Method for promoting expression of recombinant type III collagen, recombinant Pichia pastoris and application thereof

Abstract

The present invention relates to the field of genetic engineering technology, and specifically discloses a method for promoting the expression of recombinant type III collagen, a recombinant Pichia pastoris, and an application thereof. A plasmid comprising a signal peptide having a nucleotide sequence such as SEQ ID NO.3 is transferred into Pichia pastoris GS115 to obtain a signal peptide-modified Pichia pastoris. The recombinant plasmid obtained by sequentially connecting the eIF-5A gene, the DYS1 gene, and the LIA1 gene to the vector is transferred into the signal peptide-modified Pichia pastoris to obtain a recombinant Pichia pastoris. The recombinant Pichia pastoris is activated and cultured to induce expression of the target protein. The method provided by the present invention can significantly increase the expression amount of recombinant type III collagen in Pichia pastoris GS115, and the expression level of recombinant type III collagen is increased by more than 287.92%.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for promoting the expression of recombinant type III collagen, recombinant Pichia pastoris and applications. Background Art

[0002] Collagen is the most abundant structural protein in animals, present extensively in tissues such as skin, bones, tendons, and blood vessels, and possesses important biological functions. Type III collagen, a key member of the collagen family, is primarily secreted by fibroblasts and participates in the formation of the extracellular matrix and tissue repair. Due to its excellent biocompatibility and biodegradability, type III collagen holds broad application prospects in medicine, cosmetics, food, and other fields.

[0003] Traditional collagen extraction methods primarily rely on isolation and purification from animal tissues, but this approach has numerous limitations, including limited sources, potential pathogen contamination, and inconsistent quality between batches. With the advancement of molecular biology and genetic engineering, the production of recombinant collagen using recombinant DNA technology has become an effective approach to address these issues. As an efficient exogenous protein expression system, Pichia pastoris is widely used in recombinant protein production due to its advantages, including high expression levels, ease of large-scale fermentation, and ability to undergo post-translational modification.

[0004] However, the production of recombinant type III collagen in Pichia pastoris still faces several technical challenges. For example, collagen is rich in glycine and proline, and its unique amino acid composition can lead to low translation efficiency or even stagnation in Pichia pastoris. The secretion signal peptide sequence of the recombinant protein has a significant impact on protein secretion efficiency. Unoptimized signal peptides can lead to intracellular accumulation of the recombinant protein, affecting production. Therefore, improving the expression efficiency of recombinant collagen remains an urgent issue. Summary of the Invention

[0005] To develop a method for improving the expression efficiency of recombinant collagen, the present invention provides a method for increasing the expression of recombinant type III collagen, a recombinant Pichia pastoris, and applications. The method provided by the present invention significantly increases the expression of recombinant type III collagen in Pichia pastoris GS115, increasing the expression level of recombinant type III collagen by over 287.92%.

[0006] The present invention provides a method for promoting the expression of recombinant type III collagen, comprising the following steps:

[0007] The signal peptide with the nucleotide sequence shown in SEQ ID NO.3 was transferred into Pichia pastoris GS115 to obtain a signal peptide-modified Pichia pastoris;

[0008] The eIF-5A gene with a nucleotide sequence as shown in SEQ ID NO. 4, the DYS1 gene with a nucleotide sequence as shown in SEQ ID NO. 6, and the LIA1 gene with a nucleotide sequence as shown in SEQ ID NO. 8 are sequentially linked via a 2A peptide with a nucleotide sequence as shown in SEQ ID NO. 10 to obtain a recombinant gene fragment;

[0009] Then the recombinant gene fragment is transferred into Pichia pastoris with modified signal peptide to obtain recombinant Pichia pastoris;

[0010] The recombinant Pichia pastoris is activated and cultured to induce the expression of the target protein.

