Recombinant hydroxylated micromolecular collagen as well as expression system and preparation method thereof

Abstract

The invention provides recombinant hydroxylated micromolecular collagen as well as an expression system and a preparation method thereof, and belongs to the technical field of bioengineering. According to the invention, synthetic biology, genetic engineering and biotechnology are utilized to co-express micromolecular collagen and P4H, then amino acid modification is carried out on the micromolecular collagen and P4H, hydroxylation is carried out at a G-X-Y theoretical hydroxylation site, and post-translational modification is carried out to obtain the recombinant hydroxylated micromolecular collagen. The recombinant hydroxylated micromolecular collagen is modified after amino acid expression, so that the molecular structure is stable, compared with self-assembly after micromolecular expression, the recombinant hydroxylated micromolecular collagen is more controllable, and transdermal absorption and biological structure stability can be both considered; the recombinant hydroxylated micromolecular collagen can realize industrial production, and has good application in the fields of medicines, medical equipment, biological materials, tissue engineering products, cosmetics or health care products and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a recombinant hydroxylated small molecule collagen and an expression system and a preparation method thereof. Background Art

[0002] Collagen is an important biomaterial, which is the main component of the extracellular matrix (ECM). It can be produced by expressing recombinant collagen in insect cells or other mammalian cells, and genetic engineering is used as a technical means to increase the expression amount. However, this method has a high investment cost, low yield, and the synthesized collagen is all macromolecules. Macromolecular collagen is expensive and difficult to synthesize and separate, and it is easy to degrade, has poor stability, low yield, and is very likely to cause protein loss during production and separation, and is complicated to operate. In addition, it also has limitations in application, and it is difficult to achieve large-scale production.

[0003] In recent years, due to the high cost of mammalian cell culture, slow growth cycle, and low expression, although it is secreted into the supernatant, there are many purification steps, high product loss, and it cannot be industrialized; while the yield in the large intestine expression system has increased, it has not reached the scale of commercial production. Therefore, the above two expression yields are low, the protein stability is poor and easy to degrade, and it is limited to laboratory research scale. Therefore, it is necessary to develop a recombinant small molecule collagen with better stability.

[0004] The triple helix of collagen is composed of repeated glycine-XY motifs, and the X and Y positions can be any amino acids, usually proline or 4-hydroxyproline, respectively. Collagen is a procollagen molecule composed of an amino-terminal propeptide, a short non-helical N-terminal peptide, a central triple helix, a C-terminal peptide, and a carboxyl-terminal propeptide. The post-translational modification of the single pre-a chain (hydroxylation of proline and lysine residues, glycosylation of lysine and hydroxylysine residues, and sulfation of tyrosine residues) stops due to the formation of the triple helix. The hydroxylation of collagen proline residues catalyzed by collagen proline 4-hydroxylase (C-P4H) is essential for the stability of the collagen triple helix.

[0005] Hydroxylation mainly relies on proline hydroxylase (P4H). Currently, there is no research on the expression system of co-expression of recombinant small molecule collagen and P4H enzyme. Therefore, it is necessary to develop a recombinant hydroxylated small molecule collagen to improve the stability and activity of recombinant small molecule collagen. Summary of the invention

[0006] In view of some deficiencies in the prior art, the present invention provides a recombinant hydroxylated small molecule collagen and an expression system and a preparation method thereof; the present invention utilizes synthetic biology, genetic engineering and biotechnology to co-express small molecule collagen with P4H, and then performs amino acid modification on the small molecule collagen, performs hydroxylation at the GXY theoretical hydroxylation site, and performs post-translational modification to obtain the recombinant hydroxylated small molecule collagen; the recombinant hydroxylated small molecule collagen of the present invention stabilizes the molecular structure through amino acid post-expression modification, and is more controllable in self-assembly after small molecule expression, and can take into account both transdermal absorption and biological structure stability; the recombinant hydroxylated small molecule collagen can be industrially produced and has good applications in the fields of drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0008] The present invention first provides a recombinant hydroxylated small molecule collagen, which is a hydroxylated recombinant small molecule collagen. The recombinant small molecule collagen includes an amino acid sequence containing a hydroxylation site on the triple helix region of human type VII collagen or the α1 chain of human type III collagen.

[0009] Preferably, the amino acid sequence is as shown in i, ii or iii:

[0010] i. The amino acid sequence is as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or the amino acid sequence is obtained by repeating the sequence shown in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 as the basic unit multiple times in series;

[0011] ii. the amino acid sequence is shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6;

[0012] iii. A protein derived from i or ii, in which the amino acid sequence in i or ii is substituted, deleted or added with one or more amino acids and has human collagen activity, or an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the sequence shown in i or ii.

[0013] The present invention also provides nucleic acid encoding the above recombinant hydroxylated small molecule collagen.

[0014] Preferably, the nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO: 7 to SEQ ID NO: 9, or a degenerate sequence thereof.

[0015] The present invention also provides a recombinant expression vector, which comprises the above nucleic acid.

[0016] The present invention also provides a recombinant engineered bacterium, which comprises the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or a recombinant engineered bacterium containing a recombinant plasmid expressing P4H enzyme and capable of expressing the above-mentioned recombinant hydroxylated small molecule collagen.

[0017] Preferably, the recombinant engineered bacteria include a chassis strain for expressing the above-mentioned recombinant hydroxylated small molecule collagen, and the chassis strain expresses P4H enzyme.

[0018] Preferably, the chassis strain comprises nucleic acids encoding the amino acids shown in SEQ ID No.10 and SEQ ID No.13.

[0019] 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.33067, CGMCC NO.33068, and CGMCC NO.33069.

[0020] The present invention also provides a chassis strain for expressing the above-mentioned recombinant hydroxylated small molecule collagen, wherein the chassis strain expresses P4H enzyme.

[0021] Preferably, the host bacteria of the chassis strain includes one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula; preferably Pichia pastoris; more preferably Pichia pastoris X33 or Pichia pastoris KM71H.

[0022] The present invention also provides a method for preparing the above-mentioned recombinant hydroxylated small molecule collagen, the preparation method comprising:

[0023] (1) selecting and designing the sequence of recombinant hydroxylated small molecule collagen, and then constructing a collagen tandem repeat sequence based on the recombinant hydroxylated small molecule collagen as a basic unit tandem repeat;

[0024] (2) constructing a recombinant expression vector expressing collagen tandem repeat sequences;

[0025] Constructing a recombinant vector expressing P4H enzyme;

[0026] (3) electroporating the constructed recombinant expression vector expressing the collagen tandem repeat sequence and the recombinant vector expressing the P4H enzyme into the host bacteria, screening and verifying, and obtaining a recombinant engineered bacterium expressing a high copy of the recombinant hydroxylated small molecule collagen;

[0027] (4) fermenting and inducing the expression of the obtained recombinant engineered bacteria to obtain the recombinant hydroxylated small molecule collagen.

[0028] Preferably, in step (1), the recombinant hydroxylated small molecule collagen is a hydroxylated recombinant small molecule collagen, and the recombinant small molecule collagen includes an amino acid sequence containing a hydroxylation site on the triple helix region of human type VII collagen or the α1 chain of human type III collagen;

[0029] The amino acid sequence is shown in i, ii or iii:

[0030] i. The amino acid sequence is as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or the amino acid sequence is obtained by repeating the sequence shown in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 as the basic unit multiple times in series;

[0031] ii. the amino acid sequence is shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6;

[0032] iii. A protein derived from i or ii, in which the amino acid sequence in i or ii is substituted, deleted or added with one or more amino acids and has human collagen activity, or an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the sequence shown in i or ii.

[0033] Preferably, when the amino acid sequence of the recombinant hydroxylated small molecule collagen is as shown in i, the collagen tandem sequence contains 8 to 10 basic unit recombinant type VII collagens; and there are sites for recognition and cleavage by Kex2 enzyme, Ste13 or Kex1 enzyme between two adjacent basic units.

[0034] Preferably, the recognition and cleavage site includes KR or RR dibasic amino acid residues, followed by EA, EAEA or other amino acid residues that facilitate cleavage by Kex2 enzyme, Ste 13 enzyme or Kex1 enzyme.

[0035] Preferably, in step (1), the nucleic acid of the collagen tandem sequence comprises a nucleotide sequence as shown in SEQ ID NO: 7 to SEQ ID NO: 9, or a degenerate sequence thereof.

[0036] Preferably, in step (2), the recombinant expression vector and the recombinant plasmid vector include pPICZαB, pFLDα, and pPIC9K.

[0037] Preferably, the vector is pPIC9K or pPICZαB.

[0038] Preferably, in step (2), the recombinant plasmid expressing the P4H enzyme comprises nucleic acids encoding the amino acids shown in SEQ ID No. 10 and SEQ ID No. 13, a PTEF promoter element and a PEM7 promoter element.

[0039] Preferably, in step (3), the host bacteria includes one of Pichia pastoris, Saccharomyces cerevisiae and Hansenula.

[0040] Preferably, the host bacteria is Pichia pastoris, preferably Pichia pastoris X33 or Pichia pastoris KM71H.

[0041] Preferably, the host bacteria is CGMCC No.29601, CGMCC No.25815, or CGMCC No.258199.

[0042] Preferably, in step (3), the recombinant engineered bacteria are deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the deposit numbers CGMCC NO.33067, CGMCC NO.33068, and CGMCC NO.33069.

[0043] The present invention also provides recombinant hydroxylated small molecule collagen prepared by the above method.

[0044] The present invention also provides an expression system of the above-mentioned recombinant hydroxylated small molecule collagen, which comprises: a recombinant engineering bacterium of a recombinant plasmid expressing P4H enzyme and a collagen tandem sequence constructed with the recombinant hydroxylated small molecule collagen as a basic unit.

