A method for preparing recombinant humanized collagen with triple helical structure expressed by yeast

Abstract

The present invention discloses a method for preparing a recombinant humanized collagen protein with a triple helix structure expressed by yeast, and belongs to the field of genetic engineering technology. The present invention constructs a plasmid that can be used for multiple copies in vitro and multiple copies in vivo at the same time, then constructs a collagen protein expression vector based on the plasmid, and obtains a high-expression strain by expression amount screening, and finally introduces a P4H expression plasmid into the high-expression strain to obtain a co-expression strain, and after fermentation and purification, a recombinant humanized collagen protein with a triple helix structure expressed by yeast can be obtained, and the recombinant humanized collagen protein contains hydroxyproline, and hydroxyproline accounts for more than 30% of the total proline. The recombinant humanized collagen protein prepared by the present invention can be widely used in the fields of cosmetics, medical cosmetology, medical devices, biomedical materials, etc.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for preparing recombinant humanized collagen with triple helical structure expressed by yeast. Background Art

[0002] Collagen is the main component of the extracellular matrix and the most abundant and widely distributed protein in animals. It has a unique tissue structure and biological function, and plays an important role in maintaining the normal physiological functions and injury repair of cells, tissues, and organs.

[0003] Recombinant humanized collagen is a recombinant protein that modifies or resynthesizes a gene sequence of human collagen and then expresses the new gene sequence through a host protein expression system. Recombinant human collagen has the advantages of collagen antigenicity, biodegradability, and good biocompatibility, and does not have the disadvantages of animal collagen, such as viral risks and immune rejection, so it is widely used in the field of biomaterials.

[0004] There are a lot of literature reports on cases of recombinant expression of humanized collagen and other collagens, such as "Recombinant Microbial Systems for the Production of Human Collagen and Gelatin" published by Julio Báez et al., and "Three Decades of Research on Recombinant Collagens" published by Fertala. Although both involve the construction of recombinant human collagen, they all involve recombinant human collagen single chains. Such recombinant collagen lacks key collagen characteristics, including triple helical conformation, modification of proline and lysine residues, and resistance to enzyme degradation. Another example is "CN114853881B A recombinant human fusion collagen and its efficient hydroxylation method and application". In this patent, recombinant human collagen is co-expressed with the proline hydroxylase gene of Chlorella paramecium virus 1, but non-human P4H has different substrate specificity, and the obtained recombinant collagen may cause immune risks. That is, although a variety of recombinant collagens are disclosed in the prior art, they have the above-mentioned defects and cannot meet the diversified needs of the market, especially the market demand for triple helical recombinant humanized collagens. Therefore, the present invention intends to develop a yeast-expressed recombinant humanized collagen with a triple helical structure to achieve efficient expression of triple helical recombinant humanized collagens and meet the market demand for triple helical recombinant humanized collagens. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a recombinant humanized collagen with a triple helix structure expressed by yeast to solve the problems existing in the above-mentioned prior art. A plasmid that can be used for multiple copies in vitro and multiple copies in vivo is constructed, and then a collagen expression vector is constructed based on the plasmid and a high-expression strain is obtained by expression amount screening. Finally, a P4H expression plasmid is introduced into the high-expression strain to obtain a co-expression strain, and a triple helix structure recombinant humanized collagen can be obtained after fermentation and purification.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a recombinant humanized collagen with a triple helix structure expressed by yeast, comprising the following steps:

[0008] The recombinant expression plasmids I, II and III are transferred into yeast in any of the following ways (1) or (2):

[0009] (1) transferring any one of the recombinant expression plasmid I, recombinant expression plasmid II and recombinant expression plasmid III into yeast to obtain recombinant strain I; transferring any one of the recombinant expression plasmid I, recombinant expression plasmid II and recombinant expression plasmid III into the recombinant strain I to obtain recombinant strain II; transferring the P4H expression vector into the recombinant strain II to obtain recombinant strain III;

[0010] (2) Based on (1), any one of the recombinant expression plasmid I, the recombinant expression plasmid II and the recombinant expression plasmid III is transferred into the recombinant bacteria III to construct the recombinant bacteria IV;

[0011] Fermenting the recombinant bacteria III or the recombinant bacteria IV to obtain recombinant humanized collagen with a triple helical structure expressed by yeast;

[0012] Gene fragment I, gene fragment II or gene fragment III are used to transform pPIC9K vector respectively to construct plasmid I, plasmid II and plasmid III; the truncated sequence 5D-Cpro of the functional region of human type III collagen α1 chain is cloned into plasmid I, plasmid II and plasmid III respectively to obtain recombinant expression plasmid I, recombinant expression plasmid II and recombinant expression plasmid III; the nucleotide sequences of the gene fragment I, gene fragment II or gene fragment III are shown in SEQ ID NO:1-3 respectively.

