Highly active recombinant human type I collagen truncated form, preparation method and application

Abstract

The present invention discloses a highly active recombinant human type I collagen truncate, a preparation method and an application thereof. The truncate is obtained by optimizing and truncating the sequence of recombinant human type I collagen. The recombinant human type I collagen truncate JYC101 of the present invention can not only be efficiently secreted and expressed in Pichia pastoris, but also has better cell adhesion, cell proliferation and cell migration activities than the original recombinant human type I collagen, and has broad application prospects in the fields of skin care products, medical devices, tissue engineering and regenerative medicine.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to a highly active recombinant human type I collagen truncate, a preparation method and an application thereof. Background Art

[0002] Collagen is an important functional structural protein, accounting for about 30% of the total protein content in the human body. It is widely distributed in the skin, bones, tendons and connective tissues of the human body. Type I collagen is the most abundant, and it plays an important role in maintaining the stability and integrity of tissues and organs. Type I procollagen is the precursor of mature type I collagen. Compared with mature collagen, procollagen also has two terminal propeptide structures (N-propeptide and C-propeptide) to promote the formation of a triple helix structure. After the type I procollagen folded into a triple helix is ​​sheared to remove the N-propeptide and C-propeptide, it will be secreted into the extracellular matrix and then self-assembled into a highly ordered collagen fiber structure.

[0003] In addition to structural features, there are a variety of functional binding sites in human type I collagen, such as GER (glycine-glutamic acid-arginine) or GEK (glycine-glutamic acid-lysine) with adjacent positive and negative charges that bind to extracellular integrins, and GVMGFP (glycine-valine-methionine-glycine-phenylalanine-proline) that binds to the discoidin domain receptor DDR. The biological functions of the above binding sites depend on the triple helix structure, while others only require collagen monomers. Chinese patent CN111363028A discloses a recombinant human type I collagen, an expression strain and a construction method thereof, which comprises selecting a water-soluble and stable nucleotide sequence from the helical region of type I human collagen gene of known sequence, inserting the preferred gene fragment into a Pichia pastoris expression plasmid, transforming Pichia pastoris to obtain a highly expressed Pichia pastoris genetically engineered bacterium, and obtaining a highly purified recombinant human type I collagen after preliminary fermentation and purification steps, and exhibiting good hydrophilicity and stability, and being proven to have better cell proliferation, migration and adhesion promoting properties than commercially available recombinant collagen. Summary of the invention

[0004] The present invention is based on the recombinant human type I collagen of Chinese patent CN111363028A. By removing its different functional binding domains, including cell integrins, fibronectin, sulfated glycosaminoglycans and Decorin, the corresponding truncations are obtained. The differences in the performance of different truncations in promoting cell proliferation, migration and adhesion are analyzed in combination with cell experiments, and a recombinant human type I collagen truncation with higher activity than the original recombinant human type I collagen is screened and obtained.

[0005] The technical solution of the present invention is as follows:

[0006] The highly active recombinant human type I collagen truncate is based on the recombinant human type I collagen in Chinese patent CN111363028A, obtained by removing the functional domain binding to sulfated glycosaminoglycan at its amino terminus, and is named JYC101, and its amino acid sequence is shown in SEQ ID No.2; or it is an amino acid sequence having the same or similar biological activity as the amino acid sequence shown in SEQ ID No.2 after a certain degree of amino acid insertion, substitution, addition, deletion, etc. on the basis of the sequence shown in SEQ ID No.2; or it is an amino acid sequence having greater than 80% identity with the amino acid sequence of SEQ ID No.2 and having the same or similar biological activity as the amino acid sequence shown in SEQ ID No.2.

[0007] The gene encoding the above-mentioned recombinant human type I collagen truncation JYC101.

[0008] Preferably, when Pichia pastoris is used as the host, the nucleotide sequence of the gene encoding the recombinant human type I collagen truncation JYC101 is as shown in SEQ ID No.5.

[0009] A recombinant expression vector contains the gene encoding the recombinant human type I collagen truncated form JYC101.

[0010] Furthermore, the recombinant expression vector is an expression vector containing the gene expression cassette encoding the recombinant human type I collagen truncated form JYC101, preferably a tandem two-copy expression vector containing two gene expression cassettes encoding the recombinant human type I collagen truncated form JYC101. In a specific embodiment of the present invention, the gene expression cassette encoding the recombinant human type I collagen truncated form JYC101 contains the aox1 promoter, alpha-mating factor signal peptide, JYC101 and aox1 terminator in sequence.

[0011] The method for constructing the above-mentioned recombinant expression vector comprises the following specific steps:

[0012] (1) Amplifying the gene encoding the recombinant human type I collagen truncated form JYC101, the aox1 promoter, the alpha-mating factor signal peptide, and the aox1 terminator, respectively, and then splicing the aox1 promoter, the alpha-mating factor signal peptide, the synthetic gene, and the aox1 terminator through an overlapping PCR strategy to obtain a gene expression cassette;

[0013] (2) Amplify the linearized empty pPIC9K plasmid, clone the single gene expression cassette into the vector pPIC9K using the Gibson assembly technique, and construct a recombinant expression vector 9K-C100 carrying a single gene expression cassette;

[0014] (3) The correctly sequenced recombinant expression vector 9K-C100 was enriched, and the 9K-C100 plasmid was linearized by two restriction endonucleases, NotI and AgeI. The nucleic acid fragment carrying the aox1 promoter, alpha-mating factor signal peptide and the gene encoding the recombinant human type I collagen truncate JYC101 was amplified, and the above nucleic acid fragment was integrated into the linearized 9K-C100 plasmid using Gibson assembly technology to obtain two tandem copies of the expression plasmid 9K-C101.