[0045] The present invention also provides a composition, which includes the above-mentioned recombinant hydroxylated small molecule collagen, or the recombinant hydroxylated small molecule collagen encoded by the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineering bacteria, or the recombinant hydroxylated small molecule collagen prepared by the above-mentioned method; the composition includes medical devices, biomaterials, tissue engineering products, and cosmetics.

[0046] The present invention also provides a product, which includes the above-mentioned recombinant hydroxylated small molecule collagen, or the recombinant hydroxylated small molecule collagen encoded by the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineering bacteria, or the recombinant hydroxylated small molecule collagen prepared by the above-mentioned method, or a combination; the product includes medical devices, biomaterials, tissue engineering products, and cosmetics.

[0047] The present invention also provides the use of the above-mentioned recombinant hydroxylated small molecule collagen, or the above-mentioned recombinant hydroxylated small molecule collagen encoded by the nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineering bacteria, or the recombinant hydroxylated small molecule collagen prepared by the above-mentioned method, or the above-mentioned composition, or the above-mentioned product in the preparation of medical devices, biomaterials, tissue engineering products, and cosmetics.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The recombinant hydroxylated small molecule collagen of the present invention is selected from a sequence of about 50 amino acids with hydroxylation sites on the triple helix region of human type VII collagen or the α1 chain of human type III collagen. The recombinant hydroxylated small molecule collagen can be expressed in Pichia pastoris, can be efficiently secreted and expressed outside the cell, is not easily degraded during the purification stage, and reduces the difficulty of purification. The expression method of the present invention avoids the cost and risk of exogenous protein residue caused by protease cleavage in vitro, and can also shorten the time and cost of the subsequent purification process.

[0050] (2) The recombinant hydroxylated small molecule collagen of the present invention is co-expressed with the P4H enzyme in Pichia pastoris, and the amino acid sequence is hydroxylated based on the expression of the target sequence. It is hydroxylated at the GXY theoretical hydroxylation site and then modified after translation, so that the structure of the small molecule collagen is more stable, and both transdermal absorption and biological structure stability can be taken into account. The modification of amino acids after expression in the scheme of the present invention stabilizes the molecular structure, which is more controllable than the self-assembly after small molecule expression.

[0051] (3) The recombinant hydroxylated small molecule collagen described in the present invention has similar physical and chemical properties and biological functions to the original sequence, has a smaller molecular weight, has a better effect on tissue absorption, and has better cell adhesion and migration effects than unhydroxylated small molecule collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the verification result of Pichia pastoris colony PCR after 705, 721 and 3A5D2NT were co-transfected with P4H.

[0053] Figure 2 This is the verification result of Pichia pastoris colony PCR after KM71H / P4H co-transformed P4H.

[0054] Figure 3 This is the WB verification result after the recombinant engineering bacteria were co-transformed with P4H; in the figure, a is the P4HA1 WB detection picture; b is the P4HB WB detection picture.

[0055] Figure 4This is the supernatant of recombinant hydroxylated small molecule collagen shake flask expression after 72 hours of induction; in the figure, a is X33 / pPICZαB-705-P4H; b is KM71H / pPICZαB-721-P4H; c is GS115-HCPB-PPKEX2 / 3A5D2NT-P4H.

[0056] Figure 5 The electrophoresis diagrams of the recombinant hydroxylated small molecule collagen 5L fermentation supernatant (a) and after purification (b).

[0057] Figure 6 The figure shows the cell adhesion activity detection. a is the NIH / 3T3 cell viscosity ratio, and b is the HaCat cell adhesion ratio.

[0058] Figure 7 Figure 2 is the result of cell migration area ratio detection, a is the migration rate of NIH / 3T3 cells, and b is the migration rate of HaCat cells.

[0059] Figure 8 These are pictures of cell migration; in the figure, (a) is the migration picture of NIH / 3T3 cells; (b) is the migration picture of HaCat cells.

[0060] Fig. 9 The hydroxylation modification sites (a) and secondary mass spectrum (b) of recombinant hydroxylated small molecule collagen 705.

[0061] Fig.10 The hydroxylation modification sites (a) and secondary mass spectrum (b) of recombinant hydroxylated small molecule collagen 721.

[0062] Fig.11 The hydroxylation modification sites (a) and secondary mass spectrum (b) of the recombinant hydroxylated small molecule collagen 3A5D2NT.

[0063] Fig.12 These are the results of cytotoxicity assay of recombinant hydroxylated small molecules. DETAILED DESCRIPTION

[0064] 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. In the embodiments of the present invention, all that is not described in detail is accomplished by conventional experimental methods, and all that is not described in detail in the processes involved in the embodiments is understandable and easily implemented by those skilled in the art based on the product manual or basic knowledge in the art, and therefore will not be described in detail.

[0065] Embodiment 1:

[0066] S1. Design of amino acid sequence of recombinant hydroxylated small molecule collagen:

[0067] A sequence of about 50 amino acids from the triple helix region of human type VII collagen (Q02388·CO7A1_HUMAN) or the α1 chain of human type III collagen (P02461) was selected, KR was added to the C-terminus, EA was added to the N-terminus, and 6-10 copies were connected in series to make the target sequence size about 50KD to adapt to the optimal expression range of Pichia pastoris. After expression in Pichia pastoris, kex2 contained in Pichia pastoris itself cuts the KR carboxyl end in the amino acid connection KREA, ste13 cuts the nitrogen-terminal EA, and Kex1 cuts the remaining KR residues, which are secreted into the extracellular space through the secretion signal peptide. The recombinant hydroxylated small molecule collagen is composed of multiple repeated polypeptide sequences, the sequence starts with EA, the two monomers are connected by the KEX2 restriction site, and GLEKR or ends.

[0068] (1) Select 2156-2207AA of Q02388.CO7A1-HUMAN, add LE to the C-terminus, and obtain a recombinant hydroxylated small molecule collagen, named 705, the sequence of which is shown in SEQ ID NO: 1. Based on SEQ ID NO: 1, add EA to the N-terminus and KR to the C-terminus to form a monomer, and the monomer is repeated 6 times to form a recombinant collagen sequence of amino acids shown in SEQ ID NO: 2.

[0069] SEQ ID NO: 1:

[0070] GERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLE;

[0071] SEQ ID NO:2:

[0072] KREAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEKREAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEKREAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEK REAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEKREAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEKREAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEKR.

[0073] (2) 2656-2701AA of Q02388.CO7A1-HUMAN was selected, LE was added to the C-terminus, and a recombinant hydroxylated small molecule collagen was obtained, named 721, and the sequence was shown in SEQ ID NO: 3. Based on SEQ ID NO: 3, EA was added to the N-terminus and KR was added to the C-terminus to form a monomer, and the monomer was repeated 10 times to form a recombinant collagen sequence with amino acids as shown in SEQ ID NO: 4.

[0074] SEQ ID NO:3:

[0075] GHKGEMGEPGVPGQSGAPGKEGLIGPKGDRGFDGQPGPKGDQGEKGLE;

[0076] SEQ ID NO:4:

[0077] *.

[0078] (3) The amino acid sequence of position 1036-1085 of the α1 chain of human type III collagen: P02461 was selected to form a recombinant hydroxylated small molecule collagen of 50 amino acids, named 3A5D2NT, and the sequence is shown in SEQ ID NO: 5. Based on SEQ ID NO: 5, "RGLE" amino acid was added to its C-terminus as a linker sequence, EA was added to the N-terminus, and KR was added to the C-terminus to form a monomer, and the monomer was repeated 9 times to form a collagen recombinant sequence of amino acids as shown in SEQ ID NO: 6.

[0079] SEQ ID NO:5:

[0080] GKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGH;

[0081] SEQ ID NO:6:

[0082] .

[0083] S2. Synthesis of DNA sequences and construction of recombinant plasmids:

[0084] S21. Construction of a recombinant expression vector expressing recombinant hydroxylated small molecule collagen:

[0085] Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize the nucleic acid sequences encoding the above-mentioned amino acid sequences SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, and the corresponding nucleic acid sequences are shown in SEQ ID NO:7-SEQ ID NO:9, respectively.

[0086] The gene fragments shown in SEQ ID NO:7 and SEQ ID NO:8 were cloned into pPICZαB respectively, and the gene fragment shown in SEQ ID NO:9 was cloned into the pPIC9K vector. The exogenous target protein sequence was cloned using XhoI and NotI as restriction sites, so that the target fragment was accurately inserted into the secretory vector reading frame containing the secretion signal α-factor, and recombinant expression vectors expressing SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 were obtained, and were named pPICZαB-705, pPICZαB-721, and pPIC9K-3A5D2NT, respectively.

[0087] SEQ ID NO:7-SEQ ID NO:9 are shown below, wherein "CTCGAG" is the XhoⅠ restriction site and "GCGGCCGC" is the NotⅠ restriction site.