[0013] Preferably, the construction method of the recombinant bacteria III comprises: transferring the recombinant expression plasmid I into yeast to obtain a recombinant strain I; transferring the recombinant expression plasmid II into the recombinant strain I to obtain a recombinant bacteria II; transferring the P4H expression vector into the recombinant bacteria II to obtain a recombinant bacteria III.

[0014] Preferably, the method for constructing the recombinant bacterium IV comprises: transferring the recombinant expression plasmid into the recombinant bacterium III to construct the recombinant bacterium IV.

[0015] Preferably, the P4H expression vector is obtained by replacing the KanR gene of the P4H 9K DP plasmid with the gene fragment shown in SEQ ID NO: 6.

[0016] Preferably, the nucleotide sequence of the human type III collagen α1 chain functional region truncated sequence 5D-Cpro is as shown in SEQ ID NO:5.

[0017] Preferably, the plasmid I is based on the pPIC9K vector as the backbone, and the 4648-9221 bp in the nucleotide sequence of the pPIC9K vector is replaced with the sequence shown in SEQ ID NO: 1, and the 938-943 bp in pPIC9K is deleted by point mutation, and the BamHI recognition site is introduced between 1583-1584 bp by point mutation.

[0018] Preferably, the plasmid II is obtained by replacing PpHIS4 on the plasmid I with the sequence shown in SEQ ID NO: 2;

[0019] The plasmid III is obtained by replacing PpHIS4 on the plasmid I with the sequence shown in SEQ ID NO: 3.

[0020] Preferably, the yeast includes Pichia pastoris KM71 and X33.

[0021] Preferably, the fermentation conditions are a rotation speed of 300 rpm, a ventilation volume of 4 L / min, a temperature of 30° C., and a pH of 6.0;

[0022] After the fermentation is completed, the fermentation liquid is centrifuged to collect the bacteria, the bacteria are resuspended at a mass-to-volume ratio of 1:10, crushed, centrifuged, and the precipitate is collected. The precipitate is washed, renatured, enzyme-digested, and freeze-dried to obtain recombinant humanized collagen with a triple helical structure expressed by yeast.

[0023] The present invention also provides a recombinant humanized collagen protein with a triple helix structure expressed by yeast, which is prepared by the preparation method.

[0024] The present invention discloses the following technical effects:

[0025] The present invention uses the modified plasmid to construct a recombinant humanized collagen expression vector, first screens the collagen expression to obtain a high-expression strain, then introduces the P4H expression plasmid to obtain a co-expression engineering strain, which can be used to study the hydroxylation difference of P4H from different sources on full-length collagen in Pichia pastoris. The fermentation and purification product of the co-expressed engineering strain is obtained by fermenting the mature yeast to express a recombinant humanized collagen with a triple helix structure, and the recombinant humanized collagen contains hydroxyproline, and the hydroxyproline accounts for more than 30% of the total proline.

[0026] The present invention provides a method for preparing recombinant humanized collagen with a triple-helix structure expressed by yeast. The modified plasmid can facilitate the construction of multi-copy collagen expression strains. The collagen obtained by fermenting the recombinant engineering strain has a triple-helix structure and can be used in scenarios requiring large molecular weight and high support in the fields of medical health and medical beauty. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0028] Figure 1 is a map of plasmid pPIC9K;

[0029] Figure 2 is a map of plasmid pMChZ-AOX;

[0030] Figure 3 is the map of plasmid pMCnZ-AOX;

[0031] Figure 4 is a map of plasmid pMCrZ-AOX;

[0032] Figure 5 is a map of the P4H expression vector pPIC9K-P4H(DP)-1;

[0033] Figure 6 Western Blot results; A: KM71H / 5D-Cpro-1#-Z S Collagen WB detection results after 72 hours of induction; B: Comparison of expression levels of KM71H / 5D-Cpro-2#, KM71H / 5D-Cpro-2#-P4H and KM71H / 5D-Cpro-1#; C: Comparison of expression levels at different electroporation times;

[0034] In the embodiment of the present invention, the naming rule of the obtained transformants is: starting strain name / POI-number of electroporation times-screening antibiotics 是否敏感 If the selection marker is not recovered, the antibiotic sensitivity description will be omitted. Here, Z is the abbreviation of Zeocin, S (sensitive) stands for sensitive, R (resistant) stands for resistant, the number before # indicates the number of times the collagen expression plasmid was electroporated, and the number with a circle indicates that the same plasmid was used for electroporation as in the previous round.

