Expression cassette and method for producing humanized triple helix collagen by using expression cassette

Abstract

The invention relates to an expression cassette and a method for producing humanized triple helix collagen by using the expression cassette, and belongs to the technical field of genetic engineering. The expression cassette sequentially comprises a promoter sequence, a fusion gene and a terminator sequence from upstream to downstream, the fusion gene comprises nucleotide fragments A, B and C, each fragment comprises an immutable region and a variable region, and the variable region is selected from I, II, III, IV and XVII type human collagen. A kex2 protease recognition site KR is introduced into an expression box, so that an expression product can be subjected to trisection cutting, and three equal-length peptide chains with a triple-helix basic structure are formed. The invention also provides a recombinant humanized collagen with a self-assembled triple helix structure and a production method thereof, and the method comprises the following steps: preparing a recombinant expression vector containing the expression cassette and a proline hydroxylase expression vector, and transferring the two expression vectors into competent cells for co-expression. The method can be used for producing the recombinant humanized collagen with the self-assembled triple-helix structure, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and particularly to an expression cassette, recombinant humanized collagen, and a preparation method thereof. Background Art

[0002] Collagen is the most abundant protein in the human body and plays an important role in tissue repair, wound healing, and skin health. With the development of biotechnology, the research and application of recombinant collagen have become increasingly widespread. Currently, the expression systems of recombinant collagen mainly include Escherichia coli, yeast, insect cells, and mammalian cells, etc. Among them, the Pichia pastoris expression system has become an important platform for the production of recombinant collagen due to its high expression level, correct protein folding, and strong post-translational modification ability.

[0003] In the field of recombinant protein expression, the Chinese patent "CN111094566A Multi-copy gene protein expression system" discloses a multi-copy gene protein expression system, which solves the problems of low expression level of recombinant proteins and genetic instability of cell lines by introducing several expression cassettes encoding the same mature target recombinant protein but having different nucleotide sequences into cells [1]. The expression cassette at least includes a promoter sequence, a start codon, a polynucleotide sequence encoding the target protein, a stop codon, and a terminator sequence.

[0004] In terms of collagen expression, the Chinese patent "CN118955688A A recombinant fragmented human type III collagen, its preparation method and application" discloses a recombinant fragmented human type III collagen and its preparation method, which obtains a recombinant fragmented human type III collagen without a propeptide but including telopeptides necessary for fiber formation by splicing and recombining fragments of the amino acid sequence of human type III collagen. The Chinese patent "CN118852407A Humanized type IV collagen, expression vector, preparation method and application" discloses a humanized type IV collagen, its expression vector, and a preparation method, which solves the problems of low initiation efficiency of the existing constitutive expression system and the easy residue caused by introducing harmful reagents during the expression process of the inducible expression system by optimizing and designing the nucleotide sequence of natural humanized type IV collagen and obtaining a tandem nucleotide sequence through codon optimization in Pichia pastoris.

[0005] In addition, the Chinese patent "CN111087463B A recombinant human type III collagen and its prokaryotic expression method" discloses a recombinant human type III collagen and its prokaryotic expression method, which obtains a gene sequence through codon optimization and design, inserts it into an expression vector to construct a recombinant expression vector, and transforms Escherichia coli competent cells for efficient expression.

[0006] However, there are still some technical problems in the expression of recombinant collagen. First, the collagen sequence often leads to difficult or non-expression in Pichia pastoris due to sequence repetition. Second, the technical threshold for expressing heterotrimeric collagen is high, and traditional methods cannot co-express three different heterologous chains in the same host, relying on complex in vitro assembly steps. Third, recombinant collagen lacks hydroxyproline modification, resulting in a loose triple helix structure, poor thermal stability, truncated core functional domains, insufficient mechanical properties and biological activities. In addition, the problems of low expression level and insufficient purification efficiency are also common, including codon preference affecting the expression level and difficult purification.

[0007] Therefore, it is urgent to develop a method for efficiently expressing recombinant humanized collagen with a self-assembled triple helix structure in Pichia pastoris to solve the problems of difficult expression, unstable structure and insufficient biological activity existing in the prior art. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method for efficiently expressing recombinant humanized collagen with a triple helix structure in Pichia pastoris, aiming at the defects of recombinant collagen expression and application in the prior art, to solve the problem of non-expression or difficult expression of the recombinant collagen sequence in Pichia pastoris, and to overcome the technical problems such as high technical threshold for expressing heterotrimeric collagen, lack of effective ways to produce collagen with a heterotrimeric structure, loose triple helix structure, poor thermal stability, low expression level, insufficient purification efficiency and the risk of immune response caused by some expression systems.

[0009] The technical solution adopted by the present invention to solve its technical problems is: to provide an expression cassette, which sequentially includes the following elements from upstream to downstream: a promoter sequence, a fusion gene and a terminator sequence; the fusion gene includes nucleotide fragment A, nucleotide fragment B and nucleotide fragment C.