[0088] SEQ ID NO:7:

[0089]

[0090] SEQ ID NO:8:

[0091] CTCGAGAAAAGGGAAGCCGGACATAAGGGCGAGATGGGCGAACCAGGAGTCCCA

[0092] GGCCAATCCGGTGCGCCTGGCAAGGAAGGCTTAATTGGTCCCAAGGGGGACAGGGGCT

[0093] TCGATGGTCAGCCTGGTCCAAAGGTGATCAAGGAGAGAAGGGACTAGAGAAACGCGA

[0094] GGCTGGCCATAAGGGTGAGATGGGAGAACCAGGCGTTCCAGGTCAGTCTGGCGCCCCC

[0095] GGAAAAGAAGGGCTCATCGGGCCGAAGGGCGATCGCGGTTTTGACGGGCAGCCAGGA

[0096] CCGAAAGGGGATCAGGGTGAGAAGGGCTTGGAGAAGCGGGAAGCCGGTCACAAGGGGG

[0097] GAAATGGGTGAACCTGGGGTCCCCGGGCAATCGGGTGCTCCTGGAAAGGAGGGGTTGA

[0098] TTGGGCCAAAAGGCGACCGAGGATTCGACGGGCAGCCTGGCCCTAAGGGAGATCAAGG

[0099] GGAAAAGGGCCTGGAGAAACGCGAGGCTGGTCACAAGGGTGAGATGGGGGAACCGGG

[0100] CGTACCTGGACAAAGCGGGGCCCCGGGAAAAGAGGGTCTTATAGGTCCAAAGGGCGAC

[0101] AGAGGATTCGACGGCCAACCCGGACCCAAGGGTGATCAGGGCGAAAAGGCTTAGAG

[0102] AAGCGAGAGGCAGGGCACAAAGGGGAGATGGGGGAACCCGGTGTACCTGGACAATCG

[0103] GGAGCACCCGGCAAGGAGGGACTCATTGGGCCGAAAGGGGATAGGGGTTTTGATGGAC

[0104] AACCTGGGCCGAAAGGGGACCAAGGGGAAAAGGGGCTAGAGAAACGAGAAGCAGGA

[0105] CACAAAGGTGAAATGGGTGAGCCAGGAGTGCCTGGACAAAGCGGGGCTCCCGGGAAA

[0106] GAAGGGCTGATAGGCCCGAAGGGTGACAGAGGCTTCGATGGCCAGCCGGGCCCAAAAG

[0107] GAGATCAGGGCGAAAAAGGTCTGGAGAAACGGGAGGCAGGACACAAAGGAGAGATGG

[0108] GGGAGCCTGGCGTCCCCGGGCAATCTGGTGCTCCCGGCAAGGAAGGACTAATAGGACC

[0109] CAAGGGCGATCGTGGTTTTGATGGCCAACCCGGGCCAAAAGGTGATCAAGGTGAAAAA

[0110] GGGTTGGAAAAACGTGAAGCAGGACATAAAGGTGAAATGGGCGAACCGGGAGTACCA

[0111] GGCCAGTCAGGGGCTCCAGGAAAGGAGGGCCTTATCGGACCGAAGGGAGATCGGGGCT

[0112] TCGACGGCCAGCCCGGGCCTAAAGGTGACCAGGGGGAAAAAGGATTAGAGAAGCGTG

[0113] AGGCGGGTCATAAGGGTGAGATGGGCGAGCCTGGTGTTCCTGGGCAGAGTGGAGCCCC

[0114] AGGGAAAGAAGGGCTTATTGGTCCTAAGGGAGACAGGGGGTTTGACGGTCAACCCGGG

[0115] CCGAAAGGAGATCAGGGGGAAAAGGGCTTAGAAAAACGAGAAGCGGGTCATAAAGGC

[0116] GAGATGGGTGAGCCGGGAGTGCCGGGTCAATCAGGAGCGCCGGGCAAGGAAGGTCTG

[0117] ATCGGACCTAAGGGTGATCGTGGTTTTGATGGCCAACCAGGTCCGAAGGGTGACCAGGGGGAGAAAGGTTTAGAAAAGAGATGAGCGGCCGC。

[0118] SEQ ID NO:9:

[0119] CTCGAGGAAGCTGGAAAGTCCGGCGACCGGGGAGAGTCAGGGCCTGCAGGGCCT

[0120] GCTGGTGCCCCAGGACCGGCCGGCAGTAGAGGTGCTCCTGGTCCTCAAGGACCACGGG

[0121] GCGACAAAGGTGAAACAGGTGAACGAGGTGCGGCAGGTATAAAAGGGCACAGAGGGC

[0122] TAGAAAAGCGGGAAGCAGGAAAAAGTGGCGATCGCGGGGAATCAGGTCCCGCGGGGC

[0123] CGGCGGGTGCGCCCGGACCGGCGGGGTCCAGAGGTGCCCCTGGACCGCAGGGACCTC

[0124] GGGGTGACAAAGGTGAAACTGGGGAAAGGGGGGCTGCGGGCATAAAGGGACACCGCG

[0125] GACTCGAGAAGAGAGAGGCAGGTAAAAGTGGGGATAGAGGCGAATCTGGTCCGGCAG

[0126] GACCAGCGGGCGCCCCCGGTCCTGCTGGATCACGAGGTGCCCCGGGTCCGCAAGGTCC

[0127] TAGGGGTGACAAAGGCGAGACGGGCGAACGAGGGGCAGCAGGAATTAAAGGCCACCG

[0128] AGGGCTGGAGAAGCGGGAAGCAGGTAAATCAGGAGATAGGGGGGAAAGCGGACCGGC

[0129] CGGACCCGCTGGTGCGCCGGGACCTGCTGGATCGAGAGGTGCACCGGGTCCCCAAGGG

[0130] CCTCGTGGAGATAAAGGGGAGACCGGGGAGCGCGGGGCTGCAGGGATTAAGGGACAC

[0131] CGCGGCTTGGAGAAGCGAGAGGCTGGTAAAAGCGGCGACCGCGGCGAATCGGGTCCG

[0132] GCGGGTCCCGCGGGCGCACCTGGGCCTGCTGGGTCTAGGGGAGCGCCAGGGCCTCAAG

[0133] GTCCTCGCGGTGACAAGGGAGAAACAGGCGAGCGTGGGGCAGCGGGCATTAAAGGTCA

[0134] TCGTGGGTTAGAAAAGCGTGAGGCTGGCAAATCTGGCGATCGCGGGGAGAGCGGTCCA

[0135] GCAGGTCCGGCTGGTGCTCCAGGTCCAGCTGGTAGCCGTGGCGCCCCAGGTCCTCAAG

[0136] GTCCTCGTGGCGACAAAGGGGAGACCGGCGAGCGTGGAGCGGCAGGCATCAAAGGTC

[0137] ATCGTGGACTTGAAAAGAGGGAGGCGGGAAAGTCGGGCGATCGGGGTGAATCCGGCCC

[0138] AGCGGGTCCTGCTGGAGCCCCAGGCCCCGCTGGATCGCGGGGCGCACCTGGCCCCCAA

[0139] GGACCGCGTGGGGATAAAGGGGAGACTGGCGAGAGAGGCGCCGCCGGAATCAAAGGT

[0140] CACCGGGGATTAGAAAAGAGGGAGGCAGGGAAGTCGGGTGATCGAGGCGAATCAGGT

[0141] CCTGCTGGACCCGCTGGCGCGCCGGGTCCTGCTGGTAGCAGAGGAGCCCCTGGGCCTC

[0142] AAGGCCCGAGGGGTGATAAGGGTGAAACCGGAGAACGAGGCGCCGCTGGTATAAAGG

[0143] GACATCGTGGACTCGAGAAGCGAGAAGCGGGGAAAAGTGGGGACAGAGGAGAGTCTG

[0144] GTCCTGCCGGACCCGCCGGTGCTCCTGGTCCAGCAGGCTCCCGCGGCGCGCCTGGTCC

[0145] GCAAGGTCCTAGGGGAGACAAAGGAGAAACGGGCGAGCGAGGCGCCGCCGGGATCAAGGGGCATCGGGGGTTGGAGAAGAGGTAAGCGGCCGC。

[0146] S22. Construction of recombinant plasmid expressing P4H enzyme:

[0147] A. Construction of human P4H alpha subunit (abbreviated as P4HA) vector:

[0148] The amino acid of P13674.P4HA1_HUMAN was selected and named as human P4H alpha subunit (P4HA for short), and its sequence was shown in SEQ ID NO:10. The nucleic acid sequence encoding P4HA was synthesized by Nanjing GenScript Biotech Co., Ltd. The primers P4HA Forward and P4HA Reverse were designed to amplify the P4HA fragment, and an amplified fragment containing BamHⅠ at the 5' end and EcoRⅠ at the 3' end was obtained. The amplified fragment and pPIC9K plasmid were digested with restriction endonucleases BamHⅠ and EcoRⅠ, and the digested DNA was recovered and purified. The amplified fragment and the pPIC9K plasmid were ligated overnight at 16°C using T4 ligase to obtain a ligated sample. The ligated sample was transformed into competent Escherichia coli DH5α using the CaCl2 heat shock method, and then plated, cultured, screened, and sequenced for verification, indicating that its sequence was the P4HA sequence. The human P4H alpha subunit (named P4HA) vector was successfully obtained and recorded as P4HA 9K plasmid (abbreviated as P4H 9K).

[0149] SEQ ID NO:10:

[0150] ;

[0151] P4HA Forward (SEQ ID NO:11): AGATATCGGGTCCCCGGGAT;

[0152] P4HA Reverse (SEQ ID NO: 12): CGTGCCCCGGAATTCCTATTA.

[0153] B. Construction of human P4H enzyme β subunit (PDI for short) vector:

[0154] The amino acids of P07237.PDIA1_HUMAN 18-508 were selected and named as human P4H enzyme β subunit (abbreviated as PDI), and its sequence was shown in SEQ ID NO:13. Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize the nucleic acid sequence encoding PDI. Primers PDI Forward and PDI Reverse were designed to amplify the PDI fragment, and an amplified fragment containing BamHⅠ at the 5' end and EcoRⅠ at the 3' end was obtained. The amplified fragment and pPIC9K plasmid were digested with restriction endonucleases BamHⅠ and EcoRⅠ, and the digested DNA was recovered and purified. The amplified fragment and pPIC9K plasmid were ligated overnight at 16°C using T4 ligase to obtain a ligation sample; the ligation sample was transformed into competent Escherichia coli DH5α by CaCl2 heat shock method, and the sequence was plated, screened, and sequenced for verification, indicating that the sequence was the PDI sequence, and the human P4H enzyme β subunit (abbreviated as PDI) vector was successfully obtained.

