Method for promoting expression of recombinant III-type collagen, recombinant pichia pastoris and application

Abstract

The invention relates to the technical field of gene engineering, and particularly discloses a method for promoting expression of recombinant III-type collagen, recombinant pichia pastoris and application of the recombinant pichia pastoris. Plasmids containing signal peptide with the nucleotide sequence as shown in SEQ ID NO.3 are transferred into pichia pastoris GS115 to obtain pichia pastoris modified by the signal peptide, and the recombinant III-type collagen is obtained. The method comprises the following steps: sequentially connecting an eIF-5A gene, a DYS1 gene and an LIA1 gene to a vector to obtain a recombinant plasmid, transferring the recombinant plasmid into pichia pastoris modified by signal peptide to obtain recombinant pichia pastoris, activating the recombinant pichia pastoris, culturing, and inducing target protein expression. According to the method provided by the invention, the expression quantity of the recombinant III-type collagen of the pichia pastoris GS115 can be remarkably improved, and the expression level of the recombinant III-type collagen is improved by 287.92% or above.

Description

Technical Field

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

[0002] Collagen is the most abundant structural protein in animals and is widely present in tissues such as skin, bone, tendon, and blood vessels, and has important biological functions. Type III collagen is one of the important members of the collagen family, which is mainly 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 has broad application prospects in the fields of medicine, cosmetics, food, etc.

[0003] Traditional methods for extracting collagen mainly involve isolation and purification from animal tissues, but this method has many limitations, such as limited sources, potential risks of pathogen contamination, and unstable quality between batches. With the development of molecular biology and genetic engineering technologies, using recombinant DNA technology to produce recombinant collagen has become an effective way to solve the above problems. Pichia pastoris, as an efficient heterologous protein expression system, has been widely used in the production of recombinant proteins due to its advantages such as high expression level, easy large-scale fermentation, and post-translational modification ability.

[0004] However, in the process of using Pichia pastoris to produce recombinant type III collagen, some technical challenges still remain. For example, collagen is rich in glycine and proline, and its special amino acid composition may lead to low translation efficiency or even stalling in Pichia pastoris. The secretion signal peptide sequence of recombinant proteins has an important impact on the secretion efficiency of proteins, and unoptimized signal peptides may lead to the accumulation of recombinant proteins in cells, affecting the yield. Therefore, how to improve the expression efficiency of recombinant collagen remains an urgent problem to be solved. 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 level of recombinant type III collagen, recombinant Pichia pastoris, and applications thereof. The method provided by the present invention can significantly increase the expression level 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%.

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

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

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

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

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

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

[0012] Furthermore, the process of culturing and inducing the expression of the target protein after activating the recombinant Pichia pastoris is as follows: The recombinant Pichia pastoris is activated and then inoculated into BMGY medium, and cultured with shaking until the OD 600 reaches 4-6, then transferred to BMMY liquid medium containing maltose, and the initial OD 600 is controlled to be 0.9-1, and continue to culture with shaking, and add methanol every 22h-24h to induce the expression of the target protein.

[0013] The present invention also provides a Pichia pastoris with a modified signal peptide, and the Pichia pastoris with a modified signal peptide 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 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. After linearizing the recombinant expression vector with Sac I enzyme, it is transferred into competent Pichia pastoris GS115 by electroporation to obtain Pichia pastoris with a 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 successively ligating the eIF-5A gene with the nucleotide sequence shown in SEQ ID NO.4, the DYS1 gene with the nucleotide sequence shown in SEQ ID NO.6, and the LIA1 gene with the nucleotide sequence shown in SEQ ID NO.8, and then transferring them into the Pichia pastoris with the signal peptide modified as described above.

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

[0019] The eIF-5A gene with the nucleotide sequence shown in SEQ ID NO.4, the DYS1 gene with the nucleotide sequence shown in SEQ ID NO.6, and the LIA1 gene with the nucleotide sequence shown in SEQ ID NO.8 are successively ligated through the 2A peptide shown in SEQ ID NO.10 to obtain the eIF-DYS1-LIA1 fragment, and then the eIF-DYS1-LIA1 fragment is ligated into the pPICZB vector to obtain the pPICZB-eIF-DYS1-LIA1 plasmid;

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

[0021] The present invention also provides an application of the Pichia pastoris with the signal peptide modified as described above or the recombinant Pichia pastoris in the production of recombinant type III collagen.