[0010] Further, the nucleotide fragment A includes constant region A1, variable region A2 and constant region A3, the nucleotide fragment B includes constant region B1, variable region B2 and constant region B3, and the nucleotide fragment C includes constant region C1, variable region C2 and constant region C3.

[0011] Further, the nucleotide sequence encoding the constant region A1 is SEQ ID NO:1:

[0012] GGTAGAGCCCCAACAGATCAGCACATCAAGCAGGTTTGCATGAGAGTCATCCAAGAGCACTTTGCCGAAATGGCCGCTTCCTTGAAGAGACCAGATTCAGGAGCTACAGGTCCACCAGGTCCACCAGGTCCACCAGGTCCACCA; The amino acid sequence encoding the constant region A1 is SEQ ID NO:2:

[0013] GRAPTDQHIKQVCMRVIQEHFAEMAASLKRPDSGATGPPGPPGPPGPP;

[0014] Furthermore, the nucleotide sequence encoding the constant region A3 is SEQ ID NO:3:

[0015] GGTCCACCAGGTCCACCAGGTCCACCAGGTCCACCAGGTCCACCACATCATCATCATCACCAC; The amino acid sequence encoding the constant region A3 is SEQ ID NO:4: GPPGPPGPPGPPGPPHHHHHH;

[0016] Furthermore, the nucleotide sequence encoding the constant region B1 is SEQ ID NO:5:

[0017] GGTAGAGACGCTACAGATCAGCACATCGTTGACGTTGCCTTGAAGATGCTGCAGGAACAATTGGCCGAAGTTGCTGTTTCCGCTAAGAGAGAAGCTTTGGGAGCAGTTGGTCCACCAGGTCCACCAGGTCCACCAGGTCCACCA; The amino acid sequence encoding the constant region B1 is SEQ ID NO:6:

[0018] GRDATDQHIVDVALKMLQEQLAEVAVSAKREALGAVGPPGPPGPPGPP;

[0019] Furthermore, the nucleotide sequence encoding the constant region B3 is SEQ ID NO:7:

[0020] GGTCCACCAGGTCCACCAGGTCCACCAGGTCCACCAGGTCCACCACATCATCATCACCACCAC; The amino acid sequence encoding the constant region B3 is SEQ ID NO:8: GPPGPPGPPGPPGPPHHHHHH;

[0021] Furthermore, the nucleotide sequence encoding the constant region C1 is SEQ ID NO:9:

[0022] GGTAAGGAGGCTTCCGAACAGAGAATCAGAGAGCTTTGCGGAGGTATGATCTCCGAACAAATCGCTCAATTGGCCGCTCATCTGAGAAAGCCATTAGCTCCAGGTTCTATTGGTCCACCAGGTCCACCAGGTCCACCAGGTCCACCA; the amino acid sequence encoding the constant region C1 is SEQ ID NO:10:

[0023] GKEASEQRIRELCGGMISEQIAQLAAHLRKPLAPGSIGPPGPPGPPGPP;

[0024] Furthermore, the nucleotide sequence encoding the constant region C3 is SEQ ID NO:11:

[0025] GGTCCACCAGGTCCACCAGGTCCACCAGGTCCACCAGGTCCACCACATCATCATCACCATCAT; the amino acid sequence encoding the constant region C3 is SEQ ID NO:12: GPPGPPGPPGPPGPPHHHHHH;

[0026] Furthermore, the constant region A1, the variable region A2, the constant region A3, the constant region B1, the variable region B2, the constant region B3, the constant region C1, the variable region C2, and the constant region C3 are connected in series;

[0027] Furthermore, the variable region 1, the variable region 2, and the variable region 3 are selected from any one or more of types I, II, III, IV, and XVII human collagen;

[0028] Furthermore, the nucleotide sequences encoding the variable region 1, the variable region 2, and the variable region 3 are selected from any 3 identical or different ones among SEQ ID NO:13 - SEQ ID NO:18, where SEQ ID NO:13:

[0029] GGTCCACCAGGTAGAGACGGTCAACCAGGTCATAAGGGAGAGAGAGGTTACCCAGGTAACATTGGTCCAGTT;

[0030] SEQ ID NO:14:

[0031] GGTTTTAGAGGTCCAGCAGGTCCAAACGGTATTCCAGGAGAAAAGGGTCCAGCAGGAGAAAGAGGAGATCAA;

[0032] SEQ ID NO:15:

[0033] GGAGAAAAAGGAGAAAGAGGAGCAGCAGGAGAACCAGGTCCAAAAGGATCTTCAGGTTCTCCAGGTCCACAA;

[0034] SEQ ID NO:16:

[0035] GGAGAAAGAGGTTTCCCAGGAGAAAGAGGAGTTCAAGGTCCACCAGGTCCAGCAGGTCCAAGAGGAGCTAATGGTGCTCCA;