[0035] Figure 7 The results of copy number determination of different strains;

[0036] Figure 8 The circular dichroism spectrometer test results of 5D-Cpro lyophilized powder;

[0037] Fig. 9 Transmission electron microscopy detection of 5D-Cpro freeze-dried powder. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] The present invention constructs a plasmid that can be used for multiple copies in vitro and multiple copies in vivo at the same time, then constructs a collagen expression vector based on the plasmid and obtains a high-expression strain by expression screening, and finally introduces a P4H expression plasmid into the high-expression strain to obtain a co-expression strain; after fermentation and purification, a triple helical structure type III recombinant humanized small molecule collagen can be obtained. The following is further described with specific examples.

[0044] Example 1 A method for preparing a recombinant humanized collagen protein expressing a triple helical structure in yeast

[0045] 1. Plasmid construction

[0046] The present invention transforms the plasmid to construct a plasmid pMChZ-AOX, a plasmid pMCrZ-AOX or a pMCnZ-AOX that can be used for multiple copies in vitro and multiple copies in vivo. The specific construction method is as follows:

[0047] 1.1 Construction of plasmid pMChZ-AOX

[0048] Synthesize SEQ ID NO: 1 sequence and replace pPIC9K vector (purchased from Invitrogen, see the map) Figure 1 ) sequence, i.e., the KanR resistance gene, Bom and AOX13'fragment elements in pPIC9K were replaced with the replacement fragment SEQ ID NO:1; BamHI (938-943 bp) and αMF located before αMF in pPIC9K were deleted by point mutation, and a BamHI recognition site was introduced downstream of AOX1tt (also called AOX1 transcription termination or AOX1 termination) (between 1583 and 1584 bp) by point mutation, while the sequences of other vector elements remained unchanged.

[0049] Among them, the synthesized SEQ ID NO:1 sequence contains lox71 and lox66 sequences at both ends, ori is located upstream of lox71, and a Cre transcription unit is located inside. The Cre gene is driven by the AOX1 promoter with a SacⅠ restriction site mutation; there are prokaryotic and eukaryotic promoters upstream of BleoR, which can be used for prokaryotic and eukaryotic screening, and there is a prokaryotic promoter upstream of AmpR, which can be used for prokaryotic screening.

[0050] The plasmid pMChZ-AOX (h represents the recombination site PpHIS4, and Z represents the screening marker BleoR) was constructed. The plasmid can express Cre recombinase when induced by methanol, catalyze the recombination of lox71 and lox66, and realize the recovery of the resistance gene. BamHI located after AOX1tt can be used to construct an in vitro multi-copy plasmid with the tail enzyme. The original plasmid pPIC9K and the pMChZ-AOX plasmid constructed by the present invention are shown in the figure. Figure 2 shown.

[0051] The meanings of the various parts in SEQ ID NO: 1 are explained as follows:

[0052] The lowercase letters (positions 61-649) are ori, the gray background at the 5' end (positions 782-815) is lox71, the gray background at the 3' end (positions 5384-5417) is lox66; the single underline "" (positions 854-1714) is AmpR, the double underline "" (1715-1819) is pAmpR (AmpR promoter), the wavy line (1854-2788) is the pAOX1 promoter with the mutated SacⅠ site, the dotted line "__" (2814-3842) is the Cre recombinase gene, the emphasis (3940-4186) is AOX1tt, and the bold underline (4200-4611th position) is pTEF1 (corresponding to TEF1 promoter in the figure), the wavy lowercase letters (4619-4666th position) is pEM7 (corresponding to EM7 promoter in the figure), the italic lowercase letters (4685-5059th position) is BleoR, and the underlined lowercase letters (5125-5372th position) is CYC1tt (corresponding to CYC1 terminator in the figure). The sequence information of SEQ ID NO: 1 is as follows:

[0053]

[0054]

[0055]

[0056]

[0057] 1.2 Construction of plasmid pMCnZ-AOX

[0058] The synthesized sequence SEQ ID NO: 2 (NTS) was substituted for PpHIS4 on the pMChZ-AOX obtained in 1.1 above to obtain the plasmid pMCnZ-AOX (n represents the recombination site is NTS), the map is shown in Figure 3 shown.

