA, and 1082-1102AA were selected and connected in series, and then repeated 6 times and LEKR was added in the front section to obtain recombinant type I collagen 105. The amino acid sequence of the recombinant type I collagen 105 is shown in SEQ ID NO.5.

[0076] SEQ ID NO.5:

[0077] LEKRGARGPSGPQGPGGPPPGPKGNSGEPGAPGSKGDTGPAGKEGGKGPRGETGPAGRPGEVGARGPAGPQGPRGDKGEGPRGDKGETGEQGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGPAGKESGE PGAPGSKGDTGPAGKEGGKGPRGETGPAGRPGEVGARGPAGPQGPRGDKGETGEQGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGPAGKEGGKGPRGETGPAGRPGEVGARGPAGPQGPRGDKGETGEQ

[0078] 302-322AA, 485-532AA, 740-763AA, and 1010-1039AA were selected and connected in series, and then repeated 4 times and LEKR was added in the front section to obtain recombinant type I collagen 106. The amino acid sequence of the recombinant type I collagen 106 is shown in SEQ ID NO.6.

[0079] SEQ ID NO.6:

[0080] LEKRGPRGLPGERGRPGAPGPAGARGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGPPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGPKGDRGDAGPKGADGSPGKDGVRGESGREGAPGAEGSPGRDGSPGAKGDRGETGP RGLPGERGRPGAPGPAGARGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGPKGDRGDAGPKGADGSPGKDGAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGPKGDRGDAGPKGADGSPGKDGVRGESGREGAPGAEGSPGRDGSPGAKGDRGET

[0081] 302-322AA, 485-532AA, and 776-799AA were selected and connected in series, and then repeated 5 times and LEKR was added in the front section to obtain recombinant type I collagen 107. The amino acid sequence of the recombinant type I collagen 107 is shown in SEQ ID NO.7.

[0082] SEQ ID NO.7:

[0083] LEKRGPRGLPGERGRPGAPGPAGARGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGAPGDKGESGPSGPAGPTGARGAPGPRGLPGERGRPGAPGPAGARGERGGPGSRGFPGADGVAGPKGPARGAPGPRGLPGERGRPGAPGPAGA RGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGAPGDKGESGPSGPAGPTGARGAPGPRGLPGERGRPGAPGPAGARGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGAPGDKGESGPSGPAGPTGARGAP

[0084] 668-679AA, 740-763AA, 1010-1039AA, and 1082-1102AA were selected and connected in series, and then repeated 5 times and LEKR was added in the front section to obtain recombinant type I collagen 108. The amino acid sequence of the recombinant type I collagen 108 is shown in SEQ ID NO.8.

[0085] SEQ ID NO.8:

[0086] LEKRGAPGPSGARGERGPKGDRGDAGPKGADGSPGKDGVRGESGREGAPGAAEGSPGRDGSPGAKGDRGETGARGPAGPQGPRGDKGETGEQGAPGPSGARGERGPKGDRGDAGPKGADGSPGKD GVRGESGREGAPGAEGSPGRGPAGPQGPRGDKGETGEQGAPGPSGARGERGPKGDRGDAGPKGADGSPGKDGVRGESGREGAPGAEGSPGRDGSPGAKGDRGETGARGPAGPQGPRGDKGETGEQ

[0087] Suzhou Hongxun Biotechnology Co., Ltd. was commissioned to perform codon optimization based on the preference of Pichia pastoris, and then synthesize nucleotides encoding 101, 102, 103, 104, 105, 106, 107, and 108, the sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.16, respectively.

[0088] SEQ ID NO.9:

[0089] ttggaaaagagaggaccgcccgggcccgccgggcaagatggccggccaggtccgccggggcctccaggtgcccgcggccaagcgggacctgctggacccgcgggtcctgtcggcccagtgggtgctcgaggccctgccggcccgcaaggccctaggggcgataagggcgagacaggtgaacaggggccaccagggcctgcgggtcaggacgggagacccggaccccccggacctcccggcgctagggggcaggcgggaccagcaggaccagcaggaccggttggacccgtaggcaggctggaccagcagggcccgcgggccctgtaggccctgtaggtgctcgcggtcccgcaggtccgcaaggaccgcggggagataagggtgaaactggcgagcaggggccgcccggacccgccggccaggacggccggccaggaccgcctggccctcctggagcacgcggtcaagccggtccggcgggacccgcgggccccgtcggccccgtcggcgctcgaggcccggccggcccgcagggtccccgaggggataaaggtgaaactggggagcaggggccaccagggcccgcgggacaagacggaaggccagggcctcctgggccgccaggcgcccggggtcaggcaggtccagccggtccggctgggccggtggggcctgtgggggctagaggccctgccgggcctcaggggcctagaggtgacaagggcgaaacgggggagcaa

