A type iii humanized collagen and preparation method and application thereof

Abstract

This invention discloses a type III humanized collagen, its preparation method, and its applications, relating to the field of synthetic biology. The type III humanized collagen of this invention uses a 100% human collagen amino acid sequence, is non-immunogenic and endotoxin-free; it has good water solubility, strong plasticity, and is processable; it possesses a triple helix structure, which can promote the proliferation of adult fibroblasts and HaCaT cells, and has potential value in tissue engineering and other fields; furthermore, it utilizes Pichia pastoris for secretory recombinant expression, resulting in a single product structure, simple separation and purification process, and low production cost, which is conducive to industrial-scale production.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, and in particular to a type III humanized collagen, its preparation method, and its applications. Background Technology

[0002] Collagen is the most abundant protein in the extracellular matrix of human cells and plays a vital role in the normal functioning of human cells and tissues. Currently, 28 different types of collagen have been identified in the human body, with type I, type II, and type III collagen accounting for more than 85% of the total collagen content. Type III collagen is distributed in a loose network around type I collagen, playing a supporting role in connective tissue and maintaining the smoothness and suppleness of skin tissue. After adulthood, dermal fibroblasts cease synthesizing type III collagen, leading to a gradual decline in the proportion of type III collagen in skin tissue.

[0003] Currently, industrial collagen raw materials mainly come from animal extraction, using acid, alkali, or enzymatic methods to extract collagen from animal skin or bones. However, this method suffers from problems such as insufficient raw material supply, low batch stability, high difficulty in separation and purification, and immunogenicity, leading to high costs for downstream applications. In recent years, with the development of synthetic biology, the use of microbial recombinant expression of collagen has gradually become a new trend. Commonly used microbial recombinant collagen expression systems include *Escherichia coli*, *Pichia pastoris*, and *Saccharomyces cerevisiae*. Among them, *Pichia pastoris* has attracted widespread attention due to its excellent post-translational modification and protein secretion expression capabilities. Compared to eukaryotic expression systems from plants and animals, the *Pichia pastoris* expression system has advantages such as ease of operation, high-density fermentation, high protein expression levels, short cycle time, and low cost. Compared to prokaryotes such as *E. coli*, *Pichia pastoris* can efficiently secrete exogenous proteins, which is beneficial for protein separation and purification processes and does not contain endotoxins.

[0004] The isolation and purification of recombinant collagen is one of the limiting factors for its large-scale production. Purification costs often exceed 30% of the total cost, and recombinant collagen often fails to form a complete and stable triple helix structure, resulting in poor stability and frequent degradation during the isolation and purification process, raising questions about the bioactivity of the produced recombinant collagen. Therefore, screening for collagen functional fragments capable of forming triple helix structures from natural collagen sequences and performing secretory expression, thereby simplifying the isolation and purification process, undoubtedly has significant advantages for industrial production.

[0005] Currently, reports on recombinant expression of human collagen mainly focus on the expression of full-length collagen single chains and partial fragments of the helical region, as well as tandem combinations of partial fragments. Compared with full-length single chains, tandem expression of functional fragments has advantages such as smaller molecular weight, easier secretion and expression, higher yield, and the functional activity of natural collagen, and is gradually becoming the focus of research.

[0006] Chinese invention patent CN109593126A discloses a polypeptide, its production method, and its uses. The polypeptide described in the paper exhibits excellent adhesion and high stability in aqueous solution. However, it is expressed using *E. coli*, requiring cell disruption and complex separation and purification processes. Patent CN114920826A discloses a type III human-like collagen protein. This type III human-like collagen protein has good water solubility and can be expressed solublely in *Pichia pastoris*. However, the paper does not mention whether this protein has a triple helix structure, raising questions about its stable function in the human cellular microenvironment. Patent CN102443057A discloses a recombinant human collagen protein containing 599 amino acids, expressed recombinantly using *Pichia pastoris*. However, the paper does not mention the water solubility of the protein or whether the single chains can spontaneously form a triple helix structure outside the cell. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a type III humanized collagen.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned type III humanized collagen.

[0009] Another object of the present invention is to provide the application of the above-mentioned type III humanized collagen.

[0010] The type III humanized collagen of this invention has good water solubility and can spontaneously form a triple helix structure outside the cell, which can promote the proliferation of adult fibroblasts and HaCaT cells. It can also be secreted and recombinantly expressed by Pichia pastoris, simplifying the separation and purification process and reducing production costs.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] The first objective of this invention is to provide a type III humanized collagen whose amino acid sequence comprises n repeats of the sequence shown in SEQ ID NO.1, where n is an integer ≥1, preferably an integer from 1 to 30; more preferably n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, wherein when n is an integer ≥2, there are no linkers between the repeat sequences, and they are directly connected.

[0013] Preferably, when n=1, the amino acid sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding it is as shown in SEQ ID NO.4.

[0014] Preferably, when n=10, the amino acid sequence is as shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding it is as shown in SEQ ID NO.5.

[0015] Preferably, when n=30, the amino acid sequence is as shown in SEQ ID NO.3, and the nucleotide sequence of the gene encoding it is as shown in SEQ ID NO.6.

[0016] The type III humanized collagen comprises:

[0017] 1) An amino acid sequence with more than 80% homology to the amino acid sequence shown in SEQ ID NO.1, which retains the cell proliferation effect of the amino acid sequence of SEQ ID NO.1;

[0018] 2) An amino acid sequence in which one or more amino acid residues are added, replaced, deleted, or inserted in the amino acid sequence shown in SEQ ID NO.1, while retaining the cell proliferation effect of the amino acid sequence in SEQ ID NO.1;

[0019] 3) Replace the proline (Pro, P) in the sequence shown in SEQ ID NO.1 with non-natural amino acids, such as hydroxyproline (Hyp), fluoroproline (Flp), and chloroproline (Clp). Hydroxyproline, fluoroproline (Flp), and 4-chloroproline may be in 4-cis or 4-trans configuration, which retain the cell proliferation effect of the amino acid sequence in SEQ ID NO.1;

[0020] 4) The N-terminal amino acid of the amino acid sequence shown in SEQ ID NO.1 or the n repeat sequences of the sequence shown in SEQ ID NO.1 is acetylated and the C-terminal amino acid is amidated, which retains the cell proliferation effect of the amino acid sequence of SEQ ID NO.1;

[0021] Another object of the present invention is to provide the encoding gene for the aforementioned type III humanized collagen.

[0022] A third objective of the present invention is to provide a recombinant expression vector containing the coding gene.

[0023] The recombinant expression vector is pPIC9K.

[0024] A fourth objective of this invention is to provide recombinant bacteria expressing the aforementioned type III humanized collagen.

[0025] Furthermore, the recombinant bacteria are preferably Pichia pastoris GS115 as the host.

[0026] The fifth objective of this invention is to provide a method for constructing the recombinant strain, comprising the following steps: transforming a recombinant expression vector containing a type III humanized collagen coding gene into Pichia pastoris GS115 competent cells, culturing and screening positive transformants to obtain the recombinant strain.

[0027] The sixth objective of this invention is to provide a method for rapidly preparing type III humanized collagen, comprising the following steps: inoculating the recombinant bacteria of type III humanized collagen into a fermentation medium at an inoculation rate of 2-10%, adding methanol to induce fermentation for 48-120 h, centrifuging to remove the bacterial cells, obtaining a supernatant containing type III humanized collagen, and purifying and desalting to obtain the type III humanized collagen.

[0028] Furthermore, the fermentation medium is BMMY medium, with a formulation of YNB 13.4 g / L and biotin 4.0 × 10⁻⁶. -4 g / L, KH2PO4 11.8g / L, K2HPO4·3H2O 4.0g / L, yeast extract 10.0g / L, peptone 20.0g / L.

[0029] The concentration of methanol is 0.5% to 3% v / v; more specifically, 1% v / v.

[0030] The conditions for induced fermentation are 28-30℃ and 200-250rpm for 48-120h; further, 28℃ and 250rpm for 96h.

[0031] Preferably, the purification and desalting process involves: adding ammonium sulfate I for treatment, centrifuging, collecting the supernatant, adding ammonium sulfate II for treatment, centrifuging to collect the precipitate and dialysis, and collecting the supernatant to obtain the type III humanized collagen.

[0032] The final concentration of ammonium sulfate I is 10-50%, and the final concentration of ammonium sulfate II is 55-95% (preferably 70%).

[0033] The ammonium sulfate I treatment time is 1 to 10 hours, and the ammonium sulfate II treatment time is 1 to 10 hours.

[0034] The dialysis membrane has a molecular weight cutoff of 3 to 20 kDa, preferably 5 kDa.

[0035] The number of dialysis sessions is 1 to 6, preferably 4 to 6.

[0036] The dialysis buffer is one of deionized water, phosphate buffer, or acetate buffer.

[0037] The application of the aforementioned type III humanized collagen in biomedical materials or cosmetics.

[0038] The present invention has the following advantages and effects compared with the prior art:

[0039] The type III humanized collagen amino acid sequence provided by this invention uses 100% human collagen sequence, is free of immunogenicity and endotoxin; has good water solubility, strong plasticity, and is processable; has a triple helix structure, and has potential value in the fields of tissue engineering; and uses Pichia pastoris for secretory recombinant expression, the product has a single structure, the separation and purification process is simple, which is conducive to industrial-scale production. Attached Figure Description

[0040] Figure 1 This is a flowchart of the preparation process for type III humanized collagen.

[0041] Figure 2 The results were obtained by SDS-PAGE analysis of the fermentation supernatant of recombinant type III humanized collagen; lane 1: HC1, lane 2: HC10, lane 3: HC30.

[0042] Figure 3 This is a prediction of the hydrophilicity and hydrophobicity of recombinant expression of type III humanized collagen; where, respectively, they correspond to HC1, HC10, and HC30.

[0043] Figure 4 It is a circular dichroism chromatographic assay of purified type III humanized collagen.

[0044] Figure 5 This study tested the activity of purified type III humanized collagen on human fibroblast proliferation. Note: Compared with the blank control group (Blank), ***p<0.001.

[0045] Figure 6 This study tested the activity of purified type III humanized collagen on HaCaT cell proliferation. Note: Compared with the blank control group (Blank), ***p<0.001. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0047] Unless otherwise specified, the test methods in the following examples are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.

[0048] The culture media involved in the following examples are as follows:

[0049] LB medium (g·L) -1 Yeast extract 5.0 g, peptone 10.0 g, NaCl 10.0 g. LB solid medium is prepared by adding 20.0 g / L of the above. -1 Agar powder.

[0050] YPD medium (g·L) -1 Yeast extract 10.0 g, peptone 20.0 g, glucose 20.0 g. YPD solid medium is prepared by adding 20.0 g / L of these components. -1 Agar powder.

[0051] MD solid culture medium (g·L) -1 ): Glucose 20.0, YNB 13.4, Agar powder 20.0, 4×10 -4 Biotin.

[0052] BMGY medium (g·L) -1 ): Glycerin 10.0, YNB 13.4, Biotin 4.0×10 -4 KH2PO4 11.8, K2HPO4·3H2O 4.0, yeast extract 10.0, peptone 20.0.

[0053] BMMY medium (g·L) -1 YNB 13.4, Biotin 4.0×10 -4 KH2PO4 11.8, K2HPO4·3H2O 4.0, yeast extract 10.0, peptone 20.0.

[0054] All culture media were prepared using deionized water and sterilized at 121°C for 15–20 minutes after preparation.

[0055] In this invention, the amino acid sequence used is GKDGPRGPTGPIGPPGPAGQPGDKGE G (SEQ ID NO. 1), and its corresponding nucleic acid sequence is shown in SEQ ID NO. 4. The type III humanized collagen of this invention may contain multiple repeating sequences shown in SEQ ID NO. 1, provided that there are no linkers between the repeating sequences. This invention does not limit the number of repeating sequences, as long as they retain the proliferative effect of the amino acid sequence in SEQ ID NO. 1. Preferably, the number of repeating sequences is 10, that is, a protein containing the sequence shown in SEQ ID NO. 2, and its corresponding nucleic acid sequence is shown in SEQ ID NO. 5.

[0056] The protein of the present invention comprises the sequence shown in SEQ ID NO.1 or an amino acid sequence having more than 80% homology with its amino acid sequence, or an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid residues are added, substituted, deleted or inserted in n repeating sequences of the sequence shown therein, and retains the proliferation effect of the amino acid sequence of SEQ ID NO.1.

[0057] In this invention, homology refers to the overlap rate of sequences, which can be a direct quantitative relationship between two sequences, such as the percentage of partial similarity; it also includes the proportion of sequences where two sequences have identical nucleotide or amino acid residues at the same site; or it can be different sequences formed through divergent evolution from a common ancestor. This can be obtained by comparing sequence information using conventional bioinformatics methods.

[0058] Amino acid addition refers to adding several amino acids to the N-terminus or C-terminus of the protein amino acid sequence of this invention, which retains the proliferative effect of the amino acid sequence in SEQ ID NO.1. Specifically, Chen et al. found that extending several amino acids at both ends of the active sequence of human type III collagen had no significant effect on 3T3 cell activity (Hua, Chen et al. “Characterization by high-resolution crystal structure analysis of a triple-helix region of human collagen type III with potent cell adhesion activity.” Biochemical and Biophysical Research Communications 508(2018):1018-1023.).

[0059] Amino acid substitution refers to the replacement of one, two, or three amino acids in the amino acid sequence of the protein of this invention with amino acids of similar or related properties to form a polypeptide or protein. The forms of amino acid substitution can include replacing Gly with Pro or Ala; replacing Lys with Arg, Gln, Asn, or His; replacing Asp with Glu; replacing Pro with Ala; replacing Arg with Lys, Gln, Asn, or His; replacing Thr with Ser; replacing Ile with Leu, Val, Met, Ala, or Phe; replacing Ala with Val, Leu, or Ile; replacing Gln with Asn; and replacing Glu with Asp, all of which retain the proliferative effect of the amino acid sequence in SEQ ID NO. 1.

[0060] Amino acid deletion refers to the removal of one, two, or three or more amino acids from the amino acid sequence of the protein in this invention, while retaining the proliferative effect of the amino acid sequence in SEQ ID NO.1. Specifically, Yu et al. found that deleting several amino acids, such as arginine, alanine, leucine, and aspartic acid, from the N-terminus of collagen-like peptides had no significant effect on the triple helix structure and stability of collagen-like peptides (Yu, LeTracy et al. "Hollow Octadecameric Self-Assembly of Collagen-like Peptides." Journal of the American Chemical Society (2023): n.pag.).

[0061] Amino acid insertion refers to inserting amino acid residues at appropriate positions in the amino acid sequence of the protein of the present invention. The inserted amino acid residues may be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other, thus preserving the proliferation effect of the amino acid sequence of SEQ ID NO.1.

[0062] The amino acids described in this invention are preferably L-type amino acids; however, one or more of these amino acids (e.g., 2-7, 2-6, 2-5, or 2-4) can be replaced with D-type amino acids, artificially modified amino acids, or naturally occurring rare amino acids to improve the bioavailability, stability, and biological activity of the protein. D-type amino acids refer to amino acids corresponding to the L-type amino acids that make up proteins; artificially modified amino acids refer to common L-type amino acids that make up proteins after modification such as methylation or phosphorylation; naturally occurring rare amino acids include uncommon amino acids that make up proteins and amino acids that do not make up proteins, such as 5-hydroxylysine, methylhistidine, γ-aminobutyric acid, and homoserine. This also includes replacing proline with non-natural amino acids, such as hydroxyproline (Hyp), fluoroproline (Flp), and 4-chloroproline (Clp) (PubChem CID: 69997622). Hydroxyproline (Hyp), fluoroproline (Flp), and chloroproline (Clp) may be in 4-cis or 4-trans configurations, which retain the proliferative effect of the amino acid sequence of SEQ ID NO.1.

[0063] The protein of the present invention comprises the sequence shown in SEQ ID NO.1 or n repeating sequences of the sequence shown therein, wherein the N-terminal amino acid of the amino acid sequence is acetylated and the C-terminal amino acid is amidated, and the protein retains the proliferative effect of the amino acid sequence of SEQ ID NO.1.

[0064] The flowchart for the preparation of type III humanized collagen of this invention is as follows: Figure 1 As shown.

[0065] Example 1: Construction of gene expression vector

[0066] When n=1 (denoted as HC1), the amino acid sequence is shown in SEQ ID NO.1. Codon optimization was performed for the Pichia pastoris expression platform, and the specific nucleotide sequence is shown in SEQ ID NO.4.

[0067] When n=10 (denoted as HC10), the amino acid sequence is shown in SEQ ID NO.2. Codon optimization was performed for the Pichia pastoris expression platform, and the specific nucleotide sequence is shown in SEQ ID NO.5.

[0068] When n=30 (denoted as HC30), the amino acid sequence is shown in SEQ ID NO.3. Codon optimization was performed for the Pichia pastoris expression platform, and the specific nucleotide sequence is shown in SEQ ID NO.6.

[0069] The complete genome sequences of SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 were synthesized by Nanjing GenScript Biotech Co., Ltd. The synthesized complete genome sequences were ligated and integrated into the pPIC9K vector via SnaBI (TACGTA) and EcoRI (GAATTC) restriction enzyme digestion. The vector was then transformed into the host strain *E. coli* TOP10 using a heat shock method. Positive transformants were screened using LB agar plates containing ampicillin, and the transformed strains were then cultured in LB liquid medium containing ampicillin. Plasmids were extracted using a plasmid miniprep kit to obtain the recombinant expression vectors pPIC9K-HC1, pPIC9K-HC10, and pPIC9K-HC30.

[0070] Example 2 Construction of recombinant expression strain

[0071] The recombinant plasmids pPIC9K-HC1, pPIC9K-HC10, and pPIC9K-HC30 were linearized by digestion with Sac I restriction endonuclease at 37°C for 2 hours. The digestion system is as follows:

[0072]

[0073] After the enzyme digestion reaction was completed, the fragments were purified using a PCR product purification kit, and the concentration of the enzyme digestion products was detected.

[0074] The purified linearized fragments of pPIC9K-HC1, pPIC9K-HC10, and pPIC9K-HC30 were transformed into Pichia pastoris GS115 competent cells via electroporation. The specific steps are as follows:

[0075] 1) Take 20 μL of linearized plasmid and add it to 80 μL of Pichia pastoris GS115 competent cells, then transfer it to a pre-cooled 0.2 cm electroporation vessel and incubate it on ice for 5 min;

[0076] 2) Electric shock conditions: 1.5kV, 2mm inner diameter electric rotary cup, electric shock time 3.5~4.5ms;

[0077] 3) Immediately add 1 mL of 1 M sorbitol solution to resuspend the bacterial cells and transfer them to a sterile 2 mL centrifuge tube;

[0078] 4) Resuspend the bacterial cells and let them stand at 30°C for 2 hours;

[0079] 5) The yeast suspension after electroconversion was plated on MD plates for screening and cultured at 30°C for 2 days.

[0080] Single colonies were selected for colony PCR and sequencing verification. Positive transformants with correct sequencing results were preserved for subsequent experiments.

[0081] Example 3: Recombinant Pichia pastoris shake-flask fermentation

[0082] 1) Select positive transformants and inoculate them into 10 mL / 50 mL YPD medium. Incubate at 30°C and 250 rpm for 18 h. Then, transfer them to 25 mL / 250 mL BMGY medium at a 1% inoculation rate and incubate at 28°C and 250 rpm for bacterial OD. 600 From phases 2 to 6, the cells are in the logarithmic growth phase.

[0083] 2) Collect bacterial cells by centrifugation at 5000g for 5 min at room temperature, and resuspend the bacterial cells in BMMY medium to OD. 600 The solution was dispensed into 50 mL / 500 mL BMMY medium and fermented at 28 °C and 250 rpm.

[0084] 3) Add 1% methanol every 24 hours;

[0085] 4) After 96 hours of fermentation, the protein expression in the fermentation supernatant was detected by SDS-PAGE. The SDS-PAGE results are as follows: Figure 2 As shown, there are obvious bands around 4.6kDa, 33kDa, and 95kDa.

[0086] Example 4: Rapid purification of type III humanized collagen

[0087] Because the protein bands in the fermentation supernatant are singular and the protein is relatively water-soluble ( Figure 3 Therefore, the ammonium sulfate fractionation precipitation method was used to purify the target protein. The specific operation steps are as follows:

[0088] 1) Under ice bath conditions, add ammonium sulfate with a final concentration of 50% to precipitate the fermentation supernatant collected in Example 4 for 1 hour, then centrifuge at 10000g for 10 minutes at 4°C to remove impurities from the precipitate and retain the supernatant.

[0089] 2) Add ammonium sulfate to the supernatant to make the final concentration 70%, precipitate for 10 h, centrifuge at 10000g for 10 min at 4℃ to obtain protein precipitate;

[0090] 3) Dissolve the protein precipitate in ultrapure water and add it to a 5 kDa dialysis bag. Dialyze the protein in ultrapure water under ice bath conditions, changing the ultrapure water (dialysis solution) every 4-6 hours for a total of 4-6 times to remove ammonium sulfate ions. Then freeze-dry the purified protein for storage.

[0091] Example 5: Circular dichroism Detection of Purified Type III Humanized Collagen

[0092] The above-mentioned lyophilized protein powder was dissolved in ultrapure water to a final concentration of 0.2 mg / mL. Before detection, the sample was equilibrated at 4℃ for 24 h, and scanned at 4℃ in the range of 190–260 nm (step 1 nm, averaging time 5 s). Results are as follows... Figure 4 As shown, the circular dichroism chromatogram of type III humanized collagen shows a significant negative peak at a wavelength near 200 nm and a positive peak at a wavelength near 226 nm. This indicates that type III humanized collagen has formed a triple helix structure.

[0093] Example 6 Cell proliferation assay

[0094] The effects of type III humanized collagen on the proliferation of human fibroblasts and HaCaT cells (human immortalized fibroblasts) were detected using the CCK8 assay. The specific experimental steps are as follows:

[0095] 1) Digest, centrifuge, and count the pre-cultured human fibroblasts and HaCaT cells;

[0096] 2) Inoculate with 100 μL of 10 4 Cells per well were cultured in 96-well plates at 37°C, 5% CO2, and 90% humidity for 24 hours.

[0097] 3) Observe the cell growth status and density under a microscope, and take cells with good growth status and uniform cell distribution and density for subsequent experiments;

[0098] 4) Prepare a 10 μg / mL sample solution, with three replicates for each sample, and incubate at 37℃, 5% CO2, and 90% humidity for 24 hours;

[0099] 5) Add 10 μL of CCK-8 solution to each well, and then incubate at 37°C, 5% CO2, and 90% humidity for 2 hours. Measure the absorbance at 450 nm using a microplate reader.

[0100] Using no sample as a blank control, PBS as a control, and bovine type III collagen (Southern Biotech, Bovine Type III Collagen-Solution, catalog number: 1240-02S) as a positive control, the results are as follows: Figure 5 and 6 As shown, after 24 hours of culture, type III humanized collagen HC1, HC10 and HC30 can significantly promote the proliferation of adult fibroblasts and HaCaT cells, with a proliferation effect comparable to that of natural bovine type III collagen.

[0101] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A type III humanized collagen, characterized in that: Its amino acid sequence is n repeats of the sequence shown in SEQ ID NO.1, where n is an integer from 1 to 30, and when n is an integer ≥2, there is no linker between the repeat sequences.

2. A gene encoding type III humanized collagen as described in claim 1.

3. The encoding gene according to claim 2, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.

6.

4. A recombinant expression vector or recombinant bacterium comprising the encoding gene of any one of claims 2 to 3.

5. The recombinant expression vector or recombinant bacteria according to claim 4, characterized in that: The recombinant expression vector described above uses pPIC9K as the vector; The recombinant strain used Pichia pastoris GS115 as the host.

6. A method for constructing the recombinant bacteria according to claim 4 or 5, characterized in that: The process includes the following steps: transforming a recombinant expression vector containing the encoding gene of any one of claims 2 to 3 into Pichia pastoris GS115 competent cells, culturing and screening positive transformants to obtain the recombinant strain.

7. A method for rapidly preparing the type III humanized collagen according to claim 1, characterized in that: The process includes the following steps: inoculating the recombinant bacteria described in claim 4 or 5 into a fermentation medium at an inoculation rate of 2-10%, adding methanol to induce fermentation for 48-120 hours, centrifuging to remove the bacterial cells, obtaining a supernatant containing type III humanized collagen, and purifying and desalting to obtain the type III humanized collagen.

8. The method according to claim 7, characterized in that: The fermentation medium was BMMY medium, with a formulation of YNB 13.4 g / L and biotin 4.0 × 10⁻⁶. -4 g / L, KH2PO4 11.8 g / L, K2HPO4·3H2O 4.0 g / L, yeast extract 10.0 g / L, peptone 20.0 g / L; The concentration of methanol is 0.5% to 3% v / v; The purification and desalting process involves: adding ammonium sulfate I for treatment, centrifuging, collecting the supernatant, adding ammonium sulfate II for treatment, centrifuging to collect the precipitate and dialysis, and collecting the supernatant to obtain the type III humanized collagen. The final concentration of ammonium sulfate I is 10-50%, and the final concentration of ammonium sulfate II is 55-95%. The ammonium sulfate I treatment time is 1–10 h, and the ammonium sulfate II treatment time is 1–10 h; The dialysis membrane used in the dialysis has a molecular weight cutoff of 3–20 kDa.

9. The use of the type III humanized collagen as described in claim 1 in the preparation of biomedical materials or cosmetics.

Images