Recombinant humanized type V collagen, its expression vector and genetically engineered bacteria

Abstract

The present invention discloses a recombinant humanized type V collagen protein and its expression vector and genetically engineered bacteria. The present invention screens the functional short peptides that bind to cell integrins and heparin in human type V collagen protein, designs 10 tandem repeats to constitute the recombinant humanized type V collagen protein JYC501, and amplifies and clones it into the pPIC9K vector after codon optimization, and transforms it into Pichia pastoris cells after enzyme linearization. The high-copy recombinants are screened by G418 resistance gradient to obtain the Pichia pastoris genetically engineered bacteria that can express the recombinant humanized type V collagen protein JYC501. The recombinant humanized type V collagen protein JYC501 of the present invention exhibits excellent functions of promoting the expression of collagen and elastin genes, anti-inflammatory and soothing, promoting cell adhesion, cell proliferation and migration, and can be used to promote the repair of tissues such as skin, enhance the resistance of the skin, and is suitable for preparing skin care products or medical devices.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to a recombinant humanized type V collagen and an expression vector and genetic engineering bacteria thereof. Background Art

[0002] Type V collagen (COLV) is a dermal collagen that regulates fibril formation and is composed of three different polypeptide α chains (α1, α2 and α3) forming at least three different homo- or heterotrimers α1(V) 3 , α1(V) 2 α2(V) and α1(V)α2(V)α3(V). As a relatively low-content collagen in the extracellular matrix (accounting for only 2-4% of the total collagen in adult skin), COLV often assembles with type I collagen (COLI) in the form of microfilaments or 12nm diameter fibers to form heterotypic fibrils in the cornea and skin dermis, acting as the main regulator of collagen fibril formation. COLV can also bridge type VI collagen (COLVI) in the dermal matrix to type IV collagen (COLIV) in the epidermal basement membrane, regulating the stability between the epidermis and dermis. Studies have found that the loss of the COLV gene can lead to impaired collagen fibril formation, which is related to the loss of corneal transparency and the occurrence of classic congenital connective tissue dysplasia (Ehlers-Danlos) syndrome. In 2020, Professor Arjun Deb's research group at the University of California, Los Angeles published a research paper in the journal Cell on how type V collagen in scar tissue regulates the size of scar after heart injury. The study found that the absence of COLV would lead to an abnormal increase in scar size after infarction and further deterioration of heart function, indicating that COLV is a key driving factor in repairing damaged heart function (Yokota, T., McCourt, J., Ma, F., Ren, S., & Deb, A.. (2020). Type V collagen in scar tissueregulates the size of scar after heart injury. Cell, 182(3).).

[0003] The α1(V) chain consists of a main collagen domain (COL1), an N-terminal domain, and a C-terminal globular propeptide. The N-terminal domain contains a signal peptide (SP), non-collagenous domain 3 (NC3), collagen domain 2 (COL2), and non-collagenous domain 2 (NC2) subdomains. The NC3 subdomain contains a thrombin-1 N-terminal domain-like (TSPN-1) and a variable region (VAR). Studies have shown that 823-1143aa located in the COL1 region is the heparin binding domain. Heparin is a highly sulfated glycosaminoglycan produced by mast cells and secreted into the extracellular matrix. Heparin can bind to antithrombin III and enhance its activity, indirectly exerting its anticoagulant function. Therefore, heparin is widely used as an anticoagulant in clinical practice. In 2021, a study found that low molecular weight heparin (LMWH) can bind to two key cytokines IFN-γ and IL-6 and inhibit their activity, thereby blocking the normal transmission of the inflammatory pathway and exerting an anti-inflammatory effect. It plays an important role in preventing and suppressing severe autoimmune diseases and is an anti-inflammatory agent with broad application prospects. Summary of the invention

[0004] The present invention obtains a functional peptide segment with high activity by screening the functional domains binding to cell integrins and heparin in human type V collagen, and repeats the functional peptide segment 10 times in series to form a recombinant humanized type V collagen JYC501, amplifies and clones the peptide segment into a pPIC9K vector after codon optimization, linearizes the peptide segment after enzyme digestion, and transforms the peptide segment into a Pichia pastoris cell after linearization, and screens high-copy recombinants through a G418 resistance gradient, thereby obtaining a high-copy Pichia pastoris genetic engineering strain capable of expressing the recombinant humanized type V collagen JYC501.

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

[0006] A functional short peptide, whose sequence is 100% identical to that of human type V collagen and has functional sites for binding to cell integrins and heparin, and whose amino acid sequence is shown in SEQ ID No.1.

[0007] The recombinant humanized type V collagen JYC501 is composed of 10 tandemly repeated functional short peptides, and its amino acid sequence is shown in SEQ ID No.2.

[0008] The nucleotide sequence of the gene encoding the above-mentioned recombinant humanized type V collagen JYC501 is shown in SEQ ID No.3.

[0009] A recombinant expression vector is a recombinant expression vector containing the gene encoding the recombinant humanized type V collagen JYC501. In a specific embodiment of the present invention, the recombinant expression vector pPIC9K-JYC501 containing the gene encoding the recombinant humanized type V collagen JYC501 is taken as an example.

[0010] The method for constructing the above-mentioned recombinant expression vector comprises the following specific steps:

[0011] The gene sequence encoding the recombinant humanized type V collagen JYC501 shown in SEQ ID No. 3 was amplified by high-fidelity PCR, and the target fragment after gel recovery and purification was seamlessly cloned into the downstream of the α-mating factor secretion signal peptide of the pPIC9K empty vector. After verification by colony PCR and sequencing, it was enriched to obtain the recombinant expression vector pPIC9K-JYC501.

[0012] The genetically engineered Pichia pastoris bacteria for producing the above-mentioned recombinant humanized type V collagen JYC501 contains the above-mentioned recombinant expression vector pPIC9K-JYC501.

[0013] The construction method of the above-mentioned Pichia pastoris genetically engineered bacteria comprises the following steps:

[0014] The above-mentioned recombinant expression vector pPIC9K-JYC501 was linearized by restriction digestion and then transformed into Pichia pastoris GS115 competent cells, positive transformants were selected, and high-copy recombinants were screened through G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria that produce recombinant humanized type V collagen JYC501.

[0015] The preparation method of the above-mentioned recombinant humanized type V collagen JYC501 comprises the following steps:

[0016] The Pichia pastoris genetically engineered bacteria producing the recombinant humanized type V collagen JYC501 were inoculated into the BMMY induction medium, induced to express with methanol, and the fermentation supernatant was purified to obtain high-purity recombinant humanized type V collagen JYC501.

[0017] A composition comprising the above-mentioned recombinant humanized type V collagen JYC501, wherein the composition includes but is not limited to skin care products, pharmaceutical compositions, etc.

[0018] A product comprising the above-mentioned recombinant humanized type V collagen JYC501, the product including but not limited to medical devices, biomaterials, tissue engineering products, etc.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The present invention obtains a highly active functional short peptide sequence by screening the functional domains that bind to cell integrins and heparin in human type V collagen, and repeats it 10 times in series to obtain the recombinant humanized type V collagen JYC501 that has the functions of promoting the formation of collagen and elastin, anti-inflammatory and soothing, and promoting cell adhesion, proliferation and migration. Cell experiments have found that the recombinant humanized type V collagen JYC501 not only significantly up-regulates the expression of type I collagen gene and elastin gene in human fibroblasts, but also helps to down-regulate the expression of the inflammatory factor TNF-α gene, which has the effect of anti-inflammatory and soothing. In addition, the present invention optimizes the coding gene sequence of the recombinant humanized type V collagen JYC501 according to the codon preference of Pichia pastoris, constructs a recombinant expression vector, and transfers it into the Pichia pastoris strain, and screens the high-copy recombinant by G418 resistance gradient, thereby obtaining a high-copy Pichia pastoris genetic engineering bacterium capable of producing the recombinant humanized type V collagen JYC501, and realizing the large-scale production of the recombinant humanized type V collagen JYC501. The recombinant humanized type V collagen JYC501 of the present invention can be used to promote the repair of tissues such as skin, improve the resistance of the skin, and is suitable for preparing products such as skin care or medical devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the plasmid structure for expressing recombinant humanized type V collagen JYC501.

[0022] Figure 2 The electrophoresis diagram of the plasmid expressing the recombinant humanized type V collagen JYC501, wherein lane 1: linearized fragment of pPIC9K empty plasmid; lane 2: linearized fragment of pPIC9K+recombinant humanized type V collagen JYC501 plasmid.

[0023] Figure 3 It is an SDS-PAGE diagram of the supernatant of the culture medium of Pichia pastoris genetically engineered bacteria (methanol induced for 72 hours) for producing recombinant humanized type V collagen JYC501, wherein lanes 1 to 6: SDS-PAGE of the supernatant of the high-copy strain JYC501, and lane 7: the fermentation supernatant of the blank control strain.

[0024] Figure 4 This is the sequence coverage of the recombinant humanized type V collagen JYC501.

[0025] Figure 5 This is a graph showing the molecular weight determination results of the recombinant humanized type V collagen JYC501.

[0026] Figure 6 This figure shows the effect of recombinant humanized type V collagen JYC501 on the proliferation of epidermal cells and fibroblasts.

[0027] Figure 7 This figure shows the effect of recombinant humanized type V collagen JYC501 on the migration of epidermal cells and fibroblasts.

[0028] Figure 8 This figure shows the effect of recombinant humanized type V collagen JYC501 on the adhesion of epidermal cells and fibroblasts.

[0029] Fig. 9 This figure shows the effect of recombinant humanized type V collagen JYC501 on elastin gene expression.

[0030] Fig.10 This figure shows the effect of recombinant humanized type V collagen JYC501 on the gene expression of type I collagen α1 chain.

[0031] Fig.11 This is a graph showing the effect of recombinant humanized type V collagen JYC501 on TNF-α gene expression. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below through the preferred embodiments of the present invention, but the following embodiments do not limit the protection scope of the present invention.

[0033] In the embodiments of the present invention, conventional molecular biological experimental methods are used without further explanation. The processes such as PCR, enzyme cutting, seamless cloning, codon optimization, etc. involved in the embodiments are understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore will not be described in detail.

[0034] Example 1: Design and construction of high-copy Pichia pastoris genetic engineering bacteria of recombinant humanized type V collagen JYC501

[0035] (1) Bioinformatics and protein structure prediction database tools (NCBI, AlphaFold) were used to screen functional short peptides that can bind to cell integrins and heparin from human type V collagen. The amino acid sequence of the peptides is shown in SEQ ID No. 1. After 10 tandem repeats, the recombinant humanized type V collagen JYC501 was constructed. The amino acid sequence of the peptides is shown in SEQ ID No. 2.

[0036] (2) The amino acid sequence of the recombinant humanized type V collagen JYC501 obtained in step (1) is codon-optimized according to the codon preference of Pichia pastoris to obtain the optimized nucleotide sequence of the recombinant humanized type V collagen JYC501 as SEQ ID No. 3.

[0037] (3) The nucleotide sequence in step (2) was entrusted to Shanghai Qingke Biotechnology Co., Ltd. for gene synthesis, and the recombinant humanized type V collagen JYC501 gene sequence shown in SEQ ID No. 3 was amplified using primer P1 (nucleotide sequence as shown in SEQ ID No. 4) and primer P2 (nucleotide sequence as shown in SEQ ID No. 5). The high-fidelity PCR amplification product was purified by nucleic acid gel and seamlessly cloned into the downstream of the α-mating factor secretion signal peptide of the pPIC9K plasmid using Gibson assembly technology. After colony PCR and sequencing verification, the recombinant plasmid pPIC9K-JYC501 was enriched. The structure of the recombinant plasmid pPIC9K-JYC501 is as follows: Figure 1 As shown, the plasmid electrophoresis diagram is as follows Figure 2 As shown. Then, the recombinant plasmid pPIC9K-JYC501 was digested with SalI restriction endonuclease (purchased from NEB, the specific operation was carried out according to the kit instructions) at 37°C overnight, and then the linearized recombinant expression plasmid was purified using a gel recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) and then electrotransformed into Pichia pastoris GS115 competent cells. The electrotransformed cells were activated and cultured with fresh YPD medium for 2-3 hours, and then spread on YNB selection medium and placed in a 30°C incubator for inverted culture for 2-3 days to grow transformants. First, the transformant monoclone was verified by colony PCR, and the verified transformants were spread on screening plates containing 1g / L, 2g / L, 3g / L and 4g / L G418, respectively. After 2-4 days of culture, the transformants that grew well on the high-concentration G418 plates were selected for purification and rescreening verification. The genome of the rescreened transformants was extracted and verified to be correct, and then glycerol was preserved.

[0038] (4) The obtained high-copy Pichia genetically engineered strain was inoculated into 30 mL of BMGY medium (the medium consisted of 20 g / L peptone, 10 g / L yeast extract, 10× YNB 100 ml, 10× potassium phosphate buffer pH 6.0 100 ml, 10× glycerol 100 ml, and the balance was water), cultured for 16-20 hours, and then the initial OD was calculated. 600The inoculum size was 1 and inoculated into 30 mL BMMY medium (the medium consisted of 20 g / L peptone, 10 g / L yeast extract, 10×YNB 100 ml, 10× potassium phosphate buffer pH 6.0 100 ml, and the balance was water) for cultivation. 300 μL of filtered and sterilized methanol was added every 24 hours. After 72 hours, 2 mL of sample was collected by centrifugation and the supernatant was collected. 500 μL of supernatant was concentrated in an ultrafiltration tube with a size of 10 kDa to obtain about 50 μL of concentrated solution. 24 μL of concentrated solution and 6 μL of 5×loading buffer were mixed evenly and placed in boiling water for 10-15 min. After centrifugation for 15 min, 10-15 μL of supernatant was collected and loaded into SDS-PAGE to complete the qualitative analysis of recombinant humanized type V collagen JYC501. The results of SDS-PAGE detection can be found in Figure 3 ,from Figure 3 It can be seen that a high-copy Pichia pastoris genetic engineering strain that can successfully express the recombinant humanized type V collagen JYC501 was obtained.

[0039] The target protein bands at the corresponding cutting positions were recovered and digested with trypsin, and the peptides after digestion were detected by LC-MS, and the detected peptides were compared with the library.

[0040] Figure 4 This is the total ion current chromatogram of the peptides of pure JYC501 after trypsin digestion. It can be seen from the figure that the peptides of JYC501 after digestion all belong to the region of the human type V collagen sequence selected during design, and the peptide coverage rate is 100%, proving that the peptides in the target band are all derived from the collagen designed in the present invention, and JYC501 is successfully and efficiently secreted outside the cell in Pichia pastoris.

[0041] Figure 5 This is the result of mass spectrometry determination of molecular weight. It can be seen from the figure that the actual molecular weight of JYC501 is 26.9 kDa, which is consistent with the theoretical molecular weight.

[0042] Example 2 Detection of the cell proliferation activity of recombinant humanized type V collagen JYC501

[0043] The MTT method was used to detect the ability of recombinant humanized type V collagen JYC501 to promote cell proliferation using human epidermal cells and human fibroblasts as experimental cells. Figure 6 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that 10ppm, 100ppm, and 1000ppm of recombinant humanized type V collagen JYC501 all show cell proliferation promoting activity, and the promoting effect increases with the increase of the concentration of recombinant humanized type V collagen JYC501.

[0044] Example 3 Detection of the cell migration promoting activity of recombinant humanized type V collagen JYC501

[0045] The cell scratch method was used to measure cell migration, and human epidermal cells and human fibroblasts were used as experimental cells to detect the ability of recombinant humanized type V collagen JYC501 to promote cell migration. Figure 7 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that 1ppm, 10ppm, 100ppm and 1000ppm of recombinant humanized type V collagen JYC501 show cell migration promoting activity, and the promotion effect increases with the increase of the concentration of recombinant humanized type V collagen JYC501.

[0046] Example 4 Detection of cell adhesion promoting activity of recombinant humanized type V collagen JYC501

[0047] The centrifugation method was used to detect the ability of recombinant humanized type V collagen JYC501 to promote cell adhesion using human epidermal cells and human fibroblasts as experimental cells. Figure 8 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that 1ppm, 100ppm, 100ppm and 1000ppm of recombinant humanized type V collagen JYC501 all show cell adhesion promoting activity, and the promoting effect increases with the increase of the concentration of recombinant humanized type V collagen JYC501.

[0048] Example 5 Detection of the expression of elastin gene in zebrafish promoted by recombinant humanized type V collagen JYC501

[0049] 1. Test method: According to the standard "T / ZHCA 015-2022 Evaluation of the Firming Effect of Cosmetics by Relative Expression of Elastin Gene in Zebrafish Fry", the effect of recombinant humanized type V collagen JYC501 on promoting elastin gene expression in zebrafish was detected to study its firming effect.

[0050] Elastin is the main component of elastic fibers in skin tissue, which can provide structural support for the skin and protect the skin from aging and sagging. Elastin is composed of two types of short peptides arranged alternately. Eln1 and eln2 are responsible for encoding different peptides of elastin. The two genes are jointly responsible for regulating the expression level of elastin, making the skin firm and elastic. Zebrafish have elastin (eln1, eln2) genes similar to humans. Therefore, by detecting the relative expression of eln1 or (and) eln2 genes, it can be shown whether the sample has a firming effect.

[0051] 2. Experimental Procedure

[0052] (1) Zebrafish were randomly selected and placed in a 6-well plate, with 30 zebrafish in each well.

[0053] (2) Water-soluble administration samples and a normal control group were set up at the same time, with a volume of 3 mL per well. Three biological replicates were performed.

[0054] (3) Incubate at 28°C in the dark for 24 h.

[0055] (4) Total RNA was extracted from zebrafish in each experimental group, cDNA was synthesized, and the gene expression of β-actin and target genes was detected by q-PCR.

[0056] (5) Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene.

[0057] 3. Test results

[0058] Table 1

[0059]

[0060] Table 1 and Fig. 9 The recombinant type V collagen JYC501 in