[0090] SEQ ID NO.10:

[0091] ttggaaaagagaggtgcgcccggaccatctggtgctcgtggtgagagaggtgctcctggggataaaggtgaaagcggaccaagtggccctgcgggcccgacaggtgccagaggcgcacccggggcgcgtggtcccgccggaccgcaaggccctcgaggagacaaaggagaaaccggcgaacagggtgctcctggaccatcgggggcccggggagaaagaggggcccccggtgacaagggtgagtcaggtccctcagggccagctggacccacgggtgcaagaggcgctccgggcgcgcggggtcctgcagggccgcaagggcctcgtggagacaagggggagactggcgagcagggtgcaccggggccttccggcgcgaggggagagcggggtgcaccgggcgataagggtgagtcgggtccaagtgggccggcgggtcctacaggtgcccgcggcgctccaggggcacgagggcctgccgggccccagggacctcgaggcgacaaaggcgagacaggggaacaaggagcgcctggcccctcgggagcacggggagaacgcgtctggccccagcggacctgctggccccacgggggctaggggcgctccaggggcccgtggaccggcaggtccacagggcccccgcggggataaaggtgaaaccggtgaacaa

[0092] SEQ ID NO.11:

[0093] ttggaaaagaggggaaaagccggggagagaggcgtacctgggccacctggtgcagtcggaccggcggggaaggacggcgccccgggggacaagggtgaatcggggccaagcggtccggcgggcccgaccggtgcacggggtgctcctggcctccccggccaacgtggagaaagggggttcccaggactccctggaccaaccgagcggaaaagcgggggagcggggtgttcccgggcccccaggtgccgtcggtccggctggcaaagacggtgcaccaggcgataaaggagaatctggtccatccggtcctgcaggacccacaggggcgcggggcgcgccaggactgcccggccagcgaggcgaaagaggctttcccggtctgcctggcccatctggtaaggccggggaaagaggagtacccggaccaccgggggcagtaggtccggcgggaaaggatggcgcgcccggcgacaaaggcgaatcaggaccctcgggacctgcagggcctaccggcgctcgtggggcccctggccttccaggccaaaggggggagcgaggcttccccggattaccagggccctcc

[0094] SEQ ID NO.12:

[0095] ttggaaaagaggggagcgccaggaccttccggtgctaggggagagcgtggggctcgcggtcccgcgggtcctcagggtccacgaggagataagggtgaaaccggggaacagggcaaggcgggcgaacggggcgtgccaggtcctccaggtgcggttgggcctgctgggaaagacggactgccgggtcagcggggggagcgaggcttccccggattaccgggcccctcgggtgcaccaggcgacaagggggagtctgggccgtccgggccagcagggccgacaggcgcgcgcgacaagggagagaccggggagcagggaaaagcgggtgagagaggagttcccgggccgccgggcgcggtcggtccagccggtaaagatggcctcccaggccaaagaggtgaacgtggctttccgggactaccgggtccttccggggctccgggtgacaagggtgaatccggaccaagcggacctgccgggccgactggcgcccggggggcgccc

[0096] SEQ ID NO.13:

[0097] ttggaaaagaggggtgcgaggggtccctcaggaccgcaaggaccaggtggaccaccaggcccaaagggaaattccggagagccaggagcgccaggatcgaagggggacactgggcctgcgggcaaggaaggcggtaaaggcccgcgtggagagaccgggcctgcaggtaggccgggcgaggtcggggcacgaggacctgccgattccggcgaaccaggtgctcctggcagtaagggggacacggggccagctggcaaagaagggggcaagggccctcggggtgaaactggccctgcaggccggccaggagaggttggggctagaggtcctgcaggaccacaaggaccccgtggcgacaagggtgagacaggcgagcaaggggcgcgcgggccgtctggcccccagggcccaggaggtccgcctggtccgaaaggaaactctggagagcccggtgctcctgggagtaagggtgacactggccccgccggaaaggaaggcggtaaagggccccgtggggaaacgggtccagcggggaggcctggcgaagtgggagcgagaggcccagcgggaccccagggacctcgaggggataagggcgaaacaggagaacaaggtgcacgtggtccgagcggaccccagggtcctgggggtccccccggtcctaaaggtaattctggtgaaccgggagcgcccggttcgaaaggtgacaccggtcctgcaggcaaagaagggaggacccaagggtaacagcggagagcccggtgccccgggttcgaaaggtgacacaggacctgccgggaaagagggtggtaagggcccgcgtggcgagaccggaccagcgggacggccgggagaagtaggtgctcgtgggcctgccggtccgcaggggcctcgcggggataagggtgaaaccggcgaacaa