[0036] SEQ ID NO:17:

[0037] GGAGCTAGAGGTCAAGCAGGAGTTATGGGTTTCCCAGGTCCAAAAGGAGCAGCAGGAGAACCAGGTAAAGCAGGAGAAAGA;

[0038] SEQ ID NO:18:

[0039] GGAGTTATGGGTTTTCCAGGTCCAAAAGGAGCTAACGGAGAACCAGGTAAGGCAGGAGAAAAAGGTTTGCCAGGAGTTATG。

[0040] Further, the amino acid sequence corresponding to SEQ ID NO:13 is SEQ ID NO:19: GPPGRDGQPGHKGERGYPGNIGPV; the amino acid sequence corresponding to SEQ ID NO:14 is SEQ ID NO:20: GFRGPAGPNGIPGEKGPAGERGDQ; the amino acid sequence corresponding to SEQ ID NO:15 is SEQ ID NO:21: GFRGPAGPNGIPGEKGPAGERGDQ; GEKGERGAAGEPGPKGSSGSPGPQ; the amino acid sequence corresponding to SEQ ID NO:16 is SEQ ID NO:22: GERGFPGERGVQGPPGPAGPRGANGAP; the amino acid sequence corresponding to SEQ ID NO:17 is SEQ ID NO:23: GARGQAGVMGFPGPKGAAGEPGKAGER; the amino acid sequence corresponding to SEQ ID NO:18 is SEQ ID NO:24: GVMGFPGPKGANGEPGKAGEKGLPGVM.

[0041] Preferably, the promoter sequence of the expression cassette is the AOX1 promoter, the terminator sequence is the AOX1 terminator, the fragment 1, the fragment 2 and the fragment 3 are guided by the non-collagenous region sequence NC2 of type IX collagen and are tandemly formed into the expression cassette, and the nucleotide sequence encoding the expression cassette is SEQ ID NO:25:

[0042]

[0043] The amino acid sequence corresponding to the nucleotide sequence SEQ ID NO:25 is SEQ ID NO:26:

[0044]

[0045] The present invention also provides a recombinant humanized collagen with a self-assembled triple helix structure, and the recombinant humanized collagen contains the above expression cassette.

[0046] The present invention also provides a method for producing the above recombinant humanized collagen, and the method includes:

[0047] S1, preparing a recombinant expression vector pPIC9k-GHKMix and / or pPIC9k-mini containing the above expression cassette;

[0048] S2, preparing a prolyl hydroxylase expression vector pPICZB-P4Hβ-P4Hα;

[0049] S3, transferring the two expression vectors obtained in S1 and S2 into competent cells for co-expression to obtain a recombinant humanized collagen with a self-assembled triple helix structure.

[0050] Further, the recombinant expression vector pPIC9k-GHKMix / pPIC9k-mini uses the pPIC9K vector as a backbone, connects the above expression cassette between the 1215th bp and the 2071st bp of the yeast genome, and performs trisecting cleavage on the expression product of the vector by introducing a kex2 protease recognition site KR in the expression cassette to form three equal-length peptide chains of the triple helix basic structure.

[0051] Further, the recombinant expression vector pPICZB-P4Hβ-P4Hα uses the pPICZB as a backbone, introduces a Pichia pastoris kex2 protease recognition site KR, and introduces a prolyl hydroxylase gene (P4Hβ and P4Hα genes) between the EcoRI and NotI of the multiple cloning sites by a tandem expression method, inserts a kex2 protease recognition site KR at the middle site between the P4Hβ and P4Hα genes, and adds an ER retention signal HDEL (His-Asp-Glu-Leu) to the C-terminus of the P4Hβ and P4Hα subunits respectively.

[0052] Further, the nucleotide sequence encoding P4Hβ is SEQ ID NO:27, and its corresponding amino acid sequence is SEQ ID NO:28:

[0053] SEQ ID NO:27:

[0054]

[0055] SEQ ID NO:28:

[0056] MQFNWNIKTVASILSALTLAQADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVHDEL

[0057] Furthermore, the nucleotide sequence encoding P4Hα is SEQ ID NO:29, and its corresponding amino acid sequence is SEQ ID NO:30;

[0058] SEQ ID NO:29:

[0059]

[0060] SEQ ID NO:30:

[0061] MQFNWNIKTVASILSALTLAQAEFFTSIGHMTDLIYAEKELVQSLKEYILVEEAKLSKIKSWANKMEALTSKSAADAEGYLAHPVNAYKLVKRLNTDWPALEDLVLQDSAAGFIANLSVQRQFFPTDEDEIGAAKALMRLQDTYRLDPGTISRGELPGTKYQAMLSVDDCFGMGRSAYNEGDYYHTVLWMEQVLKQLDAGEEATTTKSQVLDYLSYAVFQLGDLHRALELTRRLLSLDPSHERAGGNLRYFEQLLEEEREKTLTNQTEAELATPEGIYERPVDYLPERDVYESLCRGEGVKLTPRRQKRLFCRYHHGNRAPQLLIAPFKEEDEWDSPHIVRYYDVMSDEEIERIKEIAKPKLARATVRDPKTGVLTVASYRVSKSSWLEEDDDPVVARVNRRMQHITGLTVKTAELLQVANYGVGGQYEPHFDFSRRPFDSGLKTEGNRLATFLNYMSDVEAGGATVFPDLGAAIWPKKGTAVFWYNLLRSGEGDYRTRHAACPVLVGCKWVSNKWFHERGQEFLRPCGSHDEL

[0062] Preferably, the competent cell is any one of X-33, GS115 and SMD1168, preferably the competent cell of Pichia pastoris GS115.

[0063] The beneficial effects of the present invention are as follows:

[0064] 1) The present invention optimizes the sequences of type I, II, III, IV, and XVII collagens known in the NCBI database, reduces sequence repetition, and improves the codon adaptation index (CAI), solving the problem that the recombinant collagen sequence is not expressed or difficult to express in Pichia pastoris.

[0065] 2) The present invention utilizes the endogenous protease (Kex2 protease) of Pichia pastoris. By introducing the Kex2 protease recognition site KR, the condition for one-step production of three heterologous collagens is achieved, overcoming the technical problem that the traditional method cannot co-express three different heterologous chains in the same host.

[0066] 3) By introducing the non-collagenous region (NC2) of type IX collagen and fusing and expressing the collagen domain structures of different types of collagen, the present invention provides an effective way to produce collagen with a heterologous triple helix structure;

[0067] 4) The present invention constructs a prolyl hydroxylase expression vector pPICZB-P4Hβ-P4Hα, overexpresses it and localizes it to the ER, and performs post-translational modification on the mature peptide of recombinant human collagen, solving the problem that the triple helix structure of recombinant collagen is loose and the thermal stability is poor due to the lack of hydroxyproline modification;

[0068] 5) The co-expression strain provided by the present invention has a high expression level of triple helix recombinant humanized collagen and is simple to purify. The triple helix structure recombinant human collagen obtained after purification contains hydroxyproline. Compared with natural collagen from animal sources, it has the advantages of uniform molecular weight, high purity, and no risk of virus transmission;

[0069] 6) The triple helix structure recombinant human collagen provided by the present invention has a characteristic triple helix structure of collagen. When detected by CD spectroscopy at 4 °C under the condition of 198 nm, it shows a specific negative peak presented by the triple helix structure of collagen;

[0070] 7) The triple helix recombinant humanized collagen provided by the present invention has good water solubility, stable quality, and is not easily degraded. Protein bands can still be detected after storing in a 4 °C refrigerator for half a year, and it can be applied to the fields of medical health and medical aesthetics, such as beauty and skin care products, skin repair dressings, implants, bio-based materials, medical devices and other fields. Description of the Drawings

[0071] Figure 1 It is a schematic diagram of the expression cassette structure, where 1A is a schematic diagram of the single-chain structure and 1B is a schematic diagram of the full length of the expression cassette

[0072] Figure 2 It is the CD spectral absorption peak of pPIC9k-GHKMix (purple-red) and pPIC9k-mini (sky-blue) in a cuvette at 4 °C under the condition of 190 - 260 nm.

[0073] Figure 3 It is the single-chain mass spectrometry analysis and modification analysis of pPIC9k-mini B1 - B3, where 3A is pPIC9k-mini-B1; 3B is pPIC9k-mini-B2; 3C is pPIC9k-mini-B3.

[0074] Figure 4 It is the plasmid map of pPIC9k-RHC-mini (pPIC9k-mini) with KanR resistance (G418 in eukaryotes).

[0075] Figure 5It is the plasmid map of pPIC9k-RHC-GHKmix (pPIC9k-GHKmix), with KanR resistance (G418 in eukaryotes).

[0076] Figure 6 It is the plasmid map of pPICZB-P4H, with BleoR resistance.

[0077] Figure 7 It is the SDS-PAGE gel diagram. Among them, 7A is the SDS-Page gel diagram of pPIC9k-mini and pPIC9k-GHKmix. ① is the fermentation and purification product of the double-plasmid negative clone of pPIC9k and pPICZB. ② is the purified protein of pPIC9k-mini, with 10 ul loaded. ③ is the flow-through waste liquid of the fermentation supernatant of pPIC9k-mini. ④ is the purified protein of pPIC9k-mini, with 30 ul loaded. ⑤ is the purified protein of pPIC9k-GHKmix, with 30 ul loaded. ⑥ is the protein band of pPIC9k-GHKmix after being stored naturally at 4°C for more than one year. 7B is the WB band analysis of pPIC9k-mini. ① is the fermentation supernatant (unpurified) of pPIC9k-mini. ② is the flow-through waste liquid of the fermentation supernatant of pPIC9k-mini. ③ is the purified protein of pPIC9k-mini. Detailed implementation manners

[0078] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and by means of examples. Obviously, the described examples are only a part of the examples of the present invention, rather than all the examples. All other examples obtained by those of ordinary skill in the art based on the examples of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0079] Example 1:

[0080] This example provides an expression cassette, which sequentially includes the following elements from upstream to downstream: a promoter sequence, a fusion gene, and a terminator sequence. Among them, the fusion gene includes nucleotide fragment A, nucleotide fragment B, and nucleotide fragment C.