[0015] The genetically engineered bacteria for producing the above-mentioned recombinant human type I collagen truncation JYC101 contain the above-mentioned recombinant expression vector.

[0016] In the present invention, the host cells of the genetically engineered bacteria can be eukaryotic cells such as yeast, plant cells, insect cells and mammalian cells CHO series and HEK293 series; prokaryotic cells such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum. In a specific embodiment of the present invention, Pichia pastoris is used as a representative host cell.

[0017] The method for constructing the genetically engineered bacteria for producing the recombinant human type I collagen truncated form JYC101 comprises the following steps:

[0018] The above recombinant expression vector is transformed into competent cells of host cells, positive transformants are selected, and genetically engineered bacteria are obtained.

[0019] In a specific embodiment of the present invention, the genetically engineered bacteria is a Pichia pastoris engineered bacteria, and the two-copy expression plasmid 9K-C101 linearized by SalI is electroporated into Pichia pastoris GS115 competent cells, and positive transformants are selected to obtain Pichia pastoris genetically engineered bacteria.

[0020] The preparation method of the above-mentioned recombinant human type I collagen truncated body JYC101 comprises the following steps:

[0021] The genetically engineered bacteria were inoculated into a fermentation medium for cultivation to obtain the recombinant human type I collagen truncated protein JYC101.

[0022] Preferably, the Pichia pastoris engineered bacteria are inoculated into BMMY induction medium for fermentation, and the fermentation supernatant is purified to obtain a high-purity recombinant human type I collagen truncated form JYC101.

[0023] A composition comprising the above-mentioned recombinant human type I collagen truncate JYC101, wherein the composition includes but is not limited to skin care products, pharmaceutical compositions, etc.

[0024] A product comprising the above-mentioned recombinant human type I collagen truncate JYC101, the product includes but is not limited to medical devices, biomaterials, tissue engineering products, etc.

[0025] Compared with the prior art, the advantages of the present invention are:

[0026] (1) The collagen truncate of the present invention is a truncate obtained by selectively removing the functional domain binding to sulfated glycosaminoglycan at the amino terminus of the recombinant human type I collagen in Chinese patent CN111363028A, which is a combination sequence selected from the central triple helical region sequence of human type I collagen according to protein structure and function. The truncate has 100% consistency with the sequence of human type I collagen, is non-immunogenic, and exhibits more excellent biological activity in promoting cell adhesion, migration and proliferation than the recombinant human type I collagen in Chinese patent CN111363028A, and can be widely used in the fields of cosmetics, medical devices, biomaterials, tissue engineering and medicine.

[0027] (2) The recombinant human type I collagen truncate JYC101 of the present invention can be efficiently secreted and expressed in Pichia pastoris, and can be easily purified to obtain pure collagen; the nucleotide sequence encoding the amino acid sequence is optimized and adjusted according to the codon preference of Pichia pastoris, so that the constructed engineered strain has extremely high performance in producing the target protein, and a higher yield can be obtained through high-density fermentation; Pichia pastoris is easier to culture at a high density than Saccharomyces cerevisiae, and the production strain constructed and screened in the present invention has achieved 100L pilot-scale fermentation culture and separation and purification, and has the conditions for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of plasmid 9K-C101 expressing recombinant human type I collagen truncated protein JYC101.

[0029] Figure 2 This is the electrophoresis diagram of plasmid 9K-C101 expressing recombinant human type I collagen truncated form JYC101, where lane 1: DNA marker; lane 2: linearized fragment of pPIC9K empty vector plasmid; lane 3: linearized fragment of 9K-C101 plasmid.

[0030] Figure 3It is the SDS-PAGE image of the fermentation supernatant of the engineering strain during the induced culture process, wherein lane 2: protein marker; lane 4: fermentation supernatant after 24 hours of induction; lane 5: fermentation supernatant after 36 hours of induction; lane 6: fermentation supernatant after 48 hours of induction; lane 7: fermentation supernatant after 72 hours of induction.

[0031] Figure 4 This is the HPLC spectrum of pure JYC101.

[0032] Figure 5 This is the total ion current chromatogram of the peptide fragments of pure JYC101 after trypsin digestion.

[0033] Figure 6 This is the molecular weight test result of pure JYC101.

[0034] Figure 7 This is the infrared spectrum detection result of pure JYC101.

[0035] Figure 8 This is a scanning electron micrograph of the JYC101 freeze-dried sponge.

[0036] Fig. 9 The graph shows the effects of JYC101, JYC110 and JYC111 on the proliferation of human epidermal cells and human fibroblasts.

[0037] Fig.10 Graph showing the effects of JYC101, JYC110 and JYC111 on the migration of human epidermal cells and human fibroblasts.

[0038] Fig.11 The figure shows the effects of JYC101, JYC110 and JYC111 on the adhesion of human epidermal cells and human fibroblasts.

[0039] Fig.12 This is a graph showing the effects of JYC101, recombinant human type I collagen in patent CN111363028A, and natural human collagen on the proliferation of human epidermal cells and human fibroblasts.

[0040] Fig.13 This is a diagram showing the effects of JYC101, recombinant human type I collagen in patent CN111363028A, and natural human collagen on the migration of human epidermal cells and human fibroblasts.

[0041] Fig.14 This is a diagram showing the effects of JYC101, recombinant human type I collagen in patent CN111363028A, and natural human collagen on the adhesion of human epidermal cells and human fibroblasts. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below through the preferred embodiments of the present invention, but the following embodiments do not limit the protection scope of the present invention.

[0043] In the embodiments of the present invention, conventional molecular biological experimental methods are used for those not described in detail. The processes such as PCR, enzyme digestion, seamless cloning, overlapping PCR, codon optimization, etc. involved in the embodiments are understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore will not be described in detail.