[0098] SEQ ID NO.14:

[0099] ttggaaaagagggggcctagaggactgcccggtgaaagaggaagacctggcgcacccggtccggccggagcgaggggggaacgcggggggcctggatctagggggtttcccggagcagacggtgtagcgggccctaaagggccggctggtgaacggggcagcccagggcccgcagggcccaaagggtctccgggagaagcgggccgaccaggggaagccggacctaaaggagatagaggggatgcaggtcccaagggagcagacggtagtccgggcaaagatggcgtcaggggtgagagtggccgtgagggcgcgccaggagcagagggctctcccggacgcgacggctcgccaggagccaagggtgataggggtgagagccggccggtgctcgaggagagaggggcggccccgggagtcggggttttccgggggcggacggggtagctgggccaaaagggccagcaggagagagaggctcacctgggcccgcaggacctaaggggtccccaggcgaagctgggcgcccaggcgaggccggaccgaaaggcgatagaggagatgctgggcctaagggtgctgacgggtcgccgggaaaggacggtgttcggggagaatctggtcgcgaaggggcgcctggagcggagggatcacccggacgagatggcagtccaggcgccaaaggggacagaggagagacc

[0100] SEQ ID NO.15:

[0101] ttggaaaagaggggtcctcggggattgcctggcgagaggggaagaccaggcgcgccgggtcctgcgggtgctcgtggcgaacgtggagggcctgggtctcgtgggttccccggcgcggatggcgtggctggtccaaaaggcccggccggcgaaagaggttctcccggaccagccggccccaaggggtcgcctggggaagcgggtaggcctggagaagcaggtgcacccggggataagggtgaaagtggtccttccggaccggcaggcccgacgggcgcaagaggcgccccgggaccgcgagggctgcctggagagagggggagaccgggtgcccctggacctgcgggtgctcgcggagagcgcggcggtcccggatctcgccaggtgccccggggcccgcaggagcacgcggcgaaaggggggggccgggatcacgtggatttcccggcgcagatggagtcgcaggtcccaaaggcccagccggtgaaaggggatccccaggaccggccggcccaaagggctcgccaggggaggctgggcggccgggggaggccggagcaccaggggacaaaggggagtcaggcccttccgggcccgctggtccaacaggggcccgaggcgcaccc

[0102] SEQ ID NO.16:

[0103] .

[0104] Example 2: Construction of recombinant expression vector and recombinant engineering bacteria

[0105] (1) Construction of recombinant expression vector:

[0106] The nucleotide synthesized in Example 1 was cloned between EcoRⅠ and NotⅠ of the pPIC9K empty vector (purchased from Thermo Fisher Scientific), so that the target fragment was accurately inserted into the secretory vector reading frame containing the secretion signal α-factor, and a recombinant expression vector expressing 101, 102, 103, 104, 105, 106, 107, and 108 was obtained.

[0107] (2) Construction of recombinant engineering bacteria:

[0108] 10 μg of the recombinant expression vectors expressing 101, 102, 103, 104, 105, 106, 107, and 108 were respectively digested with SalⅠ (purchased from Dalian TaKaRa Company, the specific operation was carried out according to the kit instructions) at 37°C overnight to linearize them, and then a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was used to recover the linearized plasmids, and the volume was controlled to be about 10 μL.

[0109] The linearized plasmid was electrotransformed into the competent cells of the host strain Pichia pastoris GS115 (purchased from Thermo Fisher Scientific), and the electroporated bacterial solution was spread on MD plates, with 100 μL to 200 μL spread on one plate, allowed to stand at room temperature for 10 min, and inverted cultured at 30°C for 2-5 days until a single colony (positive transformant) appeared.

[0110] Add 2 mL of sterile double distilled water to the surface of the MD plate, then use a sterile triangular applicator to gently scrape off the His+ transformants on the surface of the plate and transfer them to a 50 mL centrifuge tube. Dilute the bacterial suspension with sterile double distilled water, 10 5 The cells were spread on a YPD plate containing 0.5 mg / mL G418, inverted, and cultured at 30°C for 3 to 4 days until a single colony appeared, thereby obtaining an engineered strain of Pichia pastoris expressing 101, 102, 103, 104, 105, 106, 107, and 108.