[0059] The sequence information of SEQ ID NO:2 is:

[0060] .

[0061] 1.3 Construction of plasmid pMCrZ-AOX

[0062] The synthesized sequence SEQ ID NO:3 (PpRGI2) was substituted for PpHIS4 on the pMChZ-AOX obtained in 1.1 above to obtain the plasmid pMCrZ-AOX (r represents the recombination site is PpRGI2), the map is shown in Figure 4 shown.

[0063] SEQ ID NO:3

[0064]

[0065] 2. Obtaining recombinant humanized collagen and its expression vector

[0066] The full-length sequence of type III collagen α1 chain was optimized to obtain the truncated sequence 5D-Cpro of the α1 chain functional region, and the amino acid sequence is shown in SEQ ID NO: 4. The nucleic acid sequence SEQ ID NO: 5 was obtained by reverse translation according to the Pichia codon table, and the GC content and secondary structure were optimized. 5D-Cpro was cloned into pMChZ-AOX and pMCrZ-AOX, respectively, to obtain the recombinant expression plasmids pMChZ-5D-Cpro and pMCrZ-5D-Cpro. 5D-Cpro was cloned into pMCnZ-AOX to obtain the recombinant expression plasmid pMCnZ-5D-Cpro.

[0067] SEQ ID NO:4 sequence information is:

[0068] LEKRAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPCCGENLYFQGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVQLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFLHHHHHH。

[0069] The sequence information of SEQ ID NO:5 is as follows:

[0070]

[0071] Construction of P4H expression vector pPIC9K-P4H(DP)-1:

[0072] Synthesize SEQ ID NO:6 to replace KanR of P4H 9K DP plasmid in CN114480471A, that is, add P at the 5' end of the selection marker (according to the direction of pPIC9K plasmid) TEF and P EM7 , CYC1tt was added to the 3' end to obtain the P4H expression vector pPIC9K-P4H(DP)-1. Figure 5 shown.

[0073] The sequence information of SEQ ID NO: 6 is as follows, wherein the bold underlined capital letters (positions 1203-1614) are P TEF , the shadow bottom (1148-1195) is P EM7 , the underlined lowercase letters (positions 314-1129) are KanR, and the ununderlined lowercase letters (positions 1-248) are CYC1tt):

[0074]

[0075]

[0076] 3. Construction of recombinant engineering strains, induced expression and strain screening

[0077] (1) Construction of engineered strains

[0078] Pichia KM71 was used as the starting strain. Here, KM71 was used as an example. The competent state of Pichia was prepared according to the competent state preparation method described by Lin-Cereghino (Lin-Cereghino et al, 2005). The pMChZ-5D-Cpro obtained above was linearized using SalⅠ to obtain the pMChZ-5D-Cpro linearized plasmid. The pMChZ-5D-Cpro linearized plasmid was electroporated into the competent state of Pichia according to the method of Invitrogen's Pichia Expression Kit USER GUIDE, and then coated on a YPDZ (bleomycin, 300 μg / mL) plate to obtain the strain KM71H / 5D-Cpro-1#-Z. R . Recover the screening marker and obtain strain KM71H / 5D-Cpro-1#-Z S . (Where Z R Indicates bleomycin resistance, Z S Indicates sensitivity to bleomycin; the number before # indicates the number of times the collagen expression plasmid is electroporated).

[0079] pMCnZ-5D-Cpro was linearized with SpeⅠ and then electroporated into KM71H / 5D-Cpro-1#-Z S Get KM71H / 5D-Cpro-2#-Z R , recovered the screening marker and obtained the strain KM71H / 5D-Cpro-2#-Z S .

[0080] pPIC9K-P4H(DP)-1 was linearized with SalⅠ and then electroporated into KM71H / 5D-Cpro-2#-Z S Get KM71H / 5D-Cpro-2#-Z S -P4H.

[0081] pMCrZ-5D-Cpro was linearized with SpeⅠ and then electroporated into KM71H / 5D-Cpro-2#-Z S -P4H obtains KM71H / 5D-Cpro-3#-P4H.