[0155] SEQ ID NO:13:

[0156] DAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTL PDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPK SVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKK NVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDEL.

[0157] PDI Forward (SEQ ID NO: 14):

[0158] CCGCTCGAGGTTGCTATTGAAAAAAGAGATGCTCCAGAAGAAGAAGATCA;

[0159] PDI Reverse (SEQ ID NO: 15):

[0160] GCTCTAGACCTATTATTACAATTCATCTTTAAC.

[0161] C. Construction of P4H plasmid pPIC9K-P4H (DP) with dual promoters:

[0162] Primers Forward and Reverse were designed to perform PCR amplification on the PDI and promoter and terminator fragments on the human P4H enzyme β subunit (PDI for short) vector to obtain PCR products, which were fragments complementary to the two ends of pPIC9K after AtaⅡ linearization; restriction endonuclease AtaⅡ was used to digest the plasmid P4HA 9K to obtain a linearized plasmid; the PCR product and linearized plasmid were recovered and purified for later use.

[0163] The PCR product and the obtained linearized plasmid were connected at 50°C for 30 minutes using a ready-to-use seamless cloning kit, and the sample was transformed into competent E. coli DH5α using the CaCl2 heat shock method. The transformed E. coli was spread on an LB plate containing ampicillin and cultured for 24 hours. The obtained E. coli colonies were subjected to colony PCR to screen E. coli colonies containing PDI and related elements cloned into the P4HA 9K plasmid (P4H 9K). The plasmids extracted from the strains positive for colony PCR were identified by BamHⅠ and BspEI digestion. DNA sequencing of the plasmid showed that its sequence was PDI and the promoter and terminator sequence. The obtained P4H plasmid with dual promoters was named pPIC9K-P4H (DP).

[0164] Forward (SEQ ID NO:16): CCGAAAAGTGCCACCTGACGTAGATCTAACATCCAAAGA;

[0165] Reverse (SEQ ID NO: 17):

[0166] AGGTTAATGTCATGATAATAATGGTTTCTTAGACGTGTCTCACTTAATCTCTGTACT C.

[0167] D. Construction of P4H expression vector pPIC9K-P4H(DP)-1:

[0168] The nucleic acid sequences expressing the PTEF (SEQ ID NO: 18) and PEM7 (SEQ ID NO: 19) promoters were synthesized and modified at the 5' and 3' ends of the pPIC9K-P4H (DP) plasmid KanR, i.e., PTEF and PEM7 were added to the 5' end (according to the direction of the pPIC9K plasmid) of the KanR selection marker and CYC1tt was added to the 3' end to obtain the P4H expression vector pPIC9K-P4H (DP)-1.

[0169] PTEF (SEQ ID NO: 18):

[0170] cttagattagattgctatgctttctttctaatgaacaagaagtaaaaaaagttgtaatagaacaagaaaaatgaaactgaaacttgagaaattgaagaccgtt tattaacttaaatatcaatggaggtcactgaaagagaaaaaaactaaaaaaaaaaatttcaagaaaaagaaacgtgataaaaatttttattgcctttttcgac gaagaaaaagaaacgaggcggtctcttttttcttttccaaacctttagtacgggtaattaacgacaccctagaggaagaaagagggaaaatttagtatgctgt gcttgggtgttttgaagtggtacggcgatgcgcggagtccgagaaaatctggaagagtaaaaaaggagtagaaacattttgaagctatggtgtgtgggggatc;

[0171] PEM7 (SEQ ID NO: 19): gtcgtattatactatgccgatatactatgccgatgattaattgtcaac.

[0172] S3. Construction of recombinant engineering strains:

[0173] 10 μg of recombinant expression vectors pPICZαB-705, pPICZαB-721 and pPIC9K-3A5D2NT were linearized by Sal I (purchased from Dalian TaKaRa Company, the specific operation was carried out according to the kit instructions) at 37°C, and the recombinant plasmid pPIC9K-P4H (DP) was linearized by BspEI (purchased from Dalian TaKaRa Company, the specific operation was carried out according to the kit instructions) at 37°C, and then the linearized plasmid was recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and the volume was controlled at about 10 μL to obtain linearized plasmids pPICZαB-705, pPICZαB-721, pPIC9K-3A5D2NT, and pPIC9K-P4H (DP) -1.

[0174] The linearized plasmid pPIC9K-P4H(DP)-1 was electrotransformed into the competent cells of the host strain Pichia pastoris KM71H (purchased from Thermo Fisher Scientific), and the electrotransformed bacterial solution was spread on a YPDG (500 μg / mL, G418) plate, and each 200 μL was spread on a plate, and the plate was left to stand at room temperature for 10 min, and then inverted at 30°C for 2-5 days until a single colony (positive transformant) appeared. The obtained transformant was named KM71H / P4H. The linearized plasmid pPICZαB-721 was electrotransformed into the constructed KM71H / P4H, and the electrotransformed bacterial solution was spread on a YPDZ (100 μg / mL, Zeocin) plate, and each 200 μL was spread on a plate, and the plate was left to stand at room temperature for 10 min, and then inverted at 30°C for 2-5 days until a single colony (positive transformant) appeared. The obtained transformant was named KM71H / pPICZαB-721-P4H, abbreviated as KM71H-721-Hyp.

[0175] The linearized plasmid pPICZαB-705 was electroporated into the competent cells of the host strain Pichia pastoris X33 (purchased from Thermo Fisher Scientific), and the electroporated bacterial solution was spread on YPDZ (100 μg / mL, Zeocin) plates, with one plate spread for every 200 μL, 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. The obtained transformant was named X33 / pPICZαB-705 (from strain CGMCC NO.29601 in patent CN118146354A). The linearized plasmid pPIC9K-P4H (DP) was electrotransformed into the constructed X33 / pPICZαB-705, and the electrotransformed bacterial solution was spread on a YPDG (500 μg / mL, G418) plate, with each 200 μL spread on one plate, left to stand at room temperature for 10 minutes, and inverted at 30°C for 2-5 days until a single colony (positive transformant) appeared. The obtained transformant was named X33 / pPICZαB-705-P4H, abbreviated as X33-705-Hyp.

[0176] The linearized plasmid pPIC9K-3A5D2NT was electroporated into the host strain Pichia pastoris GS115-HCPB-PPKEX2 (the strain number from patent CN116948013A is: CGMCC No.25815) competent cells, and the electroporated bacterial solution was spread on YPDG (500 μg / mL, G418) plates, with each 200 μL spread on one plate, and 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. The obtained transformant was named

[0177] GS115-HCPB-PPKEX2 / 3A5D2NT (from strain CGMCC No. 25819 in patent CN116948013A). The linearized plasmid pPIC9K-P4H(DP)-1 was electrotransformed into the constructed GS115-HCPB-PPKEX2 / 3A5D2NT, and the electrotransformed bacterial solution was spread on a YPDG (500 μg / mL, G418) plate, with each 200 μL spread on one plate, left to stand at room temperature for 10 min, and inverted at 30°C for 2-5 days until a single colony (positive transformant) appeared. The obtained transformant was named GS115-HCPB-PPKEX2 / 3A5D2NT-P4H, referred to as TTA03-Hyp.

[0178] S4. Target gene transfer verification:

[0179] Resistance plate screening can speed up the experimental process and screen out strains containing the target gene. In order to further verify from the gene level, polymerase chain reaction is used as a technical means to extract the genome using Pichia pastoris as a template for verification. The specific steps are as follows:

[0180] S41.X33 / pPICZαB-705-P4H, KM71H / pPICZαB-721-P4H,

[0181] Verification of GS115-HCPB-PPKEX2 / 3A5D2NT-P4H:

[0182] Add 200 μL YPD medium to the 96-well plate, pick 16 single colonies of X33 / pPICZαB-705-P4H, KM71H / pPICZαB-721-P4H, and GS115-HCPB-PPKEX2 / 3A5D2NT-P4H and inoculate them into the 96-well plate and culture in a 30°C biochemical incubator for 24 hours. After shaking evenly, take 50 μL of bacteria from each well into an 8-tube strip, centrifuge for 5 minutes, remove the supernatant, add 80 μL of sterilized resuspended bacteria, centrifuge for 5 minutes, remove the supernatant, and then add 10 μL of 0.02 M NaOH solution and vortex suspend the bacteria to obtain a bacterial solution.

[0183] Place the mixed bacterial solution on a PCR instrument, boil the sample at 99°C for 10 minutes, cool to room temperature, and centrifuge for 5 minutes. Take 2 μL of the supernatant as a template and use rTaq as a PCR polymerase to perform polymerase chain reaction by PCR. After the reaction, verify the target band by nucleic acid electrophoresis.

[0184] The primers in the above PCR reaction process are:

[0185] 5'AOX:GACTGGTTCCAATTGACAAGC (SEQ ID NO:20);

[0186] 3'AOX:GCAAATGGCATTCTGACATCC (SEQ ID NO:21);

[0187] P4HB veri R: CCTTAAAAGATTCAGCAGCAGTC (SEQ ID NO: 22);

[0188] P4HA veri F: CAAGAAGATGAGTGGGATAAGC (SEQ ID NO: 23);

[0189] The conditions of the PCR reaction are: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 50 s for 30 cycles. The primer sequences SEQID NO: 19 and SEQ ID NO: 21 are used in the above reaction system and conditions to amplify the P4HA target fragment by polymerase chain reaction, and the target fragment size is 756 bp. The conditions of the PCR reaction are: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min 12 s for 30 cycles. The P4HB target fragment is amplified by polymerase chain reaction under the above reaction system and conditions. The primer sequences SEQID NO: 18 and SEQ ID NO: 20 are used in the above reaction system and conditions to amplify the P4HB target fragment by polymerase chain reaction, and the target fragment size is 1152 bp.