[0111] Example 3: Induced expression and identification of recombinant type I collagen

[0112] Take the Pichia engineering strains expressing 101, 102, 103, 104, 105, 106, 107, and 108 respectively, and take the engineering strain in patent CN201911135958.0 (full-length sequence of human type I α1 chain) and place it in a 100mL Erlenmeyer flask containing 10mL BMGY medium, and culture it at 28-30℃ and 220rpm until OD600 is 10-15 (18-24h). Centrifuge at 1500-3000g for 5min at room temperature, collect the cells, resuspend the cells in 10mL BMM2 medium, and make OD 600 The cell culture medium was placed on a shaker at 28-30°C and 220 rpm for 2 days to continue growth and induction of expression. 100% methanol was added to the culture medium every 24 hours to a final concentration of 1.0%. After induction with methanol for 48 hours, the bacterial liquid sample was collected, the sample volume was 1 mL, placed in a 1.5 mL EP tube, centrifuged at 12000g for 5 minutes at 4°C, the expression supernatant was collected, and the samples to be tested were stored at -80°C for later use.

[0113] The expression supernatant was collected and added with 5× loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), heated in a 100°C metal bath for 10 min, and then subjected to SDS-PAGE detection. Figure 1 As shown. Figure 1 It can be seen that the gel image shows that the expected molecular weight is in line with the size, indicating that recombinant type I collagen was successfully obtained. And compared with the engineered strain in patent CN201911135958.0, except for 101, the other 7 sequences in 101-108 can be efficiently expressed. Under the premise of giving priority to purity, 102, 103, 104, and 105 performed better.

[0114] Combined with the small-scale experiment, three recombinant collagen type I proteins 102, 103, and 105 were selected and the three engineered bacteria samples expressing 102, 103, and 105 were sent to the General Microbiology Center of the China Microbiological Culture Collection Administration. The strain collection numbers are: CGMCC No. 31952, CGMCC No. 31953, and CGMCC No. 31954. Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Storage date: September 12, 2024, classification name: Pichia pastoris Komagataellaphaffii.

[0115] Example 4: Fermentation and purification of recombinant type I collagen

[0116] (1) Fermentation:

[0117] The Pichia pastoris engineered strain expressing 102, 103, 104, and 105 in Example 3 was subjected to a 5 L small tank high-temperature fermentation test, and freeze-dried sponge samples of the four collagen sequences were purified to carry out the next step of verification.

[0118] The culture media used include:

[0119] Seed culture medium YPG: yeast powder 10g / L, peptone 20g / L, glycerol 10g / L;

[0120] Fermentation medium: NH4H2PO4 190.4g / L, KH2PO4 10.06g / L, CaSO4·2H2O1.18g / L, K2SO418.2g / L, MgSO4·7H2O 14.9g / L, glycerol 40g / L;

[0121] Feed medium: 50% W / V glycerol, plus 12 mL PTM1 trace elements per liter;

[0122] Induction medium: 100% methanol, 12 mL of PTM1 trace elements per liter;

[0123] PTM1: Sterilize by filtering with a 0.22 μm filter membrane and store at 4°C.

[0124] After the fermentation medium is sterilized at high temperature, PTM1 is added when the temperature drops to room temperature, and the pH is adjusted with ammonia water. The preferred strains 102, 103, 104, and 105 are induced at pH 4 and 5, respectively, to confirm the appropriate induction pH.

[0125] The conditions for batch culture and induced expression of Pichia yeast engineering strains expressing 102, 103, 104, and 105 are the same except for pH: the batch feeding method is adopted, and the culture temperature is 30°C. Each engineering bacterium is inoculated into two 1L shaking bottles containing 200mL seed culture medium YPG, 220rpm, 30°C, and cultured for 18-20h to OD600 = 2-10. A 5L fermenter (Baoxing Biology) is used, with a liquid volume of 2L fermentation medium, 2% glycerol is sterilized separately, and the speed is adjusted to 300rpm before inoculation, the ventilation volume is 4L / min, and the temperature is 30°C. The pH is adjusted with an alkali solution prepared with concentrated ammonia water, and the pH is set to 4.5. Then 0.9mL PTM1 is first inoculated, and then the prepared 200ml seed solution is inoculated into the tank (flame circle inoculation), and then the dissolved oxygen electrode is clicked for calibration, and fermentation is started after calibration. When the dissolved oxygen drops to 30% for the first time during growth, use the dissolved oxygen cascade speed function to maintain 30%; wait for the glycerol to be consumed, the dissolved oxygen rebounds, and the dissolved oxygen is greater than 70% (OD 600 Value is about 20), cancel the dissolved oxygen cascade speed, increase the stirring to 650rpm, use 30% linkage feeding for glycerol, and feed 80mL. Stop feeding glycerol. After the dissolved oxygen rebounds to more than 70%, set pH4 and pH5 for each strain respectively, and other conditions are the same. The temperature is 29℃, and the induction culture is carried out with methanol and glycerol mixed carbon source (methanol: 50% glycerol = 7:3). Manually add 5ml, wait for the dissolved oxygen to rebound to more than 70%, set the feeding rate to 8mL / h, increase to 10mL / h after one hour, and increase again to 20mL / h after one hour. When the dissolved oxygen value is lower than 30%, stop feeding, wait for the dissolved oxygen to rebound, and feed linkage after the dissolved oxygen rises to 30%. Induce for 40 to 60h, and the UV measurement of protein concentration is not obvious or decreases, then the tank can be released. UV protein quantitative formula: C (mg / mL) = 0.144*(A215-A225), A215<1.5.