[0082] (2) Inducible expression

[0083] The engineered strain obtained above was inoculated into a 100 mL Erlenmeyer flask containing 10 mL of BMGY medium and cultured at 28-30°C and 220 rpm until the OD 600 Centrifuge at 1500-3000g for 5 min at room temperature to collect the cells and resuspend them in BMMY medium to a concentration of OD 600 The cell culture medium was placed on a shaker at 28-30°C and 220 rpm for 3 days. 100% methanol was added to the culture medium every 24 hours to a final concentration of 1.0%. After methanol induction, bacterial samples were collected every 24 hours. The sample volume was 1 mL and placed in a 1.5 mL EP tube. The supernatant and bacterial cells were collected separately after centrifugation at 12000 g for 5 minutes at 4°C. 80 μL of the supernatant was taken, 20 μL of the loading buffer was added, and the sample was prepared by heating at 80°C for 5 minutes. 20 μL of the supernatant was taken for SDS-PAGE. Add 200 μL cell wall breaking solution and 40% liquid volume of 0.5 mm glass beads to the cells; vortex for 1 min, place on ice for 1 min, and repeat 6-10 times; centrifuge at 12000g for 30 min at 4°C, take 80 μL of the supernatant, add 20 μL of loading buffer, heat at 99°C for 5 min to prepare the sample, dissolve the precipitate with 8M urea, and prepare the sample according to the supernatant operation steps, take 20 μL for SDS-PAGE, and then perform Western Blot. The test results are as follows Figure 6 As shown, KM71H / 5D-Cpro-1#-Z RAfter 72 hours of induction, the expression was identified using anti-6His antibody as the primary antibody. One strain had the target band in the cell. The results are as follows Figure 6 As shown in A. The strain that correctly expressed the target protein was used as the starting strain. The 5D-Cpro expression plasmid with the recombination site was replaced by electroporation twice and the P4H expression plasmid was co-transfected. The target protein was identified with anti-6His antibody and P4HA1 antibody. The expression level of 5D-Cpro in the strain co-transfected with P4H was significantly increased. The four P4H co-expression strains screened out were able to correctly express P4HA and P4HB. The results are shown in Figure 6 As shown in B. Figure 6 C shows the comparison of expression levels at different electroporation times, KM71H / 5D-Cpro-2#-Z S -P4H had the highest expression level, exceeding KM71H / 5D-Cpro-3#-P4H.

[0084] 4. Identification of target gene copy number

[0085] The strains related to 5D-Cpro were commissioned to Shanghai Sangon Biotech to detect the copy number of the target gene using digital PCR. The PCR instrument was BIO-RAD T100. The copy number of the target gene in the strain obtained by the first electroporation of the target gene was taken as 1, and the ratio of the copy number of the target gene in other strains to that of the strain was plotted, as shown in Figure 2. Figure 7 The target gene copy number of 5D-Cpro related strains was consistent with the expression level detected by WB. The copy number of KM71H / 5D-Cpro-2#-P4H was about 2.5 times that of the control. After three rounds of electroporation, the expression level decreased and the corresponding copy number was also low. Although the plasmid recombination site was replaced, the target gene was still lost.

[0086] 5. High-density fermentation and protein purification

[0087] The engineered bacteria with high expression levels identified through protein expression are subjected to high-density fermentation culture and protein purification in a fermenter.

[0088] KM71H / 5D-Cpro-2#-P4H was inoculated into a 1L shake flask containing 200mL of seed medium YPG, and cultured at 220rpm and 30℃ for 18-20h until OD6 00 =2~10.

[0089] Use a 5L fermentation tank (Baoxing Biotechnology) with 2L of fermentation medium. Before inoculation, adjust the speed to 300rpm, the ventilation volume to 4L / min, and the temperature to 30°C. Use alkali solution prepared with concentrated ammonia water to adjust the pH to 6.0. First, add 0.9mL PTM1 Pichia yeast trace element mother solution, then add the prepared 200mL seed solution into the tank (flame circle inoculation), then click on the dissolved oxygen electrode to calibrate, and start fermentation after calibration. When the dissolved oxygen drops to 30% for the first time during growth, use the dissolved oxygen cascade speed function to maintain 30%; wait for the glycerol to be consumed, the dissolved oxygen to rebound, and the dissolved oxygen to be greater than 70% (OD 600 value of about 20), cancel the dissolved oxygen cascade speed, increase the stirring speed to 650rpm, use 30% linkage feeding of glycerol, and feed 150mL. Stop feeding glycerol, after the dissolved oxygen rebounds to more than 70%, induce culture with methanol, and feed at a constant rate of 4mL / h. Induction for 80-90h, OD 600 When the change is not obvious or decreases, the tank can be released to obtain the fermentation liquid.