[0081] In this example, the promoter sequence is the AOX1 promoter, and the terminator sequence is the AOX1 terminator. The AOX1 promoter is a strong promoter that can efficiently express downstream genes under methanol induction conditions. The AOX1 terminator can effectively terminate the transcription process and ensure the integrity of gene expression.

[0082] The nucleotide fragment A, the nucleotide fragment B, and the nucleotide fragment C are guided by the non-collagenous region sequence NC2 of type IX collagen and are concatenated to form the expression cassette. The non-collagenous region sequence NC2 of type IX collagen serves as a linker sequence, which can ensure the correct ligation and expression of the three nucleotide fragments.

[0083] Example 2:

[0084] Based on Example 1, in this example, the nucleotide fragment A of the expression cassette includes the constant region A1, the variable region A2, and the constant region A3; the nucleotide fragment B includes the constant region B1, the variable region B2, and the constant region B3; the nucleotide fragment C includes the constant region C1, the variable region C2, and the constant region C3.

[0085] The constant region A1, the variable region A2, the constant region A3, the constant region B1, the variable region B2, the constant region B3, the constant region C1, the variable region C2, and the constant region C3 are connected in series.

[0086] In this example, the variable region 1, the variable region 2, and the variable region 3 are selected from any one or more of human collagens of types I, II, III, IV, and XVII. These types of human collagens have different biological properties and functions. By selecting different types of collagens, recombinant humanized collagens with specific functions can be obtained.

[0087] Example 3:

[0088] Based on Example 2, in this example, the nucleotide sequence of the constant region A1 of the expression cassette is SEQ ID NO:1, and the amino acid sequence encoding the nucleotide sequence of the constant region A1 is SEQ ID NO:2; the nucleotide sequence of the constant region A3 is SEQ ID NO:3, and the amino acid sequence corresponding to the nucleotide sequence of the constant region A3 is SEQ ID NO:4; the nucleotide sequence of the constant region B1 is SEQ ID NO:5, and the amino acid sequence corresponding to the nucleotide sequence of the constant region B1 is SEQ ID NO:6; the nucleotide sequence of the constant region B3 is SEQ ID NO:7, and the amino acid sequence corresponding to the nucleotide sequence of the constant region B3 is SEQ ID NO:8; the nucleotide sequence of the constant region C1 is SEQ ID NO:9, and the amino acid sequence corresponding to the nucleotide sequence of the constant region C1 is SEQ ID NO:10; the nucleotide sequence of the constant region C3 is SEQ ID NO:11, and the amino acid sequence corresponding to the nucleotide sequence of the constant region C3 is SEQ ID NO:12.

[0089] The above-mentioned invariant region sequences A1, A3, B1, B3, C1, and C3 are important components of the expression cassette and play a crucial role in the structure and function of proteins. The sequences of the invariant regions A1, A3, B1, B3, C1, and C3 are highly conserved, ensuring the stability and functionality of the expression products.

[0090] Example 4:

[0091] Based on Example 2, the nucleotide sequences of the variable regions A2, B2, and C2 of the expression cassette in this example are selected from any 3 identical or different ones among SEQ ID NO:13 - SEQ ID NO:18, and the corresponding amino acid sequences of the nucleotide sequences SEQ ID NO:13 - SEQ ID NO:18 are SEQ ID NO:19 - SEQ ID NO:24 in sequence.

[0092] The sequence diversity of the variable regions A2, B2, and C2 enables the expression cassette to produce recombinant humanized collagens with different characteristics. By selecting different combinations of variable region sequences, the physicochemical properties, biocompatibility, and biological activity of the expression products can be regulated.

[0093] In some embodiments, the nucleotide sequences of the variable regions A2, B2, and C2 are SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18. Among them, SEQ ID NO:16 is derived from a type I collagen α1 chain fragment, SEQ ID NO:17 is derived from a type I collagen α1 chain fragment, and SEQ ID NO:18 is derived from a type II collagen α1 chain fragment.