[0190] Verification results are as follows Figure 1 As shown in the figure, it can be seen that the nucleic acid size is consistent with the expectations of the PCR results, and the strains with positive P4HA1 and P4HB fragments in the PCR results are selected for shake flask induced expression.

[0191] Verification of S42.KM71H / pPICZαB-721-P4H target sequence:

[0192] Pick 16 single colonies of KM71H / pPICZαB-721-P4H into 200 mL YPD medium, place them in a 96-well plate, and culture them in a 30°C biochemical incubator for 24 hours. Take 80 μL of the bacterial solution to extract the template, take 2 μL of the extracted template and add it to a PCR tube, perform polymerase chain reaction by PCR, and verify the target band by nucleic acid electrophoresis after the reaction.

[0193] The primers in the above PCR reaction process are:

[0194] 705BB1-23 F: ggtctcaCATGaacgatgagatttccttcaatt (SEQ ID NO: 24);

[0195] 707BB1 R: ggtctcaAAGCttaattcgcggccgctca (SEQ ID NO: 25);

[0196] The conditions of PCR reaction were as follows: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min for 30 cycles.

[0197] Verification results are as follows Figure 2 As shown in the figure, it can be seen that the target band size is 1837bp, as shown by the band in the red box, and the positive strain is selected for subsequent shake flask induced expression.

[0198] If the Pichia transformant can grow on a plate containing a high concentration of Zeocin, it means that the transformant contains multiple copies of the target gene, that is, multiple recombinant fragments have entered the yeast and integrated into the yeast chromosome through homologous recombination. This step of screening can obtain high-copy, highly efficient recombinant yeast engineering strains. The strain KM71H / pPICZαB-721-P4H was used for subsequent experiments under antibiotic plate screening and Pichia colony PCR verification.

[0199] The recombinant engineered bacteria KM71H-721-Hyp, X33-705-Hyp, and TTA03-Hyp were sent to the General Microbiology Center of China Microorganism Culture Collection Administration, and the culture collection numbers are: CGMCC NO.33067, CGMCC NO.33068, and CGMCCNO.33069. Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: December 13, 2024, classification name: Pichia pastoris Komagataella phaffii.

[0200] S43.WB identification of P4H:

[0201] Cell wall breaking: Induce strain expression, collect fermentation liquid after induction, centrifuge and place the precipitate on ice, add glass beads of equal cell volume, cell wall breaking solution (1mM EDTA, 5% glycerol, NaOH adjusted to pH 7.4) of three times the cell volume, 10μL of 1mM PMSF, then shake on a tabletop shaker for 2min, place on ice for 2min, repeat 3 times, cell wall breaking for 4min for the fourth time, centrifuge at 10000rpm and 4℃ for 10min after cell wall breaking, collect supernatant, add 5× protein loading buffer, cook at 99℃ for 10min. SDS-PAGE precast gel (12%, GenScript) verifies the expression of P4HA1 and P4HB, transfer to membrane after SDS-PAGE electrophoresis of P4HA1 and P4HB, experimental methods and steps are shown in the Molecular Cloning Experiment Guide, experimental equipment is from GenScript, and operation is carried out according to equipment requirements. P4HA1: Incubate with primary antibody P4HA1 (mouse, 66101-1-Ig, 1:2000 dilution) for 2 hours. Secondary antibody: incubate with goat anti-mouse (1:5000 dilution) for 2 hours, P4HA1 target protein size: 61KDa; P4HB: primary antibody P4HB1 (rabbit, ET7110-92), 1:2000 dilution, secondary antibody: goat anti-rabbit (1:5000), P4HB molecular weight size 52.2KDa.

[0202] The experimental results are as follows Figure 3 As shown, after incubation with specific P4HA1 and P4HB antibodies, the target band sizes were consistent, P4HA1 61KDa, P4HB 52.2KDa, and the target bands were clear. There were other target bands in the X33 / pPICZαB-705-P4H and KM71H / pPICZαB-721-P4HWB results, but the antibodies were specific and determined to be P4H enzymes. It was speculated that degradation or aggregation may have occurred during the process of breaking the cell wall or placing the cells, and the positive strains were selected for subsequent experiments.

[0203] S5. Induced expression of recombinant engineering strains and identification of recombinant collagen:

[0204] Select the strains X33 / pPICZαB-705-P4H, KM71H / pPICZαB-721-P4H, and GS115-HCPB-PPKEX2 / 3A5D2NT-P4H with positive results of PCR and WB, and place them in a 100mL Erlenmeyer flask containing 10mL BMGY medium. Culture them at 28-30℃ and 220rpm until OD600 is 6-10 (16-18h). Centrifuge at 1500-3000g for 5min at room temperature, collect the cells, resuspend the cells in BMMY medium, and adjust the OD600 to 6-10. 600The cell culture temperature was about 10, and the culture was continued for 3 days in a biochemical incubator at 28-30°C and 220rpm. 1% of the final volume of methanol was added every 12 hours. Samples were taken after 48 hours and 72 hours of induction, and the supernatant was collected by centrifugation at 1500-3000g for 5 minutes at room temperature. The collected expression supernatant was added with 2× loading buffer (Coomassie Brilliant Blue, denaturant DTT, SDS buffer), placed in a 100°C metal bath for heating for 5 minutes, and GLASS gel precast gel Tricine protein gel detection was performed.

[0205] The results are as follows Figure 4 As shown in the figure, it can be seen that each target band can be secreted in the culture supernatant, indicating that the target bands of 705, 721 and 3A5D2NT sequences are secreted and expressed extracellularly, and the molecular weight is consistent with the apparent migration characteristics of theoretical collagen, and there is no degradation. The target bands were identified by protein gel mass spectrometry, and the mass spectrometry results showed that the amino acid sequence was the same as the theoretical amino acid sequence.

[0206] S6. High-density fermentation and purification test:

[0207] (1) The selected recombinant engineered bacteria X33 / pPICZαB-705-P4H, KM71H / pPICZαB-721-P4H, and GS115-HCPB-PPKEX2 / 3A5D2NT-P4H were subjected to high-density fermentation experiments to express and produce the recombinant hydroxylated small molecule collagens of 705, 721, and 3A5D2NT on a large scale, and obtain fermentation broth containing recombinant hydroxylated small molecule collagens.

[0208] Among them, seed culture medium YPG: yeast powder 10g / L, peptone 20g / L, glycerol 10g / L;

[0209] Fermentation medium: NH4H2PO4 190.4 g / L, KH2PO4 10.06 g / L, CaSO4·2H2O 1.18 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, glycerol 40 g / L; after high-temperature sterilization of the fermentation medium, add PTM1 when the temperature drops to room temperature and adjust the pH to 5.0 with ammonia water.

[0210] Feed medium: 50% W / V glycerol, plus 12 mL PTM1 trace elements per liter;

[0211] Induction medium: 100% methanol, 12 mL of PTM1 trace elements per liter;

[0212] PTM1: Sterilize by filtering with a 0.22 μm filter membrane and store at 4°C.

[0213] The batch culture conditions and induced expression conditions of the engineering strains are as follows: the batch feeding method is adopted, and the culture temperature is 30°C. The engineering bacteria are inoculated into a 1L shake flask containing 200mL of seed culture medium YPG, 220rpm, 30°C, and cultured for 18-20h to OD600 = 2-10. A 5L fermenter (Baoxing Biology) is used, the liquid volume is 2L of fermentation medium, and 2% glycerol is sterilized separately. Before inoculation, the speed is adjusted to 300rpm, 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 to calibrate, and fermentation begins after calibration.

[0214] Induction culture: 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, and the dissolved oxygen rebounds and is greater than 70% (OD 600 Value is about 20), cancel the dissolved oxygen cascade speed, increase the stirring to 650rpm, use 30% dissolved oxygen linkage feeding, and add 80mL of feeding medium. When the dissolved oxygen rebounds to more than 70%, stop adding the feeding medium, set the pH value to 5.0 and the temperature to 30℃, and flow-add induction medium for induction culture: manually add 5mL of induction medium, and after the dissolved oxygen rebounds to more than 70%, set the feeding parameters to: flow-add induction medium at a rate of 8mL / h for 1h, then flow-add induction medium at a rate of 10mL / h for 1h, and then flow-add induction medium at a rate of 20mL / h until the dissolved oxygen value is lower than 30%. Wait for the dissolved oxygen to rebound, and then link the feeding of induction medium after the dissolved oxygen rises to 30%. Induce culture for a total of 40 to 60h, measure the protein concentration by UV, and release the tank when the protein concentration does not increase significantly or decreases.

[0215] UV protein quantification formula: C (mg / mL) = 0.144*(A215-A225), A215<1.5.

[0216] The principle of UV 215nm and 225nm to detect protein content is based on the difference in light absorption of protein at 215nm and 225nm wavelengths. The difference between the absorption values ​​of protein at 215nm and 225nm and its concentration conforms to Beer's law within a certain range, so the concentration of protein can be quantitatively determined by measuring the difference in light absorption values ​​at these two wavelengths.

[0217] After the fermentation was completed, the mixture was centrifuged at 9000 g for 5 min and the supernatant was collected for protein electrophoresis. Figure 5As shown in a. As can be seen from the figure, under high-density fermentation conditions, for 705, 721 and 3A5D2NT, each sequence collagen protein has almost only the target band when induced for 24 hours or more, and the expression amount increases with the increase of induction time. The main band of optical density analysis accounts for more than 85%. This shows that 705, 721 and 3A5D2NT can express the target protein under high-density fermentation conditions and secrete it outside the cell.