[0126] The fermentation supernatant was collected for SDS-PAGE electrophoresis detection. The test results were as follows: Figure 2 As shown. Figure 2 It can be seen that under high-density fermentation conditions, each strain can efficiently express the target protein, and the expression is better under the condition of pH 5.

[0127] (2) Purification:

[0128] The supernatant of the small-scale fermentation of each strain in step (1) was collected and purified. According to the properties of the four proteins 102, 103, 104, and 105, they were all purified by ionization. The raw materials and auxiliary materials used are shown in Table 1; the purification equipment used is the chromatograph Bio-Lab100 (Hanbang), the ultrafiltration BONA-GM-18 (Bona), and the freeze dryer Pilot2-4MC (Boyikang). The chromatography buffer was prepared, BufferA: 20mM KH2PO4, pH4.0, BufferB: 20mM KH2PO4, 1M NaCl, pH4.0.

[0129] Table 1: Purification raw materials and auxiliary materials

[0130]

[0131] Among them, the purification process flow is: ultrafiltration desalination of fermentation supernatant → chromatography → ultrafiltration desalination of eluate → freeze-drying into sponge;

[0132] Pretreatment of fermentation supernatant: ultrafiltration desalination of fermentation broth to a conductivity of 7 ms / cm, and adjustment of pH to 4.0;

[0133] Chromatography process:

[0134] 1) Column equilibration: Manual mode, flow rate set to 35 ml / min, inlet A1 (Buffer A), column valve forward flushing, continuous monitoring of the Cond conductivity curve and pH curve on the spectrum interface until the conductivity curve drops to the lowest point and flattens out (about 3 column volumes). When the pH curve flattens out, it means the column has been equilibrated. Click Pause.

[0135] 2) Sample loading: Manual mode, flow rate set to 30 ml / min, inlet A2 (sample pH 4.0, 1000 ml), click Pause when the injection volume reaches the required volume.

[0136] 3) Rebalance: Manual mode, flow rate set to 35 ml / min, inlet A1 (Buffer A), click Continue, run the program to continuously monitor the spectrum curve, and continue balancing after the UV curve, conductivity curve, and pH curve are stable, and click Pause.

[0137] 4) Elution: Manual mode, flow rate set to 35 ml / min, inlet B1 (Buffer B), set 25% B and 100% B to elute and collect respectively, click Continue, when the A215 spectrum curve rises, click Outlet1, click Continue, start eluting and collecting the target component, until UV215 drops to the lowest point, and the collection ends. Observe the eluent volume and detection concentration.

[0138] Eluent treatment:

[0139] The collected eluate was ultrafiltered and desalted to a conductivity of less than 1 ms, then freeze-dried, and sponges 102 to 105 were collected. An appropriate amount of sponges was taken for SDS-PAGE verification. The results were as follows: Figure 3 As shown. Figure 3 It can be seen that the purity of 104 is relatively low, and the purity of 102, 103, and 105 can all produce freeze-dried sponges with higher purity.

[0140] Example 5: Stability test of recombinant type I collagen

[0141] (1) Stability test of recombinant type I collagen:

[0142] The sponges 102 to 105 obtained in Example 4 were respectively prepared into 1 mg / mL solutions with ddH2O, and then subjected to stability tests under repeated freeze-thaw cycles, different temperatures (4°C, 25°C, 40°C, 60°C), and different pH conditions (4, 7, 9) to determine the sequences with the best stability of the four recombinant type I collagens.

[0143] The pH of the solution containing sponges 102-105 was adjusted to 3.0, 7.0, and 9.0 respectively, and placed at 25°C. Samples were taken at different days for SDS-PAGE verification. The results are as follows: Figure 4 As shown. Figure 4 It can be seen that among the four sequences of freeze-dried sponges, sponge 102 showed the best stability at different pH levels.