[0094] Its pPIC9k - mini tandem system and the possible functional structural sites (underlined) are shown as in SEQ ID NO:31:

[0095] SEQ ID NO:31: PGPPGPPGPP GERGFPGER GVQGPPGPAGPRGANGGRAPTDQHIKQVCMRVIQEHFAEMAASLK RPDSGATGPAPGPPGPPGPPGPPGPPGPP HHHHH HKRGRDATDQHIVDVALKMLQEQLAEVAVSAKREALGAVGPPGPPGPPGPPGARGQA GVMGFP GPKGAAGEPGKA GER GPPGPPGPPGPPGPP HHHHHH KRGKEASEQRIRELCGGMISEQIAQLAAHLRKPLAPGSIGPPGPPGPPGPPGVMGFP GPKGANGEPGKAGEKGLP GVM GPPGPPGPPGPPGPP HHHHHH

[0096] In some embodiments, the nucleotide sequences of the variable regions A2, B2, and C2 are SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, where SEQ ID NO:13 is derived from a fragment of type I collagen α1 chain, SEQ ID NO:14 is derived from a fragment of type III collagen α1 chain, and SEQ ID NO:15 is derived from a fragment of type XVII collagen α1 chain.

[0097] Its pPIC9k - GHKMix tandem system and the possible functional structural sites (underlined) are shown as in SEQ ID NO:32:

[0098] SEQ ID NO:32: GRAPTDQHIKQVCMRVIQEHFAEMAASLKRPDSGATGPPGPPGPPGPPGPPGRDGQP GHKGER GYPGNIGPVGPPGPPGPPGPPGPP HHHHHH KRGRDATDQHIVDVALKMLQEQLAEVAVSAKREALGAVGPPGPPGPPGPPGFRGPAGPNGIP GEK GPA GERGD QGPPGPPGPPGPPGPP HHHHHH KRGKEASEQRIRELCGGMISEQIAQLAAHLRKPLAPGSIGPPGPPGPPGPP GEKGER GAAGEPGPKGSSGSPGPQGPPGPPGPPGPPGPP HHHHHH

[0099] Example 5:

[0100] This example provides a recombinant humanized collagen with a self - assembling triple - helix structure. The recombinant humanized collagen contains the expression cassette as described in any one of Examples 1 - 4. The nucleotide sequence of this expression cassette is SEQ ID NO:25, and the amino acid sequence is SEQ ID NO:26.

[0101] This recombinant humanized collagen has the property of self - assembling into a triple - helix structure, which is the basis for collagen to exert its biological functions. Through the kex2 protease recognition site KR introduced into the expression cassette, the expression product can be cleaved into three equal - length peptide chains, and these three peptide chains can spontaneously assemble into a stable triple - helix structure.

[0102] Example 6:

[0103] This example provides a method for producing the recombinant humanized collagen as described in Example 5, and the method includes:

[0104] Step S1: Prepare the recombinant expression vectors pPIC9k-GHKMix and / or pPIC9k-mini containing the expression cassette as described in Example 1.

[0105] In this step, first construct the recombinant expression vector containing the expression cassette. The specific operation is as follows: Using the pPIC9K vector as the backbone, connect the expression cassette into a specific position of the yeast genome, and through introducing the kex2 protease recognition site KR into the expression cassette, perform trisecting cleavage on the expression product of this vector to form three equal-length peptide chains of the triple helix basic structure.

[0106] Step S2: Prepare the prolyl hydroxylase expression vector pPICZB-P4Hβ-P4Hα. In this step, using pPICZB as the backbone, introduce the kex2 protease recognition site KR of Pichia pastoris, and through the tandem expression method, introduce the prolyl hydroxylase genes (P4Hβ and P4Hα genes) respectively at the multiple cloning sites (AOX1 promoter, Mss1 digestion), insert the kex2 protease recognition site KR at the intermediate site between the P4Hβ and P4Hα genes, and add the ER retention signal HDEL (His-Asp-Glu-Leu) to the C-terminus of the P4Hβ and P4Hα subunits respectively, which can perform post-translational modification on the mature peptide of recombinant human collagen. The hydroxylation strategy refers to the reference New strategy for expression of recombinant hydroxylated human collagen α1(III) chains in Pichia pastoris GS115 DOI:10.1002 / bab.1264.

[0107] Prolyl hydroxylase is a key enzyme for the post-translational modification of collagen. It can catalyze the hydroxylation of proline residues in collagen, and this modification is crucial for the stability of the collagen triple helix structure. The ER retention signal HDEL can ensure that prolyl hydroxylase plays a role in the endoplasmic reticulum and improve the hydroxylation efficiency.

[0108] Step S3: Transfer the two expression vectors obtained in S1 and S2 into competent cells for co-expression to obtain the recombinant humanized collagen with a self-assembled triple helix structure.

[0109] In this step, the pPIC9k-GHKMix and / or pPIC9k-mini expression vectors are co-transformed into competent cells together with the pPICZB-P4Hβ-P4Hα expression vector. Through co-expression, procollagen and prolyl hydroxylase can be produced simultaneously, ensuring that the procollagen is properly post-translationally modified during synthesis, thereby forming recombinant humanized collagen with a self-assembling triple-helix structure.