[0044] The present invention obtains different truncated proteins by removing single functional domains (cell integrins, fibronectin, sulfated glycosaminoglycans and Decorin) of the recombinant human type I collagen in Chinese patent CN111363028A, and analyzes the differences in the performance of different truncations in promoting cell proliferation, migration and adhesion in combination with corresponding cell biology experiments, and screens out truncations with higher activity than the original recombinant human type I collagen.

[0045] The truncated body with higher activity than the original recombinant human type I collagen obtained by screening in the present invention is JYC101, which is obtained by removing the functional domain of the amino terminal of the recombinant human type I collagen that binds to the sulfated glycosaminoglycan. Since the higher-order structure and biological function of the protein are controlled by the amino acid sequence of its primary structure, the high activity of the highly active recombinant human type I collagen truncated body JYC101 of the present invention is based on the optimized selected amino acid sequence, so limited sequence modification or a certain proportion of sequence consistency (greater than 80%) may still achieve the same or similar biological activity. The highly active recombinant human type I collagen truncated body JYC101 of the present invention has an amino acid sequence as shown in SEQ ID No. 2, or an amino acid sequence having the same or similar biological activity as the amino acid sequence shown in SEQ ID No. 2 after a certain degree of amino acid insertion, substitution, addition, deletion, etc., or an amino acid sequence having greater than 80% consistency with the amino acid sequence of SEQ ID No. 2 and having the same or similar biological activity as the amino acid sequence shown in SEQ ID No. 2.

[0046] The amino acid insertion described in the present invention refers to inserting an amino acid residue at an appropriate position of the amino acid sequence SEQ ID No. 2, and the inserted amino acid residues may be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other. As long as the protein has similar biological activities as JYC101 collagen, including the activities of promoting cell proliferation, migration, adhesion, and promoting tissue repair and regeneration.

[0047] The amino acid substitution described in the present invention refers to the substitution of one or several amino acid residues (including several consecutive amino acid residues or several non-continuous amino acid residues) at a certain position of the amino acid sequence SEQ ID No. 2 with other amino acid residues. As long as the protein has similar biological activities as JYC101 collagen, including the activities of promoting cell proliferation, migration, adhesion, and promoting tissue repair and regeneration.

[0048] The amino acid addition described in the present invention refers to adding an amino acid residue at a certain position of the amino acid sequence SEQ ID No. 2. As long as the protein has similar biological activities as JYC101 collagen, including the activities of promoting cell proliferation, migration, adhesion, and promoting tissue repair and regeneration.

[0049] The amino acid deletion of the present invention refers to the removal of 1, 2, 3 or more amino acids in the amino acid sequence SEQ ID No. 2. As long as the protein has similar biological activities as JYC101 collagen, including the activities of promoting cell proliferation, migration, adhesion, and promoting tissue repair and regeneration.

[0050] The gene encoding the highly active recombinant human type I collagen truncated form JYC101 of the present invention is a gene encoding the corresponding amino acid sequence, which can be adjusted according to the codon preference of the host cell. For example, when Pichia pastoris is used as the host, the gene encoding the highly active recombinant human type I collagen truncated form JYC101 having the amino acid sequence as shown in SEQ ID No.2 has a nucleotide sequence as shown in SEQ ID No.5.

[0051] The recombinant expression vector of the present invention contains the gene encoding the highly active recombinant human type I collagen truncated form JYC101.

[0052] Furthermore, the recombinant expression vector is an expression vector containing the gene expression cassette encoding the highly active recombinant human type I collagen truncated form JYC101, preferably a tandem two-copy expression vector containing two tandem two-copy gene expression cassettes encoding the highly active recombinant human type I collagen truncated form JYC101. The expression level of JYC101 is further increased by the design of two tandem copies.

[0053] The genetically engineered bacteria expressing the highly active recombinant human type I collagen truncated form JYC101 of the present invention contains the recombinant expression vector.

[0054] The host cells of the genetically engineered bacteria described in the present invention can be eukaryotic cells such as yeast, plant cells, insect cells and mammalian cells CHO series and HEK293 series; prokaryotic cells such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum. In a specific embodiment of the present invention, Pichia pastoris is used as a representative host cell. It should be understood that those skilled in the art can express the highly active recombinant human type I collagen truncated body JYC101 with the same amino acid sequence by using other expression hosts instead of the above-mentioned Pichia pastoris as the host cell.

[0055] The genetically engineered bacteria for producing the highly active recombinant human type I collagen truncated form JYC101 of the present invention are obtained by transforming the recombinant expression vector into competent cells of host cells and selecting positive transformants.

[0056] In a specific embodiment of the present invention, the eukaryotic cell Pichia pastoris is selected as the expression system, which has complete organelles and can modify the translated protein to a certain extent (such as glycosylation, etc.), which greatly ensures the realization of the biological function of the protein. Secondly, Pichia pastoris can be cultured by high-density fermentation, and has the characteristics of easy culture, short cycle and high expression, which are in line with large-scale industrial production. In addition, the highly active recombinant human type I collagen truncate JYC101 can be secreted into the extracellular space, which can avoid the endogenous impurity protein introduced by the broken thalline. Finally, the yeast cell wall does not contain endotoxins and peptidoglycans, which can reduce the pollution of immunogenic substances. Therefore, in a specific embodiment of the present invention, the genetically engineered bacteria constructed are Pichia pastoris engineering bacteria, and the two-copy expression plasmid 9K-C101 linearized in series by SalI is electrotransferred into Pichia pastoris GS115 competent cells, and positive transformants are selected to obtain Pichia pastoris genetically engineered bacteria.