[0144] Take 1 mL of the solution containing sponges 102 to 105, freeze and thaw three times, freeze at -80°C for 10 min each time, heat at 99°C for 10 min, take samples, and verify by SDS-PAGE. The results are as follows: Figure 5 As shown. Figure 5 It can be seen that among the four sequences of freeze-dried sponges, sponge 102 showed the best stability at different temperatures.

[0145] Solutions containing sponges 102 to 105 were taken respectively and placed at 4°C, 25°C, 40°C, and 60°C for 7 days. During this period, samples were taken at different time periods for SDS-PAGE verification. The results showed that among the freeze-dried sponges of the four sequences, sponge 102 showed the best stability at different temperatures.

[0146] In summary, the stability of 102 sponge meets the stability requirements, and significant degradation occurs under different conditions.

[0147] (2) Structural verification of recombinant type I collagen:

[0148] Take appropriate amount of purified 102-105 sponges and dissolve them in ddH2O to 1 mg / mL, then place them in a 4℃ refrigerator for 48 hours. After the equilibrium is completed, use a circular dichroism spectrometer to detect. Before the detection, the circular dichroism spectrometer is calibrated with air and ddH2O, and then the solutions containing sponges 102-105 are detected in turn. The test results are as follows: Figure 6 As shown. Figure 6 It can be seen that the 102 sponge has a certain high-level structure. In addition to the primary and secondary structures, circle II has a characteristic absorption peak at 221 and has a higher-level tertiary structure. The other three have no characteristic absorption peaks at 221 and are mainly primary and secondary structures.

[0149] Example 6: Cell experiment of recombinant type I collagen

[0150] In this example, three cell experiments of cytotoxicity, cell adhesion and cell migration were performed on four small test freeze-dried sponge samples of 102 to 105 with reference to "GBT16886.5-2017 In vitro cytotoxicity test", "GB / T16886.12-2017 Sample preparation and reference materials", "YY / T 1849-2022 Recombinant collagen", etc. The specific steps are as follows.

[0151] (1) Cytotoxicity test:

[0152] The whole process was performed in a clean bench to ensure aseptic operation. L-929 cells (cell line from the Chinese Academy of Sciences Cell Bank, SCSP-5039) were cultured in MEM medium (containing 10% FBS, 1% penicillin-streptomycin) at 37°C and 5% CO2. The cells grown to the logarithmic growth phase were digested with 0.25% trypsin (containing EDTA). After digestion, the cell suspension was centrifuged (1000 rpm, 5 minutes), the supernatant was discarded, and the cells were resuspended in MEM medium and counted to obtain 1×10 5 Cells / mL cell suspension. Add samples for culture, inoculate 100 μL of the cell suspension into each well of a 96-well plate, and culture in a cell culture incubator (37°C, 5% CO2, >90% humidity), and observe cell morphology under a microscope.

[0153] After 24 hours of culture, the cells adhered to the wall and grew to about 70%. The original culture medium in the 96-well plate was discarded, and 100 μL of extract (final concentration of 100%, 75%, 50% and 25%), blank control sample, negative control sample and positive control sample were added to the corresponding wells of the 96-well plate. The 96-well plate was placed in a cell culture incubator (37°C, 5% CO2, >90% humidity) for 24 hours, and six replicate wells were set for each group.

[0154] After 24 hours of culture, the 96-well plate was removed and the cell morphology was observed under a microscope. Then the liquid was removed and 50 μL MTT (final concentration was 1 mg / mL) was added to each well and cultured in a 37°C, 5% CO2 incubator. After 2 hours, the supernatant was removed and 100 μL isopropanol was added to each well to dissolve the crystals. The absorbance value at a wavelength of 570 nm was measured on an ELISA reader to calculate its cytotoxicity. The results are shown in Figure 7 shown.

[0155] Figure 7 The results showed that under the experimental conditions, the quantitative evaluation results suggested that the relative survival rate of the 100% concentration group (concentration: 1%) of sample-102, sample-103, and sample-105 extracts was greater than 70%, which indicated that sample-102, sample-103, and sample-105 had no potential cytotoxicity to L-929 cells. The relative survival rate of the 100% concentration group (concentration: 1%) of sample-104 extract was less than 70%, indicating that the test sample-104 had potential cytotoxicity to L-929 cells. In addition, 102 had a proliferation effect on cells at a certain concentration.