[0110] Example 7:

[0111] Based on Example 6, this example provides a method for producing recombinant humanized collagen. The recombinant expression vector pPIC9k-GHKMix / pPIC9k-mini in this example is based on the pPIC9K vector. The expression cassette described in any one of Examples 1-4 is ligated into a specific position of the yeast genome, and the kex2 protease recognition site KR is introduced into the expression cassette to perform trisecting cleavage on the expression product of the vector, forming three equal-length peptide chains with a triple-helix basic structure.

[0112] The introduction of the kex2 protease recognition site KR is the key to achieving trisecting cleavage of the expression product. The kex2 protease is an endoprotease in Pichia pastoris. It can specifically recognize and cleave the KR site, thereby cleaving the expression product into three equal-length peptide chains. These three peptide chains have the ability to self-assemble and can form a stable triple-helix structure, which is the basis for collagen to exert its biological functions.

[0113] Example 8: A method for producing recombinant humanized collagen

[0114] Based on Example 6, this example provides a method for producing recombinant humanized collagen. The recombinant expression vector pPICZB-P4Hβ-P4Hα in this example is based on the pPICZB vector. The kex2 protease recognition site KR of Pichia pastoris is introduced, and the prolyl hydroxylase genes (P4Hβ and P4Hα genes) are respectively introduced into the multiple cloning sites (AOX1 promoter, Mss1 digestion) by tandem expression. The kex2 protease recognition site KR is inserted at the intermediate site between the P4Hβ and P4Hα genes, and the ER retention signal HDEL (His-Asp-Glu-Leu) is added to the C-terminus of the P4Hβ and P4Hα subunits respectively.

[0115] Prolyl hydroxylase consists of two subunits, P4Hβ and P4Hα, which together catalyze the hydroxylation of proline residues in collagen. In this example, the two subunits, P4Hβ and P4Hα, are expressed in the same vector by tandem expression, and the kex2 protease recognition site KR is inserted between the two subunits, enabling the two subunits to be correctly cleaved and assembled into a functional prolyl hydroxylase after expression.

[0116] Among them, the nucleotide sequence encoding P4Hβ is SEQ ID NO:27, and its corresponding amino acid sequence is SEQ ID NO:28; the nucleotide sequence encoding P4Hα is SEQ ID NO:29, and its corresponding amino acid sequence is SEQ ID NO:30.

[0117] The addition of the ER retention signal HDEL ensures the localization of prolyl hydroxylase in the endoplasmic reticulum because the post-translational modification of collagen mainly occurs in the endoplasmic reticulum. By localizing prolyl hydroxylase in the endoplasmic reticulum, the efficiency of collagen hydroxylation can be improved, thereby enhancing the quality and yield of recombinant humanized collagen.

[0118] Example 9: A method for producing recombinant humanized collagen

[0119] Based on Examples 6-8, the competent cells in this example are any one of X-33, GS115, and SMD1168, preferably the competent cells of Pichia pastoris GS115.

[0120] Pichia pastoris is a commonly used protein expression host, which has the advantages of high expression level, strong post-translational modification ability, and simple culture conditions. Among them, the GS115 strain is a widely used Pichia pastoris strain, which has the characteristics of fast growth rate and high expression efficiency, and is particularly suitable for the expression of recombinant proteins.

[0121] In this example, the pPIC9k-GHKMix and / or pPIC9k-mini expression vectors are co-transformed into GS115 competent cells with the pPICZB-P4Hβ-P4Hα expression vector, and high-yield and high-quality recombinant humanized collagen can be obtained through methanol-induced expression.

[0122] Example 10: Application of an expression cassette

[0123] This example provides the application of the expression cassette described in any one of Examples 1-4 in the preparation of recombinant humanized collagen. This expression cassette can be highly expressed in the Pichia pastoris expression system to produce recombinant humanized collagen with a self-assembled triple helix structure.

[0124] Recombinant humanized collagen has good biocompatibility and biological activity, and can be applied in the fields of tissue engineering, drug delivery, cosmetics, and medical devices. Compared with traditional animal-derived collagen, recombinant humanized collagen has the advantages of no risk of virus infection, good batch-to-batch consistency, and customizable properties.

[0125] Through the expression cassette and production method provided by the present invention, the efficient and stable production of recombinant humanized collagen can be achieved, providing new possibilities for the application of collagen in the biomedical field.

[0126] It should be noted that Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, Example 8, Example 9, and Example 10 are all a kind of expression cassette.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An expression cassette, characterized in that, The expression cassette sequentially includes the following elements from upstream to downstream: a promoter sequence, a fusion gene, and a terminator sequence; the fusion gene includes nucleotide fragment A, nucleotide fragment B, and nucleotide fragment C.