[0090] After the fermentation, the bacterial liquid was centrifuged at 5000rpm for 30min to collect the bacterial cells, which were resuspended in the purified cell wall breaking solution at a ratio of 1:10 (W:V), broken by a homogenizer at 1000bar, and centrifuged at 12000rpm and 4℃ for 30min to separate the supernatant and precipitate. The precipitate was washed, renatured, digested with pepsin, ultrafiltered and desalted, and then freeze-dried to obtain 5D-Cpro freeze-dried powder.

[0091] 6. Structural characterization of 5D-Cpro lyophilized powder

[0092] The 5D-Cpro lyophilized powder was reconstituted and then acid hydrolyzed. The sample was analyzed by liquid chromatography-mass spectrometry. The peak area of ​​the targeted data was calculated using MassLynx quantitative software. The identification result was obtained using the standard curve method. The results showed that the hydroxyproline content reached more than 30% of the total proline.

[0093] The 5D-Cpro freeze-dried powder was dissolved in water and tested by circular dichroism spectrometer. The test results are as follows Figure 8 As shown, it is consistent with the circular dichroism spectral characteristics of triple-helical collagen.

[0094] The collagen samples were negatively stained and then tested by transmission electron microscopy. The test results were as follows: Fig. 9 Shown are the typical structural features of collagen microfibrils with alternating light and dark characteristics.

[0095] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a recombinant humanized collagen with a triple helical structure expressed by yeast, characterized in that: The following steps are involved: The recombinant expression plasmid I and recombinant expression plasmid II were transferred into yeast as follows: Transferring either the recombinant expression plasmid I or the recombinant expression plasmid II into yeast to obtain a recombinant strain I; transferring the other of the recombinant expression plasmid I or the recombinant expression plasmid II into the recombinant strain I to obtain a recombinant strain II; transferring the P4H expression vector expressing the P4H gene into the recombinant strain II to obtain a recombinant strain III; Fermenting the recombinant bacteria III to obtain recombinant humanized collagen with a triple helical structure expressed by yeast; The preparation method of the recombinant expression plasmid I is as follows: using the pPIC9K vector as a backbone, replacing the 4648-9221 bp in the nucleotide sequence of the pPIC9K vector with the sequence shown in SEQ ID NO: 1, deleting the 938-943 bp in pPIC9K by point mutation, and introducing the BamH Ⅰ recognition site between 1583-1584 bp by point mutation to obtain the first plasmid, and then cloning the truncated sequence 5D-Cpro of the functional region of the human type III collagen α1 chain into the first plasmid to obtain the recombinant expression plasmid I; The preparation method of the recombinant expression plasmid II is as follows: replacing PpHIS4 on the first plasmid with the sequence shown in SEQ ID NO: 2 to obtain a second plasmid, and then cloning the truncated sequence 5D-Cpro of the functional region of the human type III collagen α1 chain into the second plasmid to obtain the recombinant expression plasmid II; The nucleotide sequence of the human type III collagen α1 chain functional region truncated sequence 5D-Cpro is shown in SEQ ID NO:

5.

2. The preparation method according to claim 1, characterized in that The construction method of the recombinant bacteria III comprises: transferring the recombinant expression plasmid I into yeast to obtain the recombinant strain I; transferring the recombinant expression plasmid II into the recombinant strain I to obtain the recombinant bacteria II; transferring the P4H expression vector into the recombinant bacteria II to obtain the recombinant bacteria III.

3. The preparation method according to claim 1, characterized in that: The P4H expression vector is obtained by replacing the KanR gene of the P4H 9K DP plasmid with the gene fragment shown in SEQ ID NO:

6.

4. The preparation method according to claim 1, characterized in that: The yeasts include Pichia pastoris KM71 and X33.

5. The preparation method according to claim 1, characterized in that: The fermentation conditions are a rotation speed of 300 rpm, a ventilation volume of 4 L / min, a temperature of 30° C., and a pH of 6.0; After the fermentation is completed, the fermentation liquid is centrifuged to collect the bacteria, the bacteria are resuspended at a mass-to-volume ratio of 1:10, crushed, centrifuged, and the precipitate is collected. The precipitate is washed, renatured, enzyme-digested, and freeze-dried to obtain recombinant humanized collagen with a triple helical structure expressed by yeast.

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