[0156] (2) Cell adhesion test:

[0157] Add 100 μL of sample (sample / blank control) to each 96-well plate, and prepare two gradients of 0.5 mg / mL and 1 mg / mL for each sample. Prepare 4 wells for each sample coating and incubate in a 37°C, 5% CO2 incubator for 2 hours. Remove excess coating solution from the wells, add 100 μL of 1% BSA-PBS solution, and incubate in a 37°C, 5% CO2 incubator for 1 hour. After removing the liquid in the wells, wash three times with D-PBS, discard the washing solution, seal with sealing film, and place at 4°C for use.

[0158] NIH / 3T3 cells (cell line from the Chinese Academy of Sciences Cell Bank, SCSP-515) were cultured in a 37°C, 5% CO2 cell culture incubator, and the cell density and status were observed under an inverted microscope every day. When the cells grew to 80% to 90% of the culture flask, cell passage or cell inoculation was performed. The cells were diluted to 5×10 using complete culture medium premixed with Hoechst-33342 fluorescent dye (10%). 4 100 μL of cells were added to the wells, covered with aluminum foil, and incubated for 1 h at 37°C, 5% CO2. Three replicates were measured, and the fourth well was used to adjust the microscope parameters and its measurement value was not used.

[0159] Use an inverted microscope to capture at least 4×4 digital tile images (fluorescence) of each of the 3 wells. Each well is filled with D-PBS to form a "reverse meniscus", purged of bubbles and covered with sealing film. Centrifuge the plate (inverted) at a relative centrifugal force (RCF) of 300g at 22°C for 5 min. After centrifugation, discard the sealing film and remove the supernatant from the well. After washing once with D-PBS, add 100 μL of D-PBS. For each of the 3 wells, a total of 25 fluorescent tile digital images were taken (at least 4×4 matrix is ​​recommended, with 10% tile overlap), and approximately 2400 to 3600 cells were calculated for each sample (800-1200 cells / well × 3 wells). The detection results are shown in Figure 8 And Table 2. Combination Figure 8 As can be seen from Table 2, 102-105 all have a positive effect on cell adhesion, among which 102 performs better.

[0160] Table 2. Cell adhesion test results

[0161]

[0162] Note: * indicates significant difference in migration rate compared with the serum-free group (blank group) (P<0.05), chi-square test method.

[0163] (3) Cell migration assay:

[0164] First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate with a ruler, about every 0.5cm to 1cm, across the holes. Draw 3 lines in each hole and add about 5×10 5 NIH / 3T3 cells.

[0165] On the second day of cell culture, use the tip of the gun to measure the ruler and try to scratch it perpendicular to the horizontal line on the back. The tip of the gun should be vertical and not tilted. Rinse the cells with PBS three times, remove the scratched cells, add serum-free culture medium containing the test sample as the experimental group, and set the gradient concentration to 0.05% and 0.1%. Put it in a 37℃, 5% CO2 incubator and culture it. Take samples at 0h and 24h and take pictures.

[0166] The scratch area of ​​each picture was calculated using Image J image processing software, and the migration rate of each group of cells was calculated by dividing the total area of ​​migrating cells in the fixed scratch area by the initial area of ​​the fixed scratch area. The sample was used as the horizontal axis and the migration rate ratio was used as the vertical axis (unit %). The photos of the experimental group and the control group at the initial time 0 and the end of the experiment were compared. The data differences of each experimental group were analyzed using a one-way ANOVA and the chi-square test method. The results are shown in Fig. 9 As shown in the results, it can be seen that the results show that samples 102 to 105 have a positive effect on cell migration, among which 102 performs better.

[0167] In summary, recombinant type Ⅰ collagen 102-105 all have biological activity, among which 102 performs better.

[0168] Example 7: Amplification verification

[0169] Through the shake flask verification of Example 3 and the small-scale experimental verification of Example 4, it can be concluded that the freeze-dried sponge of the recombinant type I collagen of sequence 102 has a certain advanced structure, high stability, and better cell experimental performance. In this example, the recombinant engineering strain expressing recombinant type I collagen 102 was verified by fermentation at a scale of 500L tank volume for three consecutive batches to verify the stability of expression and to outsource the identification of the sponge.

[0170] Among them, the 500L fermentation process is based on the small-scale test process, and the optimal pH for fermentation induction is preferably 5.0. Three consecutive batches (batch numbers are SF2406002, SF2406003, and SF2406004) were fermented, purified, and freeze-dried to obtain 119g, 134g, and 118g of freeze-dried sponges, respectively. The final sponges were subjected to SDS-PAGE detection, and the results are as follows Fig.10 The final sponge was tested by SDS-PAGE and grayscale analysis, and the purity was 97.6%, 96.9%, and 100%, respectively. The gel image results are shown in Fig.10 As shown in the figure, it can be seen that 102 freeze-dried sponges were obtained by three batches of 500L process, and the purity was above 95%.