[0218] (2) Purification:

[0219] Buffer A: 20 mM KH2PO4, pH 4.0;

[0220] Buffer B: 20 ​​mM KH2PO4, 1 M NaCl, pH 4.0.

[0221] The fermentation broth in step (1) was collected, and the cells and the fermentation supernatant were separated by centrifugation at 2000g, 30min, and 4°C. The cation exchange medium was balanced with buffer A (the chromatographic filler was UniGel-80sp produced by Suzhou Nano, loaded on the XK50 / 30 chromatography column produced by Lisui Technology, and the GE AKTAPure protein separation chromatography purification system was used) until the A215 absorbance and conductivity values ​​remained unchanged, and then the flow rate of 100us / cm was set for loading, and the loading volume was 0.5L / time. The ultraviolet A215 absorbance value was detected. When it rose, the sample was started. When the A215 absorbance value dropped, the sample was stopped until the ultraviolet and conductivity dropped to the minimum and no longer changed. After the loading was completed, the sample was closed, and the cationic chromatography medium was balanced with buffer A. The eluate was collected, and after the components were detected and determined, dialyzed (the dialysate was ultrapure water), then concentrated and freeze-dried, and the freeze-dried collagen sponge was collected.

[0222] Take the purified freeze-dried sponge and dissolve it in ultrapure water for protein electrophoresis. The electrophoresis results are as follows: Figure 5 As shown in b. As can be seen from the figure, the 705, 721 and 3A5D2NT collagen bands are clear and not degraded, and the target protein can be efficiently separated after purification.

[0223] Example 2: Experiment on cell adhesion activity of recombinant hydroxylated small molecule collagen

[0224] In this example, natural collagen was used as a control, and recombinant 705, 721 and 3A5D2NT collagens (recombinant hydroxylated small molecule collagens with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5) were used as examples to investigate the cell adhesion activity of the recombinant hydroxylated small molecule collagens of the present invention.

[0225] The specific steps are as follows:

[0226] (1) Material preparation: mouse embryonic fibroblasts NIH / 3T3 (cell line from the Chinese Academy of Sciences Cell Bank SCSP-515, culture and subculture methods refer to the cell instructions), human immortalized keratinocytes (HaCaT), cell line from the Chinese Academy of Sciences Cell Bank SCSP-5091), recombinant 705, 721 and 3A5D2NT collagen freeze-dried sponges, positive natural collagen (Sigma, catalog number C7774-5MG).

[0227] Among them, the processing method of positive control natural collagen is as follows: weigh a 5mL sterile centrifuge tube, add human collagen in a clean bench and weigh it, subtract the weight to get the sample amount, dissolve it with ultrapure water and add acetic acid to pH 3.0 (concentration 5mg / mL), and a milky white solution can be obtained. The protein concentration is determined by the UV protein quantitative empirical formula: C (mg / mL) = 0.144*(A215-A225), and the concentration is diluted to 0.5mg / mL with serum-free DMEM culture medium when used, and sterilized by filtering with a 0.22μm sterile filter.

[0228] (2) Coating preparation:

[0229] 100 μL of samples (standard / sample / blank control) were added to the 96-well plate. Standard: positive control natural collagen 0.5 mg / mL (Sigma, catalog number C7774-5MG); Sample: 705, 721 and 3A5D2NT recombinant hydroxylated small molecule collagen 0.5 mg / mL; Blank: D-PBS phosphate buffer.

[0230] Prepare 4 wells for each sample above, add the sample to a 96-well plate and incubate in a 37°C, 5% CO2 (v / v) incubator for 1h-4h, remove excess solution from the wells, and the coating is complete. Add 100 μL of 1% BSA-PBS solution and incubate in a 37°C, 5% CO2 incubator for 1h. After removing the liquid in the wells, wash three times with D-PBS and discard the washing solution. Premix Hoechst-33342 fluorescent dye (10%) with complete culture medium, and dilute NIH / 3T3 cells or HaCaT cells to 5×10 4 100 μL of cells were added to the wells, covered with aluminum foil, and incubated at 37°C, 5% CO2 for 1 h.

[0231] The specific detection steps are as follows: Use an inverted microscope to capture at least 4×4 digital tile images (fluorescence) of each of the 3 wells. Fill each well with D-PBS to form a "reverse meniscus", blow out bubbles and cover with sealing film. Centrifuge the plate (invert the plate) at a relative centrifugal force (RCF) of 300g at 22°C for 5 minutes. After centrifugation, discard the sealing film, remove the supernatant from the well, wash once with D-PBS, and add 100μL D-PBS. For each of the 3 wells, take a total of 25 fluorescent tile digital images (at least 4×4 matrix is ​​recommended, with 10% tile overlap), calculate approximately 2400 to 3600 cells for each sample (800-1200 cells / well × 3 wells), measure 3 replicate samples, and the fourth well is used to adjust the microscope parameters. Its measurement value is not used. The measurement results are as follows. Figure 6 shown.

[0232] from Figure 6 It can be seen that the sample has stronger adhesion after coating and is better than the blank control group. In NIH / 3T3 cells, the experimental group is more obvious than the blank control group. The adhesion effect after hydroxylation modification is better than that of the unhydroxylated sample, which can be seen from the 705 hydroxylated and unhydroxylated raw materials. It can be seen that the recombinant hydroxylated small molecule collagen of the present invention has a cell adhesion similar to that of natural collagen, and the biological activity of the small molecule collagen is promoted after hydroxylation.

[0233] Example 3: Recombinant collagen cell migration experiment

[0234] This example uses the same NIH / 3T3 cells and HaCaT cells as in Example 2 to examine the cell migration ability of recombinant 705, 721 and 3A5D2NT recombinant hydroxylated small molecule collagen freeze-dried sponges and positive control natural collagen (Sigma, catalog number C7774-5MG).

[0235] (1) Experimental preparation: First, use a marker pen to draw horizontal lines evenly on the back of a 6-well plate, using a ruler. Draw lines approximately every 0.5 cm to 1 cm across the wells, with 3 lines drawn across each well. Add approximately 5 × 10 5 cells.

[0236] (2) Scratch test: On the second day of cell culture, use the tip of the gun to measure the ruler and try to scratch perpendicular to the horizontal line on the back. The tip of the gun should be vertical and not tilted. Then rinse the cells with PBS three times, remove the scratched cells, and add serum-free culture medium containing the test sample as the experimental group, with a concentration of 0.05% (mass volume ratio). Place in a 37°C, 5% CO2 incubator for culture, take samples at 0 and 24 hours, and take pictures.

[0237] (3) Data processing: The scratch area of ​​each image was calculated using ImageJ image processing software, and the migration rate of each group of cells was calculated by dividing the total area of ​​the migrating cells in the fixed scratch area by the initial area of ​​the fixed scratch area. The time was used as the horizontal axis and the migration area 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. The experimental results are shown in Figure 2. Figure 7 and Figure 8 shown.

[0238] Combination Figure 7 and Figure 8 It can be seen that the results of the sample group after 24 hours of the migration experiment of NIH / 3T3 and HaCat cells are the same as those of the cell adhesion experiment. The stability of collagen increases after hydroxylation, and the binding effect with the action site on the cell surface is stronger. This shows that as time goes by, the cells continue to grow, and under physiological conditions, the promotion effect of the experimental group samples on cell migration is more obvious, which can effectively promote the migration of cells to biomaterials.

[0239] Example 4. Hydroxylation modification detection

[0240] (1) Detection of Hydroxyproline Content:

[0241] In this example, the samples of 705, 721 and 3A5D2NT recombinant hydroxylated small molecule collagen after co-transfection with P4H enzyme were hydrolyzed after purification and before freeze-drying, and then the hydroxylation detection kit (Shenggong: D799573-0050) was used to detect the hydroxyproline content to investigate the hydroxylation rate and stability of the recombinant hydroxylated small molecule collagen.

[0242] Testing steps:

[0243] S1. Preparation of samples and reagents:

[0244] Extraction solution: 6M HCl Preparation: 6mol / L hydrochloric acid, concentrated hydrochloric acid (37%) and H2O volume ratio = 1:1

[0245] Dilution of hydroxyproline standard solution: prepare according to the instructions of the kit.

[0246] S2. Experimental steps:

[0247] After the engineered bacteria fermentation liquid is purified, 100 μL of the solution obtained before freeze drying is the sample to be tested, 1 mL of the extract is added thereto, and the solution is digested in an oven at 110°C for 4 hours until transparent, centrifuged at 16000 rpm and 25°C for 20 minutes, and then 1 mL of the supernatant after centrifugation is taken, and the pH value is adjusted to the range of 6 to 8 with 10 mol / L NaOH (about 0.5 mL). If the pH value is too high, it is adjusted with 6 mol / L hydrochloric acid, and the volume is adjusted to 2 mL with distilled water to obtain the solution to be tested. The spectrophotometer is preheated for more than 30 minutes, the wavelength is adjusted to 560 nm, and the distilled water is zeroed. The hydroxyproline standard is diluted to 7.5, 3.75, 1.875, 0.938, 0.469, 0.234, and 0.117 μg / mL standard solutions, and the solution is added according to the instructions of the kit for hydroxylation detection.

[0248] With the concentration of the standard solution as the x-axis and the ΔA standard (ΔA = A standard tube - A blank tube) as the y-axis, a standard curve is drawn to obtain the equation y = kx + b. Substitute the ΔA determination (ΔA = A determination tube - A blank tube) into the equation to obtain x (μg / mL). Calculate according to the sample protein concentration: tissue hydroxyproline content (μg / mg prot) = x × V sample ÷ (Cpr × V sample) = x ÷ Cpr. The original experimental results of the standard are shown in Table 1. The standard curve is prepared based on the experimental results in Table 1. The standard curve equation is: y = 0.0321x + 0.004, R 2 =0.9986, with a good fit. The absorbance values ​​of the experimental group were substituted into the standard curve to calculate the hydroxyproline content. The results are shown in Table 2. The results show that 705, 721 and 3A5D2NT are hydroxylated.