[0057] As a scheme for further screening Pichia pastoris engineered bacteria with high yield and high activity of recombinant human type I collagen truncations JYC101, in a specific embodiment of the present invention, the SalI-linearized two-copy tandem expression plasmid 9K-C101 is electroporated into Pichia pastoris GS115 competent cells, and after YPD activation culture for 1 hour, the cells are diluted and spread on MGY selection plates until monoclonal transformants grow out, and single colonies are picked and spread on MGY plates containing different concentrations of G418 for copy screening, and single colonies that have passed the primary screening are then spread on MGY plates containing higher concentrations of G418 for rescreening, and single colonies that have passed the rescreening are Pichia pastoris engineered bacteria with high yield and high activity of recombinant human type I collagen truncations JYC101.

[0058] The highly active recombinant human type I collagen truncated form JYC101 of the present invention is obtained by inoculating the above-mentioned genetically engineered bacteria into a fermentation medium for cultivation.

[0059] In a specific embodiment of the present invention, the Pichia pastoris engineered bacteria producing the high-yield and high-activity recombinant human type I collagen truncation JYC101 are inoculated into BMMY induction medium for fermentation, and linked batch fed fermentation is adopted to purify the fermentation supernatant to obtain high-purity recombinant human type I collagen truncation JYC101.

[0060] The recombinant human type I collagen truncate JYC101 described in the present invention has excellent biological activity of promoting cell adhesion, migration and proliferation, and can be used to promote tissue regeneration. It can be added to a composition or made into a product, such as added to a medicine or skin care product, or made into a medical device, biomaterial, tissue engineering product, etc.

[0061] Example 1 Construction of recombinant expression vector

[0062] 1. Design of recombinant human type I collagen truncations JYC101, JYC110 and JYC111 sequences

[0063] Based on the recombinant human type I collagen sequence in patent CN111363028A, its amino acid sequence is shown in SEQ ID No.1.

[0064] SEQ ID No.1:

[0065]

[0066] The underlined part of the amino acid sequence SEQ ID No.1 is the binding region with sulfated glycosaminoglycan, the dotted part is the binding region with cell integrin, and the wavy part is the binding region with fibronectin. By removing the functional domain of the amino terminal of SEQ ID No.1 that binds to sulfated glycosaminoglycan, a truncated body with an amino acid sequence as shown in SEQ ID No.2 is obtained, which is named JYC101; by removing the functional domain of SEQ ID No.1 that binds to cell integrin, a truncated body with an amino acid sequence as shown in SEQ ID No.3 is obtained, which is named JYC110; by removing the functional domain of SEQ ID No.1 that binds to fibronectin, a truncated body with an amino acid sequence as shown in SEQ ID No.4 is obtained, which is named JYC111.

[0067] 2. Synthesis of gene sequences encoding JYC101, JYC110 and JYC111 collagen truncations and construction of recombinant expression vectors

[0068] (1) Codon optimization and synthesis of gene fragments: In this example, a Pichia pastoris expression plasmid was constructed. According to the expression preference of Pichia pastoris, the coding genes of the truncated forms JYC101, JYC110 and JYC111 were codon optimized to obtain nucleotide sequences as shown in SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, respectively. The coding gene fragments of the truncated forms JYC101, JYC110 and JYC111 were commissioned to be synthesized by Nanjing GenScript Biotech Co., Ltd.

[0069] (2) Construction of recombinant expression vector:

[0070] Construction of recombinant plasmid 9K-C101 expressing JYC101: The SEQ ID No.5 sequence was integrated into the pUC plasmid to obtain the pUC-JYC101 plasmid. Using the pUC-JYC101 plasmid carrying the SEQ ID No.5 sequence as a template, primer pairs P1 (nucleotide sequence as shown in SEQ ID No.8) and P2 (nucleotide sequence as shown in SEQ ID No.9) were designed to amplify the gene fragment of JYC101; using the pPIC9K empty vector plasmid as a template, primer pairs P3 (nucleotide sequence as shown in SEQ ID No.10) and P4 (nucleotide sequence as shown in SEQ ID No.11), primer pairs P5 (nucleotide sequence as shown in SEQ ID No.12) and P6 (nucleotide sequence as shown in SEQ ID No.13) were designed to amplify the gene sequence containing the aox1 promoter and alpha-mating factor signal peptide, as well as the aox1 terminator sequence; primer pairs P3 (nucleotide sequence as shown in SEQ ID No.10) and P6 (nucleotide sequence as shown in SEQ ID No.14) were used to amplify the gene sequence containing the aox1 promoter and alpha-mating factor signal peptide, as well as the aox1 terminator sequence, respectively. No.13) was used to complete the construction of a single JYC101 gene expression cassette under an overlapping PCR program, which contained an aox1 promoter, an alpha-mating factor signal peptide, JYC101 and an aox1 terminator in sequence; then, a primer pair P7 (nucleotides as shown in SEQID No.14) and P8 (nucleotides as shown in SEQ ID No.15) were designed to amplify the linear plasmid backbone using an empty pPIC9K plasmid as a template; finally, the single JYC101 gene expression cassette and the linear plasmid backbone were seamlessly cloned using the Gibson assembly technology to obtain plasmid 9K-C100. In order to obtain a plasmid carrying two tandem repeats of the JYC101 expression cassette, the positive transformants with correct 9K-C100 sequencing were first enriched and cultured, the plasmid was extracted and double-digested with NotI and AgeI to obtain a linearized 9K-C100 plasmid backbone, and then the primer pair P3 (nucleotide sequence as shown in SEQ ID No.10) and P9 (nucleotide sequence as shown in SEQ ID No.16) were used to amplify the gene fragment carrying the aox1 promoter, alpha-mating factor signal peptide and JYC101 sequence under the overlapping PCR program. Finally, the above gene fragment and the restriction-digested linear 9K-C100 plasmid backbone were seamlessly cloned using the Gibson assembly technology to obtain the recombinant expression plasmid 9K-C101 carrying two JYC101 gene expression cassettes.