[0022] Further, the steps for producing recombinant type III collagen using the Pichia pastoris with the signal peptide modified are as follows: Inoculate the Pichia pastoris with the signal peptide modified into the BMGY medium and shake culture until the OD 600 is 4 - 6, then transfer it to the BMMY liquid medium, control the initial OD 600 to be 0.9 - 1, continue to shake culture, and add methanol every 22h - 24h to induce the expression of the target protein;

[0023] The steps for producing recombinant type III collagen using the recombinant Pichia pastoris are as follows: Inoculate the recombinant Pichia pastoris into the BMGY medium and shake culture until the OD 600 is 4 - 6, then transfer it to the BMMY liquid medium, control the initial OD 600 to be 0.9 - 1, continue to shake culture, and add methanol every 22h - 24h to induce the expression of the target protein.

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

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. By modifying the signal peptide required for expressing recombinant collagen, the pre-sequence of the α signal peptide is replaced with the pre-sequence of the signal peptide sequence of the OST1 gene of Saccharomyces cerevisiae S288C, the method of post-translational translocation of recombinant collagen can be changed to co-translational translocation, and the expression level of the target protein of the Pichia pastoris with modified signal peptide is increased by 19.42%.

[0027] 2. The recombinant Pichia pastoris is obtained by modifying the chassis cell of the Pichia pastoris with modified signal peptide. Specifically, the translation elongation factor eIF-5A gene derived from Pichia pastoris is introduced into the chassis cell of the Pichia pastoris with modified signal peptide, and at the same time, the DYS1 gene derived from Pichia pastoris and the LIA1 gene derived from Saccharomyces cerevisiae are introduced to perform post-translational modification on eIF-5A to exert its functional activity. The modified eIF-5A plays a key role in the translation elongation process, especially in translating the collagen sequence rich in glycine and proline. Therefore, the modification of this chassis cell can significantly improve the expression level of recombinant type III collagen. Compared with the Pichia pastoris with modified signal peptide, the expression level of recombinant type III collagen in the recombinant Pichia pastoris is increased by 84.62%. Compared with the initial yeast cell GS115, the expression level of recombinant type III collagen is increased by 120.46%.

[0028] 3. By optimizing the fermentation conditions, it is found that the addition of maltose can increase the expression level of recombinant type III collagen in recombinant Pichia pastoris. Especially when 1% maltose is added, compared with the recombinant Pichia pastoris without adding maltose, the expression level of recombinant type III collagen is increased by 75.95%. Compared with the original yeast cell GS115, the expression level of recombinant type III collagen in the recombinant Pichia pastoris induced by adding 1% maltose is increased by 287.92%. Brief Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 It is the SDS-PAGE diagram of the fermentation broth before and after the modification of Pichia pastoris in the present invention and the gray scale analysis diagram of the relative expression level of recombinant type III collagen

[0031] In the figure, A is the SDS-PAGE diagram of the fermentation broth of Pichia pastoris before and after modification. Among them, numbers 1, 2, and 3 represent the SDS-PAGE diagrams of the fermentation broths of strains GS115 / pPIC9K-3α1, GS115 / pPIC9K-O-3α1, and GS115 / pPIC9K-O-3α1 / eDL in sequence;

[0032] B is the gray-scale analysis diagram of the relative expression level of recombinant type III collagen of Pichia pastoris before and after modification. Among them, number 1 represents the relative expression level of recombinant type III collagen of strain GS115 / pPIC9K-3α1; 2 represents the relative expression level of recombinant type III collagen of strain GS115 / pPIC9K-O-3α1; 3 represents the relative expression level of recombinant type III collagen of strain GS115 / pPIC9K-O-3α1.