[0011] The present invention transfers the signal peptide shown in SEQ ID NO. 3 into Pichia pastoris GS115 to obtain signal peptide-modified Pichia pastoris, and then transfers recombinant fragments of the eIF-5A gene, the DYS1 gene, and the LIA1 gene into the signal peptide-modified Pichia pastoris to obtain recombinant Pichia pastoris. Compared with the initial yeast cell GS115, the recombinant Pichia pastoris increases the expression level of recombinant type III collagen by 120.46%.

[0012] Furthermore, the process of culturing the recombinant Pichia pastoris after activation and inducing the expression of the target protein is as follows: the recombinant Pichia pastoris after activation is inoculated into BMGY medium, and shaken and cultured until the OD 600 The initial OD value was 4 to 6, and the cells were transferred to BMMY liquid medium containing maltose to control the initial OD value. 600 The concentration of the saturated fatty acid was 0.9 to 1, and shaking culture was continued. Methanol was added every 22 to 24 hours to induce the expression of the target protein.

[0013] The present invention also provides a signal peptide-modified Pichia pastoris, which is obtained by transferring the signal peptide shown in SEQ ID NO. 3 into Pichia pastoris GS115.

[0014] Furthermore, the process of transferring the signal peptide into Pichia pastoris GS115 is as follows:

[0015] The pre sequence of the α signal peptide on the secretory expression vector was replaced with the pre sequence of the signal peptide sequence of the OST1 gene of Saccharomyces cerevisiae S288C, and the pro sequence of the α signal peptide remained unchanged to obtain a recombinant expression vector. After the recombinant expression vector was linearized with Sac I enzyme, it was transformed into competent Pichia pastoris GS115 by electroporation to obtain Pichia pastoris with modified signal peptide.

[0016] Furthermore, the secretory expression vector is any one of pPIC9, pPIC9K, pHIL-S1, pPICZαA, pPICZαB and pPICZαC.

[0017] The present invention also provides a recombinant Pichia pastoris, which is obtained by sequentially connecting the eIF-5A gene with a nucleotide sequence as shown in SEQ ID NO.4, the DYS1 gene with a nucleotide sequence as shown in SEQ ID NO.6, and the LIA1 gene with a nucleotide sequence as shown in SEQ ID NO.8, and then transferring the resulting nucleotides into the signal peptide-modified Pichia pastoris.

[0018] The present invention also provides a method for constructing the recombinant Pichia pastoris, comprising the following steps:

[0019] The eIF-5A gene with a nucleotide sequence as shown in SEQ ID NO.4, the DYS1 gene with a nucleotide sequence as shown in SEQ ID NO.6, and the LIA1 gene with a nucleotide sequence as shown in SEQ ID NO.8 were sequentially linked via the 2A peptide shown in SEQ ID NO.10 to obtain an eIF-DYS1-LIA1 fragment, and then the eIF-DYS1-LIA1 fragment was ligated into a pPICZB vector to obtain a pPICZB-eIF-DYS1-LIA1 plasmid;

[0020] The pPICZB-eIF-DYS1-LIA1 plasmid was linearized with Sal I enzyme and then transformed into Pichia pastoris with modified signal peptide by electroporation to obtain recombinant Pichia pastoris.

[0021] The present invention also provides a use of the signal peptide-modified Pichia pastoris or the recombinant Pichia pastoris in producing recombinant type III collagen.

[0022] Furthermore, the steps of producing recombinant type III collagen using the signal peptide modified Pichia pastoris are as follows: inoculating the signal peptide modified Pichia pastoris into BMGY medium, shaking and culturing until OD 600 When the OD value is 4 to 6, transfer to BMMY liquid medium and control the initial OD value. 600 The pH value was 0.9 to 1, and the shaking culture was continued. Methanol was added every 22 to 24 hours to induce the expression of the target protein.

[0023] The steps of producing recombinant type III collagen using recombinant Pichia pastoris are as follows: inoculate the recombinant Pichia pastoris into BMGY medium and culture with shaking until OD 600 When the OD value is 4 to 6, transfer to BMMY liquid medium and control the initial OD value. 600 The concentration of the saturated fatty acid was 0.9 to 1, and shaking culture was continued. Methanol was added every 22 to 24 hours to induce the expression of the target protein.