[0071] Construction of recombinant plasmid 9K-C110 expressing JYC110 and recombinant plasmid 9K-C111 expressing JYC111: The sequence of SEQ ID No. 6 was integrated into the pUC plasmid to obtain the pUC-JYC110 plasmid. Using the pUC-JYC110 plasmid carrying the sequence of SEQ ID No.6 as a template, primer pairs P10 (nucleotide sequence as shown in SEQ ID No.17) and P2 (nucleotide sequence as shown in SEQ ID No.9) were designed to amplify the JYC110 gene fragment; using the pPIC9K empty vector plasmid as a template, primer pairs P3 (nucleotide sequence as shown in SEQ ID No.10) and P4 (nucleotide sequence as shown in SEQ ID No.11), primer pairs P5 (nucleotide sequence as shown in SEQ ID No.12) and P6 (nucleotide sequence as shown in SEQ ID No.13) were designed to amplify the gene sequence containing the aox1 promoter and alpha-mating factor signal peptide, as well as the aox1 terminator sequence, respectively; primer pairs P3 (nucleotide sequence as shown in SEQ ID No.10) and P6 (nucleotide sequence as shown in SEQ ID No.13) was used to complete the construction of a single JYC110 gene expression cassette (containing aox1 promoter, alpha-mating factor signal peptide, JYC110 and aox1 terminator in sequence) under the overlapping PCR program; then, primer pairs P7 (nucleotides as shown in SEQ ID No.14) and P8 (nucleotides as shown in SEQ ID No.15) were designed to amplify the linear plasmid backbone using the empty pPIC9K plasmid as a template; finally, the single JYC110 gene expression cassette and the linear plasmid backbone were seamlessly cloned using the Gibson assembly technology to obtain plasmid 9K-C102. In order to obtain a plasmid carrying two tandemly repeated JYC110 expression cassettes, the positive transformants with correct sequencing of 9K-C102 were first enriched and cultured, the plasmids were extracted and double-digested with NotI and AgeI to obtain the linearized 9K-C102 plasmid backbone, and then the primer pair P3 (nucleotide sequence as shown in SEQ ID No. 10) and P9 (nucleotide sequence as shown in SEQ ID No. 16) were used to complete the gene fragment carrying the aox1 promoter, alpha-mating factor signal peptide and JYC110 sequence under the overlapping PCR program. Finally, the above gene fragment and the restriction linear 9K-C102 plasmid backbone were seamlessly cloned using the Gibson assembly technology to obtain the recombinant expression plasmid 9K-C110 carrying two target gene expression cassettes. The recombinant plasmid 9K-C103 expressing a single JYC111 gene expression cassette and the recombinant plasmid 9K-C111 expressing two JYC111 gene expression cassettes can be constructed according to the same steps and primers as above.

[0072] The specific steps for constructing the above-mentioned recombinant expression vector are as follows:

[0073] Step a, amplify the target gene and linear vector: use a high-fidelity DNA polymerase kit (purchased from Nanjing Novezan Biotechnology Co., Ltd.), designed primers (synthesized by Beijing Qingke Biotechnology Co., Ltd.) and a template for PCR amplification. The specific operation is carried out according to the kit instructions.

[0074] Step b, recovering and purifying the gene fragments amplified in step a: using a gel recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to obtain aox1 promoter, alpha-mating factor signal peptide, JYC101 gene, JYC110, JYC111 gene, aox1 terminator, and empty pPIC9K linear plasmid, respectively, and the specific operation was carried out according to the instructions of the kit.

[0075] Step c, extract the plasmid in step a: use a plasmid mini-extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to extract the empty pPIC9K plasmid, 9K-C100, 9K-C102 and 9K-C103 plasmids respectively, and the specific operation is carried out according to the instructions of the kit.

[0076] Step d: The 9K-C100, 9K-C102 and 9K-C103 plasmids obtained in step c were double-digested at 37° C. for 1-2 hours. The digestion system was as follows (the restriction endonucleases used were purchased from NEB, and the digestion process was carried out according to its instructions):

[0077]

[0078]

[0079] The purified target gene fragment or expression cassette and vector fragment were subjected to Gibson assembly. The cloning kit (purchased from NEB) was used for ligation (50°C for 30 min) to accurately insert the target gene expression cassette into the corresponding vector position. The ligation reaction system was as follows (performed according to the Gibson assembly kit instructions):

[0080]

[0081] The ligation product was transformed into competent Escherichia coli JM109 (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and positive clones were screened on LB resistance plates containing ampicillin and kanamycin. Colony PCR was first performed for primary screening and verification, and the recombinant plasmid was extracted for sequencing identification (completed by Beijing Qingke Biotechnology Co., Ltd.). Correct sequencing proved that the recombinant expression plasmids 9K-C101, 9K-C110 and 9K-C111 were successfully constructed in this example. The plasmid map of 9K-C101 is shown in Figure 1 shown.

[0082] Example 2 Construction of recombinant engineering strains and screening of high-yield strains

[0083] 1. 10 μg of recombinant expression plasmids 9K-C101, 9K-C110 and 9K-C111 obtained in Example 1 were first digested with SalI restriction endonuclease (purchased from NEB, the specific operation was carried out according to the kit instructions) at 37°C overnight, and then the linearized recombinant expression plasmids were purified using a gel recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and the fragment concentration was controlled at 100-200 ng / μL.

[0084] 2. The linearized plasmids 9K-C101, 9K-C110 and 9K-C111 were electroporated into the host cell Pichia pastoris GS115 competent cells, and the activated bacterial solution was spread on the MGY plate at a 10-fold dilution. After being blown dry in the clean bench, the plate was inverted and cultured in a 30°C incubator for 2-5 days until a single clone grew.