[0033] Figure 2 It is the SDS-PAGE diagram and the gray-scale analysis diagram of the relative expression level of the fermentation broth of strain GS115 / pPIC9K-O-3α1 / eDL expressing recombinant type III collagen under different maltose concentrations;

[0034] In the figure, A is the SDS-PAGE diagram of the fermentation broth of strain GS115 / pPIC9K-O-3α1 / eDL expressing recombinant type III collagen under different maltose concentrations; among them, 1 to 5 represent maltose concentrations of 0, 0.5%, 1%, 1.5%, and 2% in sequence;

[0035] B is the gray-scale analysis diagram of the expression level of recombinant type III collagen of strain GS115 / pPIC9K-O-3α1 / eDL under different maltose concentrations; among them, 1 to 5 represent maltose concentrations of 0, 0.5%, 1%, 1.5%, and 2% in sequence. Specific Embodiments

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

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

[0038] Example 1: Construction of a recombinant type III collagen-producing strain with signal peptide modification.

[0039] The recombinant type III collagen expression vector with signal peptide modification was commissioned to be synthesized by Shanghai Sangon Biotech Co., Ltd. After codon optimization, the nucleotide sequence of recombinant type III collagen is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2.

[0040] (1) Replace the pre sequence of the α signal peptide on the pPIC9K vector 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 remains unchanged. The nucleotide sequence of the new signal peptide after modification is shown as SEQ ID NO.3. The underlined part is the pre sequence of the replaced OST1 gene. This expression vector is named pPIC9K-O-3α1. The nucleotide sequence of the original α signal peptide is shown as SEQ ID NO.4, and the underlined part 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) After linearizing the expression vector pPIC9K-O-3α1 with Sac I enzyme, it was transformed into competent Pichia pastoris GS115 by electroporation. The electroporation parameters were 1500V, 200Ω, and 50μF. Then the transformed Pichia pastoris GS115 was spread on an MD plate and incubated upside down in a constant temperature incubator at 30°C for 72h to screen for monoclonal strains. Through PCR verification and gene sequencing, it was found that Pichia pastoris with signal peptide modification was successfully obtained, denoted as GS115 / pPIC9K-O-3α1 strain. The GS115 / pPIC9K-O-3α1 strain was inoculated into 20 mL of YPD culture medium for activation and incubated with shaking at 30°C and 200 rpm for 24h. Then it was inoculated into BMGY liquid medium at an inoculation amount of 2% (v / v) and incubated with shaking at 30°C and 200 rpm until OD 600 reached 5, and then transferred to fresh BMMY liquid medium, controlling the initial OD 600It was 1. The culture was shaken at 30 °C and 200 rpm, and 1% (v / v) methanol was added every 24 h to induce the expression of the target protein (recombinant type III collagen). After shaking culture for 48 h, 10000 rpm was centrifuged for 1 min, and the supernatant was taken for SDS-PAGE analysis. The formula of YPD culture medium was: 2% (w / v) peptone, 1% (w / v) yeast extract, 2% (w / v) glucose, and the balance was distilled water.

[0050] Analysis was carried out by gray-scale scanning of the bands. It was found that compared with the strain Pichia pastoris GS115 without signal peptide modification, the expression level of recombinant type III collagen in the GS115 / pPIC9K-O-3α1 strain with signal peptide modification increased by 19.42% (as Figure 1 ).

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

[0052] In this example, the translation elongation factor eIF-5A was introduced into the Pichia pastoris chassis cell to enhance the expression level of recombinant type III collagen rich in glycine and proline in Pichia pastoris cells. For eIF-5A to function, it requires modification by DYS1 enzyme and LIA1 enzyme. 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 successively ligated through the 2A peptide to obtain the eIF-DYS1-LIA1 fragment. Finally, the eIF-DYS1-LIA1 fragment was ligated at the EcoR I and Not I sites of the vector pPICZB vector and named the pPICZB-eIF-DYS1-LIA1 plasmid. This plasmid was commissioned to be synthesized by Shanghai Sangon Biological Engineering.