2. The expression cassette according to claim 1, wherein The nucleotide fragment A includes constant region A1, variable region A2, and constant region A3. The nucleotide fragment B includes constant region B1, variable region B2, and constant region B3. The nucleotide fragment C includes constant region C1, variable region C2, and constant region C3. The constant region A1, the variable region A2, the constant region A3, the constant region B1, the variable region B1, the constant region B3, the constant region C1, the variable region C2, and the constant region C3 are connected in series; the variable region A2, the variable region B2, and the variable region C2 are selected from any one or more of type I, II, III, IV, and XVII human collagen.

3. The expression cassette according to claim 2, wherein The nucleotide sequence of the constant region A1 is SEQ ID NO:1, and the amino acid sequence encoding the constant region A1 is SEQ ID NO:2; the nucleotide sequence encoding the constant region A3 is SEQ ID NO:3, and the amino acid sequence encoding the constant region A3 is SEQ ID NO:4; the nucleotide sequence encoding the constant region B1 is SEQ ID NO:5, and the amino acid sequence encoding the variable region B2 is SEQ ID NO:6; the nucleotide sequence encoding the constant region B3 is SEQ ID NO:7, and the amino acid sequence encoding the constant region C1 is SEQ ID NO:8; the nucleotide sequence encoding the constant region C1 is SEQ ID NO:9, and the amino acid sequence encoding the constant region C1 is SEQ ID NO:10; the nucleotide sequence encoding the constant region C3 is SEQ ID NO:11, and the amino acid sequence encoding the constant region C3 is SEQ ID NO:

12.

4. The expression cassette according to claim 2, wherein The nucleotide sequences encoding the variable region 1, variable region 2, and variable region 3 are selected from any 3 identical or different ones among SEQ ID NOs: 13 - SEQ ID NO:

18. The amino acid sequence corresponding to the nucleotide sequence SEQ ID NO: 13 is SEQ ID NO: 19, the amino acid sequence corresponding to the nucleotide sequence SEQ ID NO: 14 is SEQ ID NO: 20, the amino acid sequence corresponding to the nucleotide sequence SEQ ID NO: 15 is SEQ ID NO: 21, the amino acid sequence corresponding to the nucleotide sequence SEQ ID NO: 16 is SEQ ID NO: 22, the amino acid sequence corresponding to the nucleotide sequence SEQ ID NO: 17 is SEQ ID NO: 23, and the amino acid sequence corresponding to the nucleotide sequence SEQ ID NO: 18 is SEQ ID NO:

24.

5. The expression cassette according to any one of claims 1-5, characterized in that The promoter sequence of the expression cassette is the AOX1 promoter, and the terminator sequence is the AOX1 terminator. The fragment 1, the fragment 2, and the fragment 3 are guided by the non-collagenous region sequence NC2 of type IX collagen and are tandemly formed into the expression cassette. The nucleotide sequence encoding the expression cassette is SEQ ID NO:25, and its corresponding amino acid sequence is SEQ ID NO:

26.

6. A recombinant humanized collagen with a self-assembled triple-helix structure, characterized in that, The recombinant humanized collagen contains the expression cassette as described in any one of claims 1-5.

7. A method for producing the recombinant humanized collagen as described in claim 6, characterized in that, The method includes: S1, preparing a recombinant expression vector pPIC9k-GHKMix and / or pPIC9k-mini containing the expression cassette as described in claim 1; S2, preparing a prolyl hydroxylase expression vector pPICZB-P4Hβ-P4Hα; S3, co-expressing the two expression vectors obtained in S1 and S2 in competent cells to obtain recombinant humanized collagen with a self-assembled triple-helix structure.

8. The method according to claim 7, wherein The recombinant expression vector pPIC9k-GHKMix / pPIC9k-mini is based on the pPIC9K vector. The expression cassette as described in any one of claims 1-7 is ligated between the 1215th bp and the 2071st bp of the yeast genome, and the expression product of the vector is trisected by introducing a kex2 protease recognition site KR into the expression cassette to form three equal-length peptide chains of the triple-helix basic structure.

9. The method according to claim 7, wherein The recombinant expression vector pPICZB-P4Hβ-P4Hα is based on the pPICZB. A kex2 protease recognition site KR of Pichia pastoris is introduced, and prolyl hydroxylase genes (P4Hβ and P4Hα genes) are respectively introduced between the EcoRI and NotI of the multiple cloning sites by a tandem expression method. A kex2 protease recognition site KR is inserted at the middle site between the P4Hβ and P4Hα genes, and an ER retention signal HDEL (His-Asp-Glu-Leu) is added to the C-terminus of the P4Hβ and P4Hα subunits respectively.

10. The method according to any one of claims 7-9, characterized in that The competent cell is any one of X-33, GS115, and SMD1168, preferably the competent cell of Pichia pastoris GS115.

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