[0249] Table 1: Absorbance of Hydroxyproline Standards

[0250]

[0251] Table 2: Hydroxyproline test results

[0252]

[0253] The samples were tested for hydroxylation using a kit to detect the hydroxyproline content in the test solution. The test found that the hydroxyproline content of sample 705 reached 0.917μg / mg prot, and the hydroxyproline content of sample 721 reached 0.854μg / mg prot. 3A5D2NT was 1.414μg / mg prot. From the results of sample hydroxyproline content (μg / mg prot), 3A5D2NT had the highest result. The sample hydroxyproline content (μg / mg prot) results can better reflect the hydroxylation rate under the unit protein content. The protein content of 705 and 721 is low, which makes the calculated results low. However, 705 and 721 are human type VII collagen, and there are fewer hydroxylation sites in the original sequence. Although type III and type VII collagen have different functions, they can both stabilize the protein and improve biological performance after hydroxylation modification.

[0254] (2) Quantitative detection of amino acids

[0255] In this step, Waters ACQUITY UPLC I-CLASS ultra-high performance liquid chromatography was used to separate the 705, 721 and 3A5D2NT recombinant hydroxylated small molecule collagens after co-transfection with P4H enzyme.

[0256] Chromatographic conditions:

[0257] Chromatographic column: Waters UPLC HSS T3 (1.8 μm, 2.1 mm × 150 mm);

[0258] Mobile phase: Phase A: water, 0.1% formic acid; Phase B: acetonitrile;

[0259] Elution gradient: conditions are shown in Table 4;

[0260] Flow rate: 0.5 mL / min;

[0261] Injection volume: 5.0 μL;

[0262] Column temperature: 50℃.

[0263] Mass spectrometry conditions: Waters XEVO TQ-S Micro tandem quadrupole mass spectrometer system was used for mass spectrometry analysis, with a positive ion source voltage of 1.5 kV, a cone voltage of 20 V, a desolvation temperature of 600°C, a desolvation gas flow rate of 1000 L / h, and a cone gas flow rate of 10 L / h.

[0264] Targeted data processing: MassLynx quantitative software was used to calculate the peak area, and the retention time allowed an error of 15 s. The quantitative results were obtained by the standard curve method. The results are shown in Tables 3 and 4.

[0265] Table 3: Amino acid content

[0266]

[0267]

[0268] The hydroxylation rate was calculated using the data in Table 3. The calculation formula is: Target protein hydroxylation rate detection = C(L-hydroxyproline) / (C(L-hydroxyproline)+C(L-proline)). The calculation results are shown in Table 4.

[0269] Table 4: Hydroxylation rate of target protein (%)

[0270] Grouping 705 721 3A5D2NT Hydroxylation rate (%) 11.59％ 20.71％ 16.13％

[0271] As can be seen from Table 4, the 705, 721 and 3A5D2NT collagen sequences are hydroxylated.

[0272] (3) Hydroxylation modification analysis:

[0273] The protein samples were chromatographically separated and proline hydroxylation modification was identified by high performance liquid chromatography-mass spectrometry (LC-MS).

[0274] S1. Sample preparation:

[0275] Take 200μL of 705, 721 and 3A5D2NT to a glass bottle, add 0.8mL of water and 1mL of concentrated hydrochloric acid, react at 110℃ for 20-24h, and then acid hydrolyze the sample after the reaction. Take 1mL of the acid hydrolyzed sample and vacuum concentrate it to dryness, then take 10μL to an EP tube, add water to 100μL, and then add 1μL of 1M DTT solution to make the final concentration of DTT 10mmol / L, and then reduce it in a 56℃ water bath for 1h. After the reaction, add 2μL of 1M IAM solution to the reaction solution to make the final concentration of IAM 20mmol / L, react at room temperature in the dark for 40min, and add 1μL of 1M DTT solution to make the final concentration of DTT 10mmol / L to neutralize the unreacted IAM. Then, 3 μL of Chymotrypsin enzyme (concentration: 0.5 μg / μL) was added thereto and incubated in a 30°C constant temperature incubator for 16 h;

[0276] The peptides after enzyme cleavage were desalted using a self-filled desalting column, and the solvent was evaporated in a 45°C vacuum centrifugal concentrator to remove impurities in the solution after enzyme cleavage except the target peptide; the peptides were dissolved in a sample dissolving solution (0.1% formic acid, 2% acetonitrile), vortexed thoroughly, and centrifuged at 13200rpm, 4°C for 10min. The supernatant was transferred to a sample tube and waited for mass spectrometry analysis.

[0277] S2. Equipment parameters:

[0278] Chromatographic separation equipment parameters: mobile phase A is an aqueous solution containing 0.1% (v / v) formic acid; mobile phase B is an 80% (v / v) acetonitrile solution containing 0.1% (v / v) formic acid. Liquid phase gradient setting: 0-4min, 1-8% B; 4-23min, 8-28% B; 23-27min, 28-99% B; 27-30min, 99% B; 30-30.1min, 99-1% B; 30.1-35min, 1% B; flow rate 0.6μL / min. Chromatographic column: 75μm id×50cm, NanoViper C18 2μm, 100A.

[0279] Mass spectrometry acquisition: Primary mass spectrometry parameters: Resolution: 60,000; AGCtarget: Custom; MaximumIT: Custom; Scanrange: 300 to 1800 m / z; Secondary mass spectrometry parameters Resolution: 15,000; AGCtarget: Custom; MaximumIT: Custom; NCE / steppedNCE: 30.

[0280] S3. Software Analysis:

[0281] The data were analyzed using the Peptide Mapping analysis function of the BioPharma Finder software. The analysis results are as follows: Fig. 9 , 10 and 11.

[0282] Data processing parameters: Protease (Chymotrypsin); Static Modification (Carbamidomethylation); Variable Modification (Deamidation, Oxidation, Hyp); Mass Accuracy (10ppm).

[0283] Combination Fig. 9 It can be seen that the sequence coverage of sample 705 was 100% after mass spectrometry identification, and proline hydroxylation-modified peptide information was identified in the sample. Its P8, P12, P17, P20, P21, P33, and P36 sites were all identified to be hydroxylated, but the hydroxylation ratio of P12 and P36 theoretical hydroxylation sites was higher. Fig.10It can be seen that the sequence coverage of sample 721 was 93.8% after mass spectrometry identification; hydroxylation of proline was identified at 6 sites in the sample, namely P9, P12, P18, P26, P36, and P38. The modification ratio of sample 721 at the theoretical hydroxylation sites of P18 and P36 was higher. Fig.11 It can be seen that the sequence coverage of 3A5D2NT sample was 90.6% after mass spectrometry identification; the sample was identified to have hydroxylation modification of proline. Fig.11 a. The sample was hydroxylated at P11, P14, P18, P20, P27, and P29. The secondary fragment matching diagram and secondary mass spectrum of the modified peptides are shown in Fig.11 b.

[0284] In summary, 705, 721, and 3A5D2NT were hydroxylated at the theoretical hydroxylation sites, and hydroxylation also occurred when proline was present at other sites, which may be related to the hydroxylation efficiency of the P4H enzyme. In the intracellular biological environment of Pichia pastoris, proline was hydroxylated.

[0285] Embodiment 5:

[0286] This example refers to GBT16886.5-2017 in vitro cytotoxicity test and GB / T16886.12-2017 sample preparation and reference materials. Taking recombinant 705, 721 and 3A5D2NT collagen as examples, the cytotoxicity of recombinant hydroxylated small molecule collagen was investigated by the cytotoxicity of recombinant 705, 721 and 3A5D2NT collagen to L-929 mammalian fibroblasts (cells from the cell bank of the Chinese Academy of Sciences, catalog number: SCSP-5039).

[0287] The specific steps are as follows:

[0288] Samples of the experimental group: recombinant 705, 721 and 3A5D2NT collagen were dissolved and diluted in MEM culture medium to prepare concentrations of 10, 5, 2.5, 1.25 mg / mL.

[0289] Negative control sample: 10% (v / v) DMSO.

[0290] Blank control sample: MEM culture medium.

[0291] (1) Cell preparation:

[0292] L-929 cells were cultured in MEM medium (containing 10% FBS and 1% penicillin-streptomycin) until the logarithmic growth phase at 37°C and 5% CO2. After the culture, L-929 cells were digested with 0.25% (w / v) trypsin (containing EDTA). After the digestion, the cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells after centrifugation were resuspended in MEM medium and counted to obtain 1×10 5 / mL of cell suspension.

[0293] (2) Sample culture:

[0294] The cell suspension was inoculated into a 96-well plate at 100 μL per well, and incubated in a cell culture incubator at 37°C, 5% CO2, and >90% humidity. The cell morphology was observed under a microscope. After 24 hours of culture, the cells grew to about 70% of the cells by adhesion. The original culture medium in the 96-well plate was discarded, and 100 μL of sample working solution (blank control sample, negative control sample, and positive control sample) was added to the corresponding wells of the 96-well plate to a final concentration of 10 mg / mL. The 96-well plate was then placed in a cell culture incubator and cultured for 24 hours at 37°C, 5% CO2, and >90% humidity. Six replicate wells were set for each group.

[0295] (3) Detection:

[0296] After culturing for 24 hours, observe the cell morphology under a microscope in the 96-well plate. Then remove the liquid, add 50 μL of MTT with a final concentration of 1 mg / mL to each well, and culture in a 37°C, 5% CO2 incubator for 2 hours. Remove the supernatant, add 100 μL of isopropanol to each well to dissolve the crystals, and measure the absorbance value at a wavelength of 570 nm on an ELISA reader to calculate its cytotoxicity. The results are as follows: Fig.12 shown.