[0024] Furthermore, in the process of producing recombinant type III collagen using recombinant Pichia pastoris, maltose is added to the BMMY liquid culture medium at a mass volume ratio of maltose to BMMY culture medium of 0-2%.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention modifies the signal peptide required for expressing recombinant collagen by replacing the pre sequence of the α signal peptide with the pre sequence of the signal peptide sequence of the OST1 gene of Saccharomyces cerevisiae S288C. This can change the recombinant collagen from a post-translational transport mode to a co-translational transport method. The expression level of the target protein in Pichia pastoris with the modified signal peptide is increased by 19.42%.

[0027] 2. The present invention obtains recombinant Pichia pastoris by modifying signal peptide-modified Pichia pastoris chassis cells. Specifically, the translation elongation factor eIF-5A gene from Pichia pastoris is introduced into the signal peptide-modified Pichia pastoris chassis cells. Simultaneously, the DYS1 gene from Pichia pastoris and the LIA1 gene from Saccharomyces cerevisiae are introduced to post-translationally modify eIF-5A to enhance its functional activity. The modified eIF-5A plays a key role in the translation elongation process, particularly in translating collagen sequences rich in glycine and proline. Therefore, the modification of this chassis cell significantly improves the expression level of recombinant type III collagen. Compared with Pichia pastoris modified with the signal peptide, the expression level of recombinant type III collagen in the recombinant Pichia pastoris was increased by 84.62%. Compared with the yeast cells in the initial state GS115, the expression level of recombinant type III collagen was increased by 120.46%.

[0028] 3. By optimizing fermentation conditions, the present invention discovered that the addition of maltose can increase the expression of recombinant type III collagen in recombinant Pichia pastoris. Specifically, when 1% maltose was added, the expression level of recombinant type III collagen increased by 75.95% compared to recombinant Pichia pastoris without maltose. Furthermore, when recombinant Pichia pastoris was induced with 1% maltose, the expression level of recombinant type III collagen increased by 287.92% compared to the original yeast GS115 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The SDS-PAGE images of the fermentation broth of Pichia pastoris before and after transformation and the grayscale analysis of the relative expression of recombinant type III collagen in the present invention are shown;

[0031] In the figure, A is an SDS-PAGE image of the fermentation broth of Pichia pastoris before and after transformation, wherein numbers 1, 2, and 3 represent SDS-PAGE images of the fermentation broth of strain GS115 / pPIC9K-3α1, strain GS115 / pPIC9K-O-3α1, and strain GS115 / pPIC9K-O-3α1 / eDL, respectively;

[0032] B is a grayscale analysis of the relative expression levels of recombinant type III collagen before and after Pichia pastoris transformation, where number 1 represents the relative expression level of recombinant type III collagen of strain GS115 / pPIC9K-3α1; number 2 represents the relative expression level of recombinant type III collagen of strain GS115 / pPIC9K-O-3α1; number 3 represents the relative expression level of recombinant type III collagen of strain GS115 / pPIC9K-O-3α1.

[0033] Figure 2 The SDS-PAGE images of the fermentation broth of the strain GS115 / pPIC9K-O-3α1 / eDL expressing recombinant type III collagen at different maltose concentrations and the grayscale analysis of the relative expression levels;

[0034] In the figure, A is an SDS-PAGE image of the fermentation broth of the strain GS115 / pPIC9K-O-3α1 / eDL expressing recombinant type III collagen at different maltose concentrations; wherein 1 to 5 represent maltose concentrations of 0, 0.5%, 1%, 1.5% and 2% respectively;

[0035] B is a grayscale analysis of the expression level of recombinant type III collagen expressed by strain GS115 / pPIC9K-O-3α1 / eDL under different maltose concentrations; 1 to 5 represent maltose concentrations of 0, 0.5%, 1%, 1.5% and 2%, respectively. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0037] The plasmids pPIC9K and pPICZB used in the examples of the present invention were purchased from the NTCC Type Culture Collection. Pichia pastoris GS115 was obtained from Thermo Fisher Scientific, product number C18100.