[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] MANIEGKLPDILSESVLKSSVPVPEDFVEVKGIDYDKPESRNMKAKDLIKGMRTMGFQASSLSEACEIIDEMRQWRGKHIDDLEEHDRKGEFDGEGYQKSTIFMGYTSNLISSGLRDTLRYLVQNKMISAIVASAGGIEEDLIKCLAPTYMGDFALKGKGLRDQGMNRIGNLLVPNDNYCKFEEWIVPILDSMLEEQEQNVVSKGVDALDGDAMVWTPSTVIDRLGKEINDETSVLYWAHKNKIPVFCPSLTDGSIGDMLFFHTFKASPKQLRIDLVNDIRRINSMSMEASKAGMLILGGGLIKHHIANACLMRNGADWAVYVNTGQEFDGSDAGARPDEAVSWGKIKAEARSVKVFADVTLVFPLMVAATFASE。

[0063] SEQ ID NO.9:

[0064] ATGTCTACTAACTTTGAAAAACATTTCCAAGAAAACGTCGATGAATGCACTCTAGAGCAACTAAGGGACATCTTAGTCAACAAGTCCGGCAAAACAGTTTTGGCCAACAGATTTAGAGCTCTGTTCAACTTAAAGACTGTTGCTGAAGAATTTGCCACTAAGCCAGAGGAAGCCAAAAAGGCCATCGAATACATTGCCGAATCCTTCGTCAATGACAAGTCTGAGTTGTTGAAGCACGAAGTGGCCTACGTGTTGGGTCAAACCAAGAACTTGGACGCTGCTCCAACTTTAAGACACGTTATGTTAGATCAAAATCAAGAACCAATGGTGAGACACGAAGCCGCTGAGGCTTTGGGTGCCCTAGGTGACAAGGATTCGTTGGATGACCTAAATAAGGCTGCTAAGGAGGATCCACACGTTGCTGTGAGAGAAACCTGTGAACTGGCCATTAACAGAATCAACTGGACCCATGGAGGTGCCAAGGATAAGGAAAACTTGCAACAATCCCTATACTCGAGTATTGACCCAGCCCCACCTCTACCATTAGAAAAGGATGCTACCATCCCAGAACTACAGGCCTTATTGAATGATCCTAAGCAACCTTTGTTCCAAAGATACAGAGCCATGTTCAGACTGAGAGATATCGGTACTGATGAAGCAATCCTGGCCTTGGCCACTGGTTTCAGTGCAGAATCCTCCCTTTTCAAGCATGAAATCGCCTACGTCTTCGGTCAAATAGGTAGTCCGGCTGCTGTCCCAAGTTTGATTGAAGTTTTGGGCAGAAAGGAAGAAGCTCCAATGGTTAGGCATGAAGCTGCTGAAGCCTTGGGTGCCATTGCTTCTCCAGAAGTTGTCGACGTCTTGAAATCTTACCTCAACGATGAAGTCGATGTCGTCAGAGAATCTTGTATCGTTGCGCTAGATATGTATGATTACGAAAACAGCAACGAACTAGAATATGCTCCAACTGCTAATTAG。

[0065] SEQ ID NO.10:

[0066] MSTNFEKHFQENVDECTLEQLRDILVNKSGKTVLANRFRALFNLKTVAEEFATKPEEAKKAIEYIAESFVNDKSELLKHEVAYVLGQTKNLDAAPTLRHVMLDQNQEPMVRHEAAEALGALGDKDSLDDLNKAAKEDPHVAVRETCELAINRINWTHGGAKDKENLQQSLYSSIDPAPPLPLEKDATIPELQALLNDPKQPLFQRYRAMFRLRDIGTDEAILALATGFSAESSLFKHEIAYVFGQIGSPAAVPSLIEVLGRKEEAPMVRHEAAEALGAIASPEVVDVLKSYLNDEVDVVRESCIVALDMYDYENSNELEYAPTAN。

[0067] SEQ ID NO.11:

[0068] GGATCCGGTGCCACAAACTTTAGTTTGCTGAAGCAAGCTGGTGATGT TGAAGAAAATCCAGGACCA。

[0069] (2) After linearizing the pPICZB-eIF-DYS1-LIA1 plasmid with Sal I enzyme, it was transformed into the signal peptide-modified GS115 / pPIC9K-O-3α1 strain by electroporation. The electroporation parameters were 1500V, 200Ω, 50μF. After electroporation, it was spread on the YPDZ plate and incubated upside down in a constant temperature incubator at 30°C for 72h. Monoclonal strains were screened out, and the GS115 / pPIC9K-O-3α1 / eDL strain that produced recombinant type III collagen after chassis cell modification was successfully obtained. In this invention, it is also simply referred to as recombinant Pichia pastoris. The GS115 / pPIC9K-O-3α1 / eDL strain was inoculated into 20 mL of YPD culture medium for activation and cultured with shaking at 30°C and 200 rpm for 24h. Then, it was inoculated into fresh BMGY medium at an inoculation amount of 2% and cultured with shaking at 30°C and 200 rpm until OD 600 reached 5, and then transferred to BMMY medium, controlling the initial OD 600It was 1, and it was cultured by shaking at 30 °C and 200 rpm. Methanol at 1% (v / v) was added every 24 h to induce the expression of the target protein. After culturing by shaking for 48 h, the culture broth was taken, centrifuged, and the supernatant was taken for SDS-PAGE analysis. YPDZ plate formula: YPD liquid medium + 2% (w / v) agar powder + 0.01% (w / v) Zeocin.

[0070] Analysis was carried out by gray-scale scanning of the bands, and it was found that compared with the strain GS115 / pPIC9K-O-3α1 that was not modified with the chassis cell and only modified with the signal peptide, the expression level of recombinant type III collagen in the strain GS115 / pPIC9K-O-3α1 / eDL increased by 84.62%; compared with the original strain GS115 / pPIC9K-3α1 without modification, the expression level of recombinant type III collagen in the strain GS115 / pPIC9K-O-3α1 / eDL increased by 120.46% (as Figure 1 shown).

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

[0072] In this example, methanol and maltose were selected to be used in combination. After the strain GS115 / pPIC9K-O-3α1 / eDL was activated, it was inoculated into 20 mL of YPD culture broth for activation and cultured by shaking at 30 °C and 200 rpm for 24 h. Then it was inoculated into fresh BMGY liquid medium at an inoculation amount of 2%, and cultured by shaking at 30 °C and 200 rpm until the OD 600 was about 5, and transferred to BMMY liquid medium containing different maltose concentrations, and the initial OD 600 was 1. Then it was cultured by shaking at 30 °C and 200 rpm, and 1% (v / v) methanol was added every 24 h to induce the expression of the target protein. After culturing by shaking for 48 h, the culture broth was taken, centrifuged at 10000 rpm for 1 min, and the supernatant was taken for SDS-PAGE analysis.

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

[0074] Analysis was carried out by gray-scale scanning of the bands, and it was found that when the maltose concentration was between 0.5% and 2%, the expression level of recombinant type III collagen could be increased. Among them, when the added maltose concentration was 1%, the expression level of recombinant type III collagen was the highest, and compared with without adding maltose, the expression level increased by 75.95%.

[0075] Comparative Example 1: A method for directly inducing the expression of recombinant type III collagen in Pichia pastoris GS115 without modifying the signal peptide.

[0076] The pPIC9K-3α1 vector was directly transformed into Pichia pastoris GS115 to obtain the transformed Pichia pastoris, denoted as strain GS115 / pPIC9K-3α1. The electrotransformation parameters were 1500V, 200Ω, and 50μF. Then, strain GS115 / pPIC9K-3α1 was spread on an MD plate and incubated upside down in a constant temperature incubator at 30°C for 72h to screen for monoclonal strains. Strain GS115 / pPIC9K-3α1 was inoculated into 20 mL of YPD culture medium for activation and cultured with shaking at 30°C and 200 rpm for 24h. Then, it was inoculated into BMGY liquid medium at an inoculation amount of 2% and cultured with shaking at 30°C and 200 rpm until the OD 600 was about 5, and then transferred to fresh BMMY liquid medium, controlling the initial OD 600 to be 1, and cultured with shaking at 30°C and 200 rpm. 1% methanol was added every 24h to induce the expression of the target protein. After culturing with shaking for 48h, the culture solution was centrifuged, and it was found that the expression level of strain GS115 / pPIC9K-3α1 was 0.82 g / L.

[0077] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concept.

[0078] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and deformations.