[0085] 3. Pick the monoclonal clones on the plate into a 1.5mL centrifuge tube containing 500μL sterile water, blow it into a bacterial suspension with a pipette, and then take 1μL of the bacterial suspension and spot it on MGY replica plates containing 0.5mg / mL, 1mg / mL, 2mg / mL, 3mg / mL and 4mg / mL geneticin G418. After drying on the clean bench, invert and culture in a 30℃ incubator for 2-3 days. By comparing the growth of different transformants on the high concentration G418 screening plate, screen out the transformants that can tolerate 4mg / mL G418 transformants. Generally speaking, the tolerance of transformants to G418 resistance reflects the number of target gene copies in their genome. The stronger the tolerance, the more copies there are, that is, multiple target gene expression cassettes are site-specifically integrated into the his4 site of Pichia pastoris through homologous single exchange. After this step of screening, recombinant Pichia pastoris production strains with high copies and high production performance for producing recombinant humanized type I collagen truncations JYC101, JYC110 and JYC111 can be obtained.

[0086] 4. Inducible Expression and Identification of Recombinant Type I Collagen Truncations

[0087] (1) The high-copy transformants obtained by screening were inoculated into a 250 mL shake flask containing 50 mL of BMGY seed medium and cultured at 28°C and 220 rpm for 18-20 hours until the OD 600 The cells were collected by centrifugation at 2000 g for 5 min at room temperature and resuspended in BMMY fermentation medium to an OD of 2-6. 600 The fermentation liquid was centrifuged at 5000 g for 10 minutes at room temperature to obtain the supernatant of the fermentation liquid and filtered to sterilize. The obtained sample was stored at -80°C for later use.

[0088] (2) Add 5× loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% DTT) to the collected fermentation broth supernatant, heat in a 100° C. metal bath for 10-15 min, and then perform SDS-PAGE detection.

[0089] The present invention uses SDS-PAGE electrophoresis to preliminarily identify the expressed protein to determine whether there is a characteristic band of the target protein in the extracellular supernatant. Figure 3 The figure is an SDS-PAGE image of the fermentation supernatant of the engineering strain producing high-copy recombinant humanized type I collagen truncation JYC101 during the induced culture process, wherein lane 2: protein marker; lane 4: fermentation supernatant after 24 hours of induction; lane 5: fermentation supernatant after 36 hours of induction; lane 6: fermentation supernatant after 48 hours of induction; lane 7: fermentation supernatant after 72 hours of induction. It can be seen that after 72 hours of induction expression, more than 70% of the extracellular proteins of JYC101 are target proteins, and the apparent molecular weight of JYC101 is about 55 kDa. The above results prove that the recombinant human type I collagen truncation JYC101 can be efficiently secreted and expressed in Pichia pastoris.

[0090] (3) Recover the target protein band at the corresponding cutting position, digest it with trypsin, detect the peptide fragments after digestion by liquid chromatography-mass spectrometry, and compare the detected peptide fragments with the library.

[0091] Figure 5 This is the total ion current chromatogram of the peptides of pure JYC101 after trypsin digestion. It can be seen from the figure that the peptides of JYC101 after digestion all belong to the region of the human type I collagen sequence selected during design, and the peptide coverage rate is 100%, which proves that the peptides in the target band are all derived from the collagen truncation designed in the present invention, and JYC101 is successfully and efficiently secreted outside the cell in Pichia pastoris.

[0092] Example 3 Pilot fermentation of genetically engineered bacteria and protein purification

[0093] 1. Pilot fermentation

[0094] The constructed and screened high-yield strains were subjected to 10L-100L linked pilot fermentation to obtain fermentation broth containing recombinant human type I collagen truncations, thereby realizing large-scale production of recombinant human type I collagen truncations.

[0095] Seed medium (formula: yeast extract 10g / L, tryptone 20g / L, glycerol 10g / L, YNB 3.4g / L); fermentation medium (formula: yeast extract 10g / L, tryptone 20g / L, KH2PO4 3g / L, K2HPO4 12g / L, YNB3.4g / L, (NH4)2SO4 10g / L); feed medium (50% v / v glycerol, 20mL PTM1 / L); induction medium (100% methanol, 20mL PTM1 / L); PTM1 was sterilized by filtration with a 0.22μm aqueous membrane and stored at 4°C. After the fermentation medium was sterilized at high temperature, PTM1 was added when the temperature dropped to room temperature, and the pH was adjusted to 5.5 with ammonia water.

[0096] The batch culture conditions and induced expression conditions of the constructed engineering strain are:

[0097] The batch feeding method was used, and the culture temperature was 28°C. The engineered bacteria were inoculated into a shake flask containing seed culture medium, and the OD was raised to 2L at 220rpm and 30±2°C. 600 ≥10; transfer to the fermenter containing seed medium at 8% inoculation amount (so that OD 600 5), the fermentation tank capacity is 10L, the stirring speed is adjusted to not exceed the maximum speed, the air flux is 2vvm, DO ≥ 30%, and the inoculation is transferred to a fermentation tank containing a fermentation medium for culture. The fermentation tank capacity is 100L, and the fermentation culture conditions are: the stirring speed is not more than the maximum speed, the tank pressure is 0.05±0.01MPa, the air flux is 1vvm, DO ≥ 30%, and the dissolved oxygen rebound flow is added with feed medium until OD 600 Stop feeding when the concentration reaches 200. After 40-72 hours of induction, the tank can be released if the protein concentration measured by HPLC does not increase significantly or decreases.

[0098] HPLC protein quantification formula: C (mg / mL) = peak area / 300165176.