[0297] Data processing: mean ± standard deviation (X 均值 ±S);

[0298] Cell viability % = OD570 of test sample group / OD570 of blank control group × 100%.

[0299] from Fig.12 It can be seen that the relative survival rate of recombinant 705, 721 and 3A5D2NT collagen at a concentration of 10 mg / mL is greater than 70%. The test substance has no potential cytotoxicity to human fibroblasts. There is almost no toxicity at a concentration of 1.25 mg / mL, and it has potential application value in the field of biomaterials and cosmetics in the future.

[0300] In summary, the present invention utilizes synthetic biology, genetic engineering and biotechnology to co-express small molecule collagen with P4H, and then performs amino acid modification on it, performs hydroxylation at the GXY theoretical hydroxylation site, and performs post-translational modification to obtain the recombinant hydroxylated small molecule collagen; the recombinant hydroxylated small molecule collagen of the present invention stabilizes the molecular structure through amino acid post-expression modification, and the self-assembly after small molecule expression is more controllable, and it can take into account both transdermal absorption and biological structure stability; the recombinant hydroxylated small molecule collagen can realize industrial production and has good application in the fields of drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0301] 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. A recombinant hydroxylated small molecule collagen, characterized in that: The recombinant hydroxylated small molecule collagen is a hydroxylated recombinant small molecule collagen, and the recombinant small molecule collagen includes an amino acid sequence containing a hydroxylation site on the triple helix region of human type VII collagen or the α1 chain of human type III collagen.

2. The recombinant hydroxylated small molecule collagen according to claim 1, characterized in that: The amino acid sequence is shown in i, ii or iii: i. The amino acid sequence is as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or the amino acid sequence is obtained by repeating the sequence shown in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 as the basic unit multiple times in series; ii. the amino acid sequence is shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; iii. A protein derived from i or ii, in which the amino acid sequence in i or ii is substituted, deleted or added with one or more amino acids and has human collagen activity, or an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the sequence shown in i or ii.

3. A nucleic acid encoding the recombinant hydroxylated small molecule collagen according to claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The nucleic acid includes the nucleotide sequence shown in SEQ ID NO: 7 to SEQ ID NO: 9, or a degenerate sequence thereof.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 3 or 4.

6. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria comprises the nucleic acid described in claim 3 or 4, or comprises the recombinant expression vector described in claim 5, or comprises a recombinant engineered bacteria comprising a recombinant plasmid expressing P4H enzyme and capable of expressing the recombinant hydroxylated small molecule collagen described in claim 1 or 2.

7. The recombinant engineered bacterium according to claim 6, characterized in that The recombinant engineered bacteria include a chassis strain for expressing the recombinant hydroxylated small molecule collagen according to claim 1 or 2, and the chassis strain expresses the P4H enzyme.

8. The recombinant engineered bacterium according to claim 7, characterized in that The chassis strain comprises nucleic acids encoding the amino acids shown in SEQ ID No.10 and SEQ ID No.

13.

9. The recombinant engineered bacterium according to claim 6, characterized in that: The recombinant engineered bacteria are deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit numbers being CGMCC NO.33067, CGMCC NO.33068, and CGMCC NO.33069.

10. A chassis strain for expressing the recombinant hydroxylated small molecule collagen according to claim 1 or 2, characterized in that: The chassis strain expresses the P4H enzyme.

11. The chassis strain according to claim 10, characterized in that The host bacteria of the chassis strain includes one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula, preferably Pichia pastoris, and more preferably Pichia pastoris X33 or Pichia pastoris KM71H.

12. A method for preparing recombinant hydroxylated small molecule collagen, characterized in that: The preparation method comprises: (1) selecting and designing the sequence of recombinant hydroxylated small molecule collagen, and then constructing a collagen tandem repeat sequence based on the recombinant hydroxylated small molecule collagen as a basic unit tandem repeat; (2) constructing a recombinant expression vector expressing collagen tandem repeat sequences; Constructing a recombinant vector expressing P4H enzyme; (3) electroporating the constructed recombinant expression vector expressing the collagen tandem repeat sequence and the recombinant vector expressing the P4H enzyme into the host bacteria, screening and verifying, and obtaining a recombinant engineered bacterium expressing a high copy of the recombinant hydroxylated small molecule collagen; (4) fermenting and inducing the expression of the obtained recombinant engineered bacteria to obtain the recombinant hydroxylated small molecule collagen.

13. The method for preparing recombinant hydroxylated small molecule collagen according to claim 12, characterized in that: In step (1), the recombinant hydroxylated small molecule collagen is a hydroxylated recombinant small molecule collagen, and the recombinant small molecule collagen includes an amino acid sequence containing a hydroxylation site on the triple helix region of human type VII collagen or the α1 chain of human type III collagen; The amino acid sequence is shown in i, ii or iii: i. The amino acid sequence is as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or the amino acid sequence is obtained by repeating the sequence shown in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 as the basic unit multiple times in series; ii. the amino acid sequence is shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; iii. A protein derived from i or ii, in which the amino acid sequence in i or ii is substituted, deleted or added with one or more amino acids and has human collagen activity, or an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the sequence shown in i or ii.

14. The method for preparing recombinant hydroxylated small molecule collagen according to claim 13, characterized in that: When the amino acid sequence of the recombinant hydroxylated small molecule collagen is as shown in i, the collagen tandem sequence comprises 8 to 10 basic unit recombinant type VII collagens; and there are sites for recognition and cutting by Kex2 enzyme, Ste13 or Kex1 enzyme between two adjacent basic units.

15. The method for preparing recombinant hydroxylated small molecule collagen according to claim 12, characterized in that: The recognition and cleavage sites include KR or RR dibasic amino acid residues, followed by EA, EAEA or other amino acid residues that facilitate cleavage by Kex2 enzyme, Ste 13 enzyme or Kex1 enzyme.

16. The method for preparing recombinant hydroxylated small molecule collagen according to claim 12, characterized in that: In step (1), the nucleic acid of the collagen tandem sequence includes the nucleotide sequence shown in SEQ ID NO: 7 to SEQ ID NO: 9, or a degenerate sequence thereof.

17. The method for preparing recombinant hydroxylated small molecule collagen according to claim 12, characterized in that: In step (2), the recombinant expression vector and the recombinant plasmid vector include pPICZαB, pFLDα, and pPIC9K.

18. The method for preparing recombinant hydroxylated small molecule collagen according to claim 17, characterized in that: The vectors are pPIC9K and pPICZαB.

19. The method for preparing recombinant hydroxylated small molecule collagen according to claim 12, characterized in that: In step (2), the recombinant plasmid expressing the P4H enzyme comprises nucleic acid encoding the amino acids shown in SEQ ID No. 10 and SEQ ID No. 13, a PTEF promoter element and a PEM7 promoter element.

20. The method for preparing recombinant hydroxylated small molecule collagen according to claim 12, characterized in that: In step (3), the host bacteria includes one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula.

21. The method for preparing recombinant hydroxylated small molecule collagen according to claim 20, characterized in that: The host bacteria is Pichia pastoris.

22. The method for preparing recombinant hydroxylated small molecule collagen according to claim 21, characterized in that: The host bacteria is Pichia pastoris X33 or Pichia pastoris KM71H.

23. The method for preparing recombinant hydroxylated small molecule collagen according to claim 22, characterized in that: The host bacteria are CGMCC No.29601, CGMCC No.25815, and CGMCC No.25819.

24. The method for preparing recombinant hydroxylated small molecule collagen according to claim 12, characterized in that: In step (3), the recombinant engineered bacteria are deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the deposit numbers CGMCC NO.33067, CGMCC NO.33068 and CGMCC NO.33069.

25. Recombinant hydroxylated small molecule collagen prepared by the method according to any one of claims 12 to 24.

26. An expression system for recombinant hydroxylated small molecule collagen, characterized in that: The expression system comprises: a recombinant engineering bacterium of a recombinant plasmid expressing the P4H enzyme as claimed in claim 6 and a collagen tandem sequence constructed with the recombinant hydroxylated small molecule collagen as a basic unit as claimed in any one of claims 1 or 2.

27. A composition, characterized in that The composition includes the recombinant hydroxylated small molecule collagen described in claim 1 or 2, or the recombinant hydroxylated small molecule collagen encoded by the nucleic acid of claim 3 or 4, or the recombinant expression vector described in claim 5, or the recombinant engineered bacteria described in any one of claims 6 to 11, or the recombinant hydroxylated small molecule collagen prepared by the method described in any one of claims 12 to 24; the composition includes medical devices, biomaterials, tissue engineering products, and cosmetics.

28. A product, comprising the recombinant hydroxylated small molecule collagen described in claim 1 or 2, or the recombinant hydroxylated small molecule collagen encoded by the nucleic acid of claim 3 or 4, or the recombinant expression vector described in claim 5, or the recombinant engineered bacteria described in any one of claims 6 to 11, or the recombinant hydroxylated small molecule collagen prepared by the method described in any one of claims 12 to 24, or the composition described in claim 27; the product includes medical devices, biomaterials, tissue engineering products, and cosmetics.

29. Use of the recombinant hydroxylated small molecule collagen described in claim 1 or 2, or the recombinant hydroxylated small molecule collagen encoded by the nucleic acid of claim 3 or 4, or the recombinant expression vector described in claim 5, or the recombinant engineered bacteria described in any one of claims 6 to 11, or the recombinant hydroxylated small molecule collagen prepared by the method described in any one of claims 12 to 24, or the composition described in claim 27, or the product described in claim 28 in the preparation of medical devices, biomaterials, tissue engineering products, and cosmetics.

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