Claims

1. A method for promoting the expression of recombinant type III collagen, characterized in that, It includes the following steps: Transfer the signal peptide with the nucleotide sequence shown in SEQ ID NO.3 into Pichia pastoris GS115 to obtain Pichia pastoris with modified signal peptide; Sequentially ligate the eIF-5A gene with the nucleotide sequence shown in SEQ ID NO.4, the DYS1 gene with the nucleotide sequence shown in SEQ ID NO.6, and the LIA1 gene with the nucleotide sequence shown in SEQ ID NO.8 through the 2A peptide with the nucleotide sequence shown in SEQ ID NO.10 to obtain a recombinant gene fragment; Then transfer the recombinant gene fragment into Pichia pastoris with modified signal peptide to obtain recombinant Pichia pastoris; Activate the recombinant Pichia pastoris and then culture it to induce the expression of the target protein.

2. The method for promoting the expression of recombinant type III collagen according to claim 1, wherein The process of culturing and inducing the expression of the target protein after activating recombinant Pichia pastoris is as follows: After activating recombinant Pichia pastoris, inoculate it into BMGY medium and shake-culture until the OD 600 reaches 4 - 6. Then transfer it to BMMY liquid medium containing maltose, and control the initial OD 600 to be 0.9 - 1. Continue to shake-culture and add methanol every 22 h - 24 h to induce the expression of the target protein.

3. A signal peptide-modified Pichia pastoris, characterized in that, The Pichia pastoris with modified signal peptide is obtained by transferring the signal peptide shown in SEQID NO.3 into Pichia pastoris GS115 in Claim 1.

4. The signal peptide-modified Pichia pastoris according to claim 3, wherein The process of transferring the signal peptide into Pichia pastoris GS115 is as follows: Replace the pre sequence of the α signal peptide on the secretory expression vector with the pre sequence of the signal peptide of the OST1 gene of Saccharomyces cerevisiae S288C, and keep the pro sequence of the α signal peptide unchanged to obtain a recombinant expression vector. After linearizing the recombinant expression vector with Sac I enzyme, it is transferred 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, wherein The secretory expression vector is any one of pPIC9, pPIC9K, pHIL-S1, pPICZαA, pPICZαB, and pPICZαC.

6. A recombinant Pichia pastoris, characterized in that, It is obtained by sequentially ligating the eIF-5A gene with the nucleotide sequence shown in SEQ ID NO.4, the DYS1 gene with the nucleotide sequence shown in SEQ ID NO.6, and the LIA1 gene with the nucleotide sequence shown in SEQ ID NO.8 and then transferring them into Pichia pastoris with modified signal peptide described in Claim 3.

7. The construction method of the recombinant Pichia pastoris as described in claim 6, characterized in that, It includes the following steps: Sequentially ligate the eIF-5A gene with the nucleotide sequence shown in SEQ ID NO.4, the DYS1 gene with the nucleotide sequence shown in SEQ ID NO.6, and the LIA1 gene with the nucleotide sequence shown in SEQ ID NO.8 through the 2A peptide shown in SEQ ID NO.10 to obtain an eIF-DYS1-LIA1 fragment, and then ligate the eIF-DYS1-LIA1 fragment into the pPICZB vector to obtain the pPICZB-eIF-DYS1-LIA1 plasmid; The pPICZB-eIF-DYS1-LIA1 plasmid is linearized with Sal I enzyme and then electrotransformed into Pichia pastoris with modified signal peptide to obtain recombinant Pichia pastoris.

8. Use of the Pichia pastoris with modified signal peptide 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 application according to claim 8, wherein The steps for producing recombinant type III collagen using Pichia pastoris modified with a signal peptide are as follows: Inoculate the Pichia pastoris modified with the signal peptide into BMGY medium and shake culture until the OD 600 reaches 4 - 6, then transfer it to BMMY liquid medium, control the initial OD 600 to be 0.9 - 1, continue shaking culture, and add methanol every 22 h - 24 h to induce the expression of the target protein; The steps for producing recombinant type III collagen using recombinant Pichia pastoris are as follows: inoculate the recombinant Pichia pastoris into BMGY medium and shake culture until the OD 600 reaches 4 - 6, transfer it to BMMY liquid medium, control the initial OD 600 to be 0.9 - 1, continue to shake culture, and add methanol every 22 h - 24 h to induce the expression of the target protein.

10. The application according to claim 9, characterized in that, During the process of using recombinant Pichia pastoris to produce recombinant type III collagen, maltose is added to the BMMY liquid medium at a mass-volume ratio of maltose to BMMY medium of 0 to 2%.

Images