[0099] After high-density pilot fermentation of the engineered bacteria of the high-yield recombinant humanized type I collagen truncated form JYC101, it was found that at 72 hours of induction, the bacterial concentration (OD 600 ) can reach more than 250, and the volume concentration of the target protein in the fermentation broth is greater than 10g / L (HPLC quantitative). Figure 4 .

[0100] 2. Purification

[0101] (1) Pretreatment of fermentation supernatant: First, 100 L of culture broth at the end of fermentation was separated into supernatant and bacterial cells by a tubular centrifuge. The supernatant was then concentrated by an organic membrane with a pore size of 10 kDa to reduce the supernatant volume. The concentrated supernatant was sterilized by a 0.45 μm filter membrane to finally obtain a fermentation supernatant sample that can be used for purification.

[0102] (2) Ion exchange chromatography: In order to obtain a high-purity target protein, the fermentation supernatant after pretreatment is separated and purified by ion exchange chromatography. First, the Q column (Bojin Bio) needs to be pretreated and balanced, and the chromatography column is rinsed with 3-5 column volumes of phosphate buffer to place the filler in the same buffer system as the target protein to protect the protein. Then, the fermentation supernatant after pretreatment is loaded onto the balanced Q column at a flow rate of 1 mL / min, and the effluent is collected. Then, it is washed with 10-15 column volumes of phosphate buffer to remove non-specifically adsorbed impurities. Finally, the target protein is eluted with 5-10 column volumes of phosphate buffer containing 0.5 M sodium chloride, and the eluate is collected in equal parts and verified by HPLC to obtain a sample solution with higher purity. In order to obtain a freeze-dried collagen sponge, the above pure solution is dialyzed, and the solvent is replaced with ultrapure water, followed by membrane concentration and freeze-drying.

[0103] Dissolve JYC101 collagen freeze-dried sponge in ultrapure water to 2 mg / mL, filter with 0.22 μm filter membrane, and inject 10 μL ( SEC-300, 5μm, 7.8*300mm chromatographic column, Aligent LC1260 high performance liquid chromatography), analyze the purity of purified collagen JYC101, such as Figure 4 As shown, the purified JYC101 has an obvious single peak in the liquid phase spectrum, and the purity is as high as over 95%, indicating that the purification method is reliable and effective, and the obtained collagen has high purity.

[0104] 3. Physicochemical and structural characterization of recombinant human type I collagen truncated protein JYC101

[0105] (1) Peptide map coverage detection: Dissolve pure JYC101 in 50mM NH4HCO3, take the dissolved sample and adjust it to a final volume of 100μL with 50mM NH4HCO3; add dithiothreitol DTT solution to make its final concentration 10mM, and reduce it in a 56℃ water bath for 1 hour; add IAM solution to make its final concentration 50mM, and react in the dark for 40 minutes; add trypsin at a mass ratio of trypsin to substrate of 1:100, and digest at 37℃ for 4 hours, and continue to add trypsin at a mass ratio of 1:100, and digest at 37℃ for 16 hours; after digestion, use a desalting column to desalt the peptides, and evaporate the solvent in a 45℃ vacuum centrifugal concentrator; dissolve the peptides in sample dissolving solution (0.1% formic acid, 2% acetonitrile), centrifuge at 12000rpm, 4℃ for 15 minutes, and transfer the supernatant to a sample tube for mass spectrometry analysis. Figure 5 As shown, according to the total ion current chromatogram analysis of pure JYC101 after trypsin treatment, its peptide map coverage is 100%.

[0106] (2) Molecular weight mass spectrometry: Take an appropriate amount of pure JYC101 and dissolve it in ultrapure water. Centrifuge at high speed and take the supernatant for ultra-high performance liquid chromatography (Ultimate 3000, Thermo Fisher Scirntific) coupled with electrospray-quadrupole time-of-flight mass spectrometry (AB SCIEX TripleTOF 5600 Mass Spectrometer, AB SCIEX). The main deconvoluted molecular weight is obtained by measuring the mass spectrum of the sample, such as Figure 6 As shown in Figure 1, the molecular weight of pure JYC101 is 35.5 kDa. Due to the presence of post-translational modifications such as glycosylation in Pichia pastoris, the absolute molecular weight of JYC101 is higher than the theoretical molecular weight (the apparent molecular weight of JYC101 is about 55 kDa), and according to the molecular weight test results, there are 2 mannose groups in JYC101.

[0107] (3) Fourier transform infrared spectroscopy (FT-IR) analysis: Figure 7 As shown, at 4000-400cm -1 Infrared spectrum scanning of JYC101 was performed within the range, and the wave numbers of amide A, amide B, amide I, amide II, and amide III were consistent with the structural characteristics of recombinant collagen.

[0108] (4) SEM analysis of JYC101 freeze-dried sponge samples: Figure 8 As shown in the figure, scanning electron microscopy analysis showed that the JYC101 freeze-dried sponge exhibited a lamellar structure and had the potential to be applied in the field of biomedical materials.

[0109] Example 4 Detection of cell proliferation promoting activity of JYC101, JYC110 and JYC111

[0110] The MTT method was used to detect the ability of JYC101, JYC110 and JYC111 to promote cell proliferation using human epidermal cells and human fibroblasts as experimental cells. Fig. 9 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that JYC101 exhibits the best cell proliferation promoting activity.

[0111] Example 5 Detection of the cell migration promoting activity of JYC101, JYC110 and JYC111

[0112] The cell scratch method was used to measure cell migration. Human epidermal cells and human fibroblasts were used as experimental cells to detect the ability of JYC101, JYC110 and JYC111 to promote cell migration. Fig.10 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that JYC101 exhibits the best ability to promote cell migration.