[0171] Example 8: Characterization of High Stability Recombinant Type I Collagen

[0172] In this example, the 103 and 105 recombinant type I collagen freeze-dried sponges obtained in Example 4 were subjected to gel electrophoresis experiments. After staining and decolorization, the main bands were cut off and sent to Suzhou Putai Biomedicine Co., Ltd. for mass spectrometry detection and verification. The detection results of 103 are as follows: Fig.11 As shown, the 105 test results are as follows Fig.12 shown.

[0173] from Fig.11 and 12 It can be seen that the mass spectrometry detection results of 103 and 105 are highly consistent with the theoretical sequences SEQ ID NO.3 and SEQ ID NO.5, indicating that the results are in line with expectations.

[0174] In this example, three batches of 102 recombinant type I collagen freeze-dried sponges obtained in Example 7 were sent to Beijing Biotech Biotechnology Co., Ltd. for mass spectrometry detection. The test results are as follows: Fig.13 As shown. Fig.13It can be seen that the mass spectrometry identification results of the full sequences of the three batches of trial production samples of 102 are completely consistent with the theoretical sequence, indicating that the results are in line with expectations and that its engineered strain can stably express the target product.

[0175] In summary, the present invention avoids potential enzyme cleavage sites and unstable sequence fragments during sequence design through directional selection of existing amino acid sequences, so that the sequences can be efficiently expressed in Pichia pastoris, and the recombinant type I collagen obtained after purification has high stability; the recombinant type I collagen takes into account relevant biological active sites, as well as hydrophilicity and other factors, has high stability and relevant biological activity, and has good practicality.

[0176] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0177] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. Highly stable recombinant type I collagen, characterized in that: The high-stability recombinant type I collagen includes a repeated series of fragments A, and the fragment A includes a plurality of sequence fragments with high stability, easy to be efficiently expressed by Pichia pastoris, and not easily cleaved by enzymes. The sequence fragments are one or more of 302-322AA, 413-445AA, 485-532AA, 554-577AA, 593-613AA, 668-679AA, 740-763AA, 776-799AA, 899-922AA, 962-976AA, 1010-1039AA, and 1070-1102AA in the human type I collagen CO1A1.

2. The high-stability recombinant type I collagen according to claim 1, characterized in that: The fragment A comprises any one of the following sequence fragment combinations: a) 554-577AA, 1070-1102AA; b)668-679AA, 776-799AA, 1082-1102AA; c)593-613AA, 776-799AA, 962-976AA; d)593-613AA, 668-679AA, 776-799AA, 962-976AA, 1082-1102AA; e)413-445AA, 899-922AA, 1082-1102AA; f)302-322AA, 485-532AA, 740-763AA, 1010-1039AA; g)302-322AA, 485-532AA, 776-799AA; h)668-679AA, 740-763AA, 1010-1039AA, 1082-1102AA.

3. The high-stability recombinant type I collagen according to claim 1, characterized in that: The size of the highly stable recombinant type I collagen is 30-50 kDa.

4. The high-stability recombinant type I collagen according to claim 1, characterized in that: LEKR is added to the N-terminus of the high-stability recombinant type I collagen.

5. The highly stable recombinant type I collagen according to any one of claims 1 to 4, characterized in that: The high-stability recombinant type I collagen includes the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, or amino acid sequences that are more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identical to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.

8.

6. A nucleotide encoding the highly stable recombinant type I collagen according to any one of claims 1 to 5.

7. A recombinant expression vector comprising nucleotides encoding the highly stable recombinant type I collagen according to claim 6.

8. A recombinant engineered bacterium, comprising a nucleotide encoding the highly stable recombinant type I collagen according to claim 6 or a recombinant expression vector according to claim 7.

9. The recombinant engineered bacterium according to claim 8, characterized in that The host bacteria of the recombinant engineering bacteria are eukaryotic bacteria or prokaryotic bacteria.

10. The recombinant engineered bacterium according to claim 8, characterized in that: The host bacterium is Pichia pastoris.

11. The recombinant engineered bacterium according to claim 8, characterized in that: The recombinant engineered bacteria are deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit numbers being CGMCC No.31952, CGMCC No.31953 and CGMCC No.31954.

12. A composition comprising the high-stability recombinant type I collagen according to any one of claims 1 to 5.

13. A product comprising the high-stability recombinant type I collagen according to any one of claims 1 to 5 or the composition according to claim 11.

14. Use of the high-stability recombinant type I collagen according to any one of claims 1 to 5, the composition according to claim 12 or the product according to claim 13 in the preparation of medical devices, biomaterials, tissue engineering products and cosmetics.

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