[0038] Example 1: Construction of a recombinant type III collagen production strain with modified signal peptide.

[0039] The recombinant type III collagen expression vector modified with a signal peptide was commissioned to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. The nucleotide sequence of the recombinant type III collagen after codon optimization is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0040] (1) The pre sequence of the α signal peptide on the pPIC9K vector was replaced with the pre sequence of the signal peptide sequence of the OST1 gene (NC_001142.9) of Saccharomyces cerevisiae S288C, and the pro sequence of the α signal peptide remained unchanged. The nucleotide sequence of the modified new signal peptide is shown in SEQ ID NO. 3. The underlined portion is the pre sequence of the replaced OST1 gene, and the expression vector is named pPIC9K-O-3α1. The original α signal peptide nucleotide sequence is shown in SEQ ID NO. 4, and the underlined portion is the pre sequence of the α signal peptide nucleotide sequence.

[0041] SEQ ID NO.1:

[0042]

[0043] SEQ ID NO.2:

[0044]

[0045] SEQ ID NO.3:

[0046] ATGAGGCAGGTTTGGTTCTCTTGGATTGTGGGATTGTTCCTATGTTTTTTCAACGTGTCTTCTGCT GCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTTAC.

[0047] SEQ ID NO.4:

[0048] ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCT GCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTTAC.

[0049] (2) The expression vector pPIC9K-O-3α1 was linearized with Sac I enzyme and then transformed into competent Pichia pastoris GS115 by electroporation. The electroporation parameters were 1500V, 200Ω, and 50μF. The transformed Pichia pastoris GS115 was then spread on an MD plate and inverted in a 30°C constant temperature incubator for 72 hours to screen out a single clone strain. PCR verification and gene sequencing revealed that the signal peptide-modified Pichia pastoris was successfully obtained and recorded as the GS115 / pPIC9K-O-3α1 strain. The GS115 / pPIC9K-O-3α1 strain was inoculated into 20 mL of YPD culture medium for activation and cultured at 30°C, 200 rpm, and shaken for 24 hours. It was then inoculated into BMGY liquid culture medium at a 2% (v / v) inoculation rate and cultured at 30°C, 200 rpm, and shaken until the OD 600 5, transferred to fresh BMMY liquid medium, and controlled the initial OD 600The culture medium was shaken at 30°C and 200 rpm, with 1% (v / v) methanol added every 24 hours to induce expression of the target protein (recombinant type III collagen). After 48 hours of shaking, the culture medium was centrifuged at 10,000 rpm for 1 minute, and the supernatant was analyzed by SDS-PAGE. The YPD culture medium formulation is: 2% (w / v) peptone, 1% (w / v) yeast extract, 2% (w / v) glucose, and the balance distilled water.

[0050] By grayscale scanning band analysis, it was found that the expression level of recombinant type III collagen in the GS115 / pPIC9K-O-3α1 strain with signal peptide modification increased by 19.42% compared with the strain Pichia pastoris GS115 without signal peptide modification (e.g. Figure 1 ).

[0051] Example 2: Construction of GS115 / pPIC9K-O-3α1 / eDL strain after Pichia pastoris chassis cell transformation.

[0052] This example introduces the translation elongation factor eIF-5A into Pichia pastoris chassis cells to enhance the expression of recombinant type III collagen, which is rich in glycine and proline. To function, eIF-5A requires modification of the DYS1 and LIA1 enzymes. The specific steps are as follows:

[0053] (1) The translation elongation factor eIF-5A gene from Pichia pastoris, the DYS1 gene from Pichia pastoris, and the LIA1 gene from Saccharomyces cerevisiae were sequentially linked through 2A peptide to obtain the eIF-DYS1-LIA1 fragment. Finally, the eIF-DYS1-LIA1 fragment was ligated to the EcoR I and Not I sites of the pPICZB vector to obtain the pPICZB-eIF-DYS1-LIA1 plasmid. The plasmid was synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0054] The nucleotide sequence of the eIF-5A gene is shown in SEQ ID NO. 5, and its amino acid sequence is shown in SEQ ID NO. 6; the nucleotide sequence of the DYS1 gene is shown in SEQ ID NO. 7, and its amino acid sequence is shown in SEQ ID NO. 8; the nucleotide sequence of the LIA1 gene is shown in SEQ ID NO. 9, and its amino acid sequence is shown in SEQ ID NO. 10. The nucleotide sequence of the 2A peptide is shown in SEQ ID NO. 11.

[0055] SEQ ID NO.5:

[0056] ATGGCTGAAGAAGAACACACCTTTGAGACCGCCGATGCCGGATCCTCTTTGACCTTCCCTATGCAATGTTCCGCTCTTAGAAAGAACGGACACGTTGTCATTAAAGGTAGACCATGTAAGATCGTTGATATGTCTACCTCTAAGACTGGTAAGCACGGTCACGCTAAGGTCCACTTGGTCGCCATCGACATCTTCACTGGTAAGAAGTTGGAAGATCTTTCTCCATCCACCCACAACATGGAGGTTCCTAACGTTAGAAGATCTGAATACCAACTGTTGGACATTGATGATGGGTACTTGAACTTGATGAACAACGATGGTGAGACCAAGGATGACGTCAAGGCTCCAGAGGGAGAGGTTGGTGAGAAATTGCAAAGTGAGTTCGATGAGGGCAAAGACTTGCTGGTCACCATTATTTCTTCCATGGGTGAGGAAGCTGCTATCTCTTTCAAGGAGGCTCCTAAA。

[0057] SEQ ID NO.6:

[0058] MAEEEHTFETADAGSSLTFPMQCSALRKNGHVVIKGRPCKIVDMSTSKTGKHGHAKVHLVAIDIFTGKKLEDLSPSTHNMEVPNVRRSEYQLLDIDDGYLNLMNNDGETKDDVKAPEGEVGEKLQSEFDEGKDLLVTIISSMGEEAAISFKEAPK。

[0059] SEQ ID NO.7:

[0060]

[0061] SEQ ID NO.8:

[0062] MANIEGKLPDILSESVLKSSVPVPEDFVEVKGIDYDKPESRNMKAKDLIKGMRTMGFQASSLSEACEIIDEMRQWRGKHIDDLEEHDRKGEFDGEGYQKSTIFMGYTSNLISSGLRDTLRYLVQNKMISAIVASAGGIEEDLIKCLAPTYMGDFALKGKGLRDQGMNRIGNLLVPNDNYCKFEEWIVP ILDSMLEEQEQNVVSKGVDALDGDAMVWTPSTVIDRLGKEINDETSVLYWAHKNKIPVFCPSLTDGSIGDMLFFHTFKASPKQLRIDLVNDIRR INSMSMEASKAGMLILGGGLIKHHIANACLMRNGADWAVYVNTGQEFDGSDAGARPDEAVSWGKIKAEARSVKVFADVTLVFPLMVAATPHASE.

[0063] SEQ ID NO.9:

[0064] ATGTCTACTAACTTTGAAAAACATTTCCAAGAAAACGTCGATGAATGCACTCTAGAGCAACTAAGGGACATCTTAGTCAACAAGTCCGGCAAAACAGTTTTGGCCAACAGATTTAGAGCTCTGTTCAACTTAAAGACTGTTGCTGAAGAATTTGCCACTAAGCCAGAGGAAGCCAAAAAGGCCATCGAATACATTGCCGAATCCTTCGTCAATGACAAGTCTGAGTTGTTGAAGCACGAAGTGGCCTACGTGTTGGGTCAAACCAAGAACTTGGACGCTGCTCCAACTTTAAGACACGTTATGTTAGATCAAAATCAAGAACCAATGGTGAGACACGAAGCCGCTGAGGCTTTGGGTGCCCTAGGTGACAAGGATTCGTTGGATGACCTAAATAAGGCTGCTAAGGAGGATCCACACGTTGCTGTGAGAGAAACCTGTGAACTGGCCATTAACAGAATCAACTGGACCCATGGAGGTGCCAAGGATAAGGAAAACTTGCAACAATCCCTATACTCGAGTATTGACCCAGCCCCACCTCTACCATTAGAAAAGGATGCTACCATCCCAGAACTACAGGCCTTATTGAATGATCCTAAGCAACCTTTGTTCCAAAGATACAGAGCCATGTTCAGACTGAGAGATATCGGTACTGATGAAGCAATCCTGGCCTTGGCCACTGGTTTCAGTGCAGAATCCTCCCTTTTCAAGCATGAAATCGCCTACGTCTTCGGTCAAATAGGTAGTCCGGCTGCTGTCCCAAGTTTGATTGAAGTTTTGGGCAGAAAGGAAGAAGCTCCAATGGTTAGGCATGAAGCTGCTGAAGCCTTGGGTGCCATTGCTTCTCCAGAAGTTGTCGACGTCTTGAAATCTTACCTCAACGATGAAGTCGATGTCGTCAGAGAATCTTGTATCGTTGCGCTAGATATGTATGATTACGAAAACAGCAACGAACTAGAATATGCTCCAACTGCTAATTAG。

[0065] SEQ ID NO.10:

[0066] MSTNFEKHFQENVDECTLEQLRDILVNKSGKTVLANRFRALFNLKTVAEEFATKPEEAKKAIEYIAESFVNDKSELLKHEVAYVLGQTKNLDAAPTLRHVMLDQNQEPMVRHEAAEALGALGDKDSLDDLNKAAKEDPHVAVRETCELAINRINWTHGGAKDK ENLQQSLYSSIDPAPPLPLEKDATIPELQALLNDPKQPLFQRYRAMFRLRDIGTDEAILALATGFSAESSLFKHEIAYVFGQIGSPAAVPSLIEVLGRKEEAPMVRHEAAEALGAIASPEVVDVLKSYLNDEVDVVRESCIVALDMYDYENSNELEYAPTAN.

[0067] SEQ ID NO.11:

[0068] GGATCCGGTGCCACAAACTTTAGTTTGCTGAAGCAAGCTGGTGATGT TGAAGAAAATCCAGGACCA.

[0069] (2) The pPICZB-eIF-DYS1-LIA1 plasmid was linearized with Sal I enzyme and then transformed into the GS115 / pPIC9K-O-3α1 strain with modified signal peptide by electroporation. The electroporation parameters were 1500V, 200Ω, and 50μF. After electroporation, it was spread on a YPDZ plate and inverted in a constant temperature incubator at 30°C for 72 hours. Monoclonal strains were screened and the GS115 / pPIC9K-O-3α1 / eDL strain that produces recombinant type III collagen after chassis cell transformation was successfully obtained. It is also referred to as recombinant Pichia pastoris in the present invention. The GS115 / pPIC9K-O-3α1 / eDL strain was inoculated into 20mL of YPD culture medium for activation and cultured at 30°C and 200rpm for 24 hours. Then, it was inoculated into fresh BMGY medium at a 2% inoculum amount and cultured at 30°C and 200rpm for OD 600 5, transferred to BMMY medium, control the initial OD 600The culture medium was shaken at 30°C and 200 rpm, with 1% (v / v) methanol added every 24 hours to induce target protein expression. After 48 hours of shaking culture, the culture medium was collected, centrifuged, and the supernatant was analyzed by SDS-PAGE. YPDZ plate formulation: YPD liquid medium + 2% (w / v) agar powder + 0.01% (w / v) Zeocin.

[0070] Grayscale scanning band analysis showed that compared with the strain GS115 / pPIC9K-O-3α1 that had not been transformed with chassis cells but only with signal peptides, the expression level of recombinant type III collagen in the GS115 / pPIC9K-O-3α1 / eDL strain increased by 84.62%; compared with the original strain GS115 / pPIC9K-3α1 that had not been transformed, the expression level of recombinant type III collagen in the GS115 / pPIC9K-O-3α1 / eDL strain increased by 120.46% (as shown in Figure 2). Figure 1 shown).

[0071] Example 3: Optimization of fermentation conditions of recombinant Pichia pastoris strains.

[0072] In this example, methanol and maltose were used in combination. After the GS115 / pPIC9K-O-3α1 / eDL strain was activated, it was inoculated into 20 mL of YPD culture medium for activation and cultured at 30°C and 200 rpm for 24 h. Then, a 2% inoculum was inoculated into fresh BMGY liquid medium and cultured at 30°C and 200 rpm for OD 0. 600 When the OD value is about 5, transfer to BMMY liquid medium containing different maltose concentrations to control the initial OD value. 600 The cells were then shaken at 30°C and 200 rpm, with 1% (v / v) methanol added every 24 hours to induce target protein expression. After shaking for 48 hours, the culture medium was collected and centrifuged at 10,000 rpm for 1 minute. The supernatant was analyzed by SDS-PAGE.

[0073] The different maltose concentrations were 0, 0.5%, 1%, 1.5% and 2% (w / v).

[0074] Grayscale scanning band analysis showed that the expression level of recombinant type III collagen was increased when the maltose concentration was between 0.5% and 2%. Among them, the expression level of recombinant type III collagen was the highest when the maltose concentration was 1%. Compared with the condition without adding maltose, the expression level increased by 75.95%.

[0075] Comparative Example 1: A method for directly inducing Pichia pastoris GS115 to express and produce recombinant type III collagen without modifying the signal peptide.

[0076] The pPIC9K-3α1 vector was directly transformed into Pichia pastoris GS115 to obtain the transformed Pichia pastoris, which was recorded as strain GS115 / pPIC9K-3α1. The electroporation parameters were 1500V, 200Ω, and 50μF. The strain GS115 / pPIC9K-3α1 was then spread on an MD plate and inverted in a 30°C constant temperature incubator for 72 hours to screen out a monoclonal strain. The strain GS115 / pPIC9K-3α1 was inoculated into 20mL of YPD culture medium for activation and cultured at 30°C, 200rpm, and shaking for 24 hours. It was then inoculated into BMGY liquid culture medium at a 2% inoculation rate and cultured at 30°C, 200rpm, and shaking until the OD 600 When the OD value is about 5, transfer to fresh BMMY liquid medium and control the initial OD value. 600 The expression level of the target protein was 0.82 g / L after 48 h of shaking culture and centrifugation of the culture medium.

[0077] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0078] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A method for promoting the expression of recombinant type III collagen, characterized in that: The steps include: The signal peptide encoding nucleotide sequence shown in SEQ ID NO.3 is transferred into Pichia pastoris GS115 to obtain a signal peptide-modified Pichia pastoris; the signal peptide encoding nucleotide sequence is connected to the type III collagen encoding nucleotide sequence shown in SEQ ID NO.1; The eIF-5A gene with a nucleotide sequence as shown in SEQ ID NO.4, the DYS1 gene with a nucleotide sequence as shown in SEQ ID NO.6, and the LIA1 gene with a nucleotide sequence as shown in SEQ ID NO.8 are sequentially connected via nucleotides encoding a 2A peptide with an amino acid sequence as shown in SEQ ID NO.10 to obtain a recombinant gene fragment; Then the recombinant gene fragment is transferred into Pichia pastoris with modified signal peptide to obtain recombinant Pichia pastoris; The recombinant Pichia pastoris is activated and cultured to induce the expression of the target protein.

2. The method for promoting the expression of recombinant type III collagen according to claim 1, characterized in that: The process of culturing and inducing the expression of the target protein after the recombinant Pichia pastoris is as follows: the recombinant Pichia pastoris is activated and inoculated into BMGY medium, and shaken and cultured until the OD 600 The initial OD value was 4 to 6, and the cells were transferred to BMMY liquid medium containing maltose to control the initial OD value. 600 The concentration of the saturated fatty acid was 0.9 to 1, and shaking culture was continued. Methanol was added every 22 to 24 hours to induce the expression of the target protein.

3. A signal peptide modified Pichia pastoris, characterized in that: Obtained by the following steps: The signal peptide encoding nucleotide sequence shown in SEQ ID NO.3 was transferred into Pichia pastoris GS115 to obtain signal peptide-modified Pichia pastoris; the signal peptide encoding nucleotide sequence was connected to the type III collagen encoding nucleotide sequence shown in SEQ ID NO.

1.

4. The signal peptide modified Pichia pastoris according to claim 3, characterized in that The process of transferring the signal peptide into Pichia pastoris GS115 is as follows: The pre sequence of the α signal peptide of the secretory expression vector is replaced with the pre sequence of the signal peptide sequence of the OST1 gene of Saccharomyces cerevisiae S288C, and the pro sequence of the α signal peptide remains unchanged to obtain a recombinant expression vector. Sac After the I enzyme was linearized, it was transformed into competent Pichia pastoris GS115 by electroporation to obtain Pichia pastoris with modified signal peptide.

5. The signal peptide modified Pichia pastoris according to claim 4, characterized in that The secretory expression vector is any one of pPIC9, pPIC9K, pHIL-S1, pPICZαA, pPICZαB and pPICZαC.

6. A recombinant Pichia pastoris, characterized in that The eIF-5A gene, the DYS1 gene, and the LIA1 gene are sequentially connected and then transferred into the signal peptide modified Pichia pastoris according to claim 3.

7. A method for constructing the recombinant Pichia pastoris according to claim 6, characterized in that: The steps include: The eIF-5A gene with a nucleotide sequence as shown in SEQ ID NO.4, the DYS1 gene with a nucleotide sequence as shown in SEQ ID NO.6, and the LIA1 gene with a nucleotide sequence as shown in SEQ ID NO.8 were sequentially linked via the nucleotide encoding the 2A peptide as shown in SEQ ID NO.10 to obtain an eIF-DYS1-LIA1 fragment, and then the eIF-DYS1-LIA1 fragment was ligated into a pPICZB vector to obtain a pPICZB-eIF-DYS1-LIA1 plasmid; pPICZB-eIF-DYS1-LIA1 plasmid Sal After the I enzyme was linearized, the signal peptide-modified Pichia pastoris was transformed by electroporation to obtain recombinant Pichia pastoris.

8. Use of the signal peptide-modified Pichia pastoris according to any one of claims 3 to 5 or the recombinant Pichia pastoris according to claim 6 in the production of recombinant type III collagen.

9. The use according to claim 8, characterized in that The steps for producing recombinant type III collagen using signal peptide modified Pichia pastoris are as follows: inoculate the signal peptide modified Pichia pastoris into BMGY medium and culture with shaking until OD 600 When the OD value is 4 to 6, transfer to BMMY liquid medium and control the initial OD value. 600 The pH value was 0.9 to 1, and the shaking culture was continued. Methanol was added every 22 to 24 hours to induce the expression of the target protein. The steps of producing recombinant type III collagen using recombinant Pichia pastoris are as follows: inoculate the recombinant Pichia pastoris into BMGY medium and culture with shaking until OD 600 When the OD value is 4 to 6, transfer to BMMY liquid medium and control the initial OD value. 600 The concentration of the saturated fatty acid was 0.9 to 1, and shaking culture was continued. Methanol was added every 22 to 24 hours to induce the expression of the target protein.

10. The use according to claim 9, characterized in that In the process of producing recombinant type III collagen using recombinant Pichia pastoris, maltose is added to the BMMY liquid culture medium at a mass volume ratio of maltose to BMMY culture medium of 0.5% to 2%.

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