[0113] Example 6 Detection of cell adhesion promoting activity of JYC101, JYC110 and JYC111

[0114] Using centrifugation method, human epidermal cells and human fibroblasts were used as experimental cells to detect the ability of JYC101, JYC110 and JYC111 to promote cell adhesion. Fig.11 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that JYC101 exhibits the best cell adhesion promoting activity.

[0115] Example 7 Detection of cell proliferation activity of JYC101, recombinant human type I collagen and natural human collagen

[0116] The MTT method was used to detect the ability of JYC101, recombinant human type I collagen in patent CN111363028A and natural human collagen to promote cell proliferation using human epidermal cells and human fibroblasts as experimental cells. Fig.12 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that the order of cell proliferation promoting activity is JYC101>recombinant human type I collagen>natural human collagen, and JYC101 exhibits cell proliferation promoting activity that is superior to recombinant human type I collagen.

[0117] Example 8 Detection of the cell migration promoting activity of JYC101, recombinant human type I collagen and natural human collagen

[0118] The cell scratch method was used to measure cell migration, using human epidermal cells and human fibroblasts as experimental cells to detect the ability of JYC101, recombinant human type I collagen in patent CN111363028A, and natural human collagen to promote cell migration. Fig.13 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that the order of the activity of promoting cell migration is JYC101>recombinant human type I collagen>natural human collagen, and JYC101 exhibits the best ability to promote cell migration.

[0119] Example 9 Detection of cell adhesion promoting activity of JYC101, recombinant human type I collagen and natural human collagen

[0120] Using centrifugation method, human epidermal cells and human fibroblasts were used as experimental cells to detect the ability of JYC101, recombinant human type I collagen in patent CN111363028A and natural human collagen to promote cell adhesion. Fig.14 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that the order of cell adhesion activity is JYC101>recombinant human type I collagen>natural human collagen, and JYC101 exhibits the best cell adhesion activity.

Claims

1. A highly active recombinant human type I collagen truncated form, characterized in that: It is a truncated form of JYC101, and its amino acid sequence is shown in SEQ ID No.

2.

2. A gene encoding the recombinant human type I collagen truncated form JYC101 as claimed in claim 1.

3. The gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID No.

5.

4. A recombinant expression vector, characterized in that: It contains the gene encoding the recombinant human type I collagen truncated form JYC101 as claimed in claim 2.

5. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector is an expression vector containing the gene expression cassette encoding the recombinant human type I collagen truncated form JYC101 according to claim 2; the gene expression cassette encoding the recombinant human type I collagen truncated form JYC101 contains in sequence aox1 Promoter, alpha-mating factor signal peptide, JYC101 and aox1 Terminator.

6. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector is a tandem double-copy expression vector containing two gene expression cassettes encoding the recombinant human type I collagen truncated form JYC101 as described in claim 2.

7. The method for constructing a recombinant expression vector according to claim 5, characterized in that: The specific steps are as follows: (1) Amplify the gene encoding the recombinant human type I collagen truncated form JYC101, aox1 Promoter, alpha-mating factor signal peptide, aox1 terminator, and then through the overlapping PCR strategy aox1 Promoter, alpha-mating factor signal peptide, synthetic gene and aox1 The terminator is spliced ​​to obtain a gene expression cassette encoding the recombinant human type I collagen truncated form JYC101 according to claim 2; (2) Amplify the linearized empty pPIC9K plasmid, clone the single gene expression cassette into the vector pPIC9K using the Gibson assembly technique, and construct a recombinant expression vector 9K-C100 carrying a single gene expression cassette; (3) Enrich the correctly sequenced recombinant expression vector 9K-C100 and Not I and Age I Two restriction endonucleases linearized the 9K-C100 plasmid and amplified the aox1 The promoter, alpha-mating factor signal peptide and nucleic acid fragment encoding the gene of recombinant human type I collagen truncation JYC101 were integrated into the linearized 9K-C100 plasmid using Gibson assembly technology to obtain two tandem copies of the expression plasmid 9K-C101.

8. The genetically engineered bacteria for producing the recombinant human type I collagen truncated form according to claim 1, characterized in that: It contains the recombinant expression vector as claimed in claim 7, and the host cell of the genetically engineered bacteria is a eukaryotic cell or a prokaryotic cell.

9. The genetically engineered bacterium according to claim 8, characterized in that: The eukaryotic cells are selected from yeast, insect cells, mammalian cells CHO series or HEK293 series, and the prokaryotic cells are selected from Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum.

10. The genetically engineered bacterium according to claim 8, characterized in that: The host cell of the genetically engineered bacteria is Pichia pastoris.

11. The method for constructing a genetically engineered bacterium according to claim 8, characterized in that: Here are the steps: The recombinant expression vector as claimed in claim 7 is transformed into competent cells of host cells, positive transformants are selected, and genetically engineered bacteria are obtained.

12. The method for constructing a genetically engineered bacterium according to claim 8, characterized in that: The steps are as follows: Sal I linearized two-copy tandem expression plasmid 9K-C101 was electroporated into Pichia pastoris GS115 competent cells, and positive transformants were selected to obtain Pichia pastoris genetically engineered bacteria.

13. The method for preparing a truncated recombinant human type I collagen according to claim 1, characterized in that: Here are the steps: The genetically engineered bacteria as claimed in claim 8 are inoculated into a fermentation medium for cultivation to obtain the recombinant human type I collagen truncated protein JYC101.

14. The preparation method according to claim 13, characterized in that: The steps are as follows: inoculating Pichia pastoris engineered bacteria into BMMY induction medium for fermentation culture, purifying the fermentation supernatant, and obtaining high-purity recombinant human type I collagen truncated body JYC101.

15. A composition or product, characterized in that It comprises the recombinant human type I collagen truncated body JYC101 as claimed in claim 1, the composition is a skin care product or a medicine, and the product is a medical device, a biomaterial, or a tissue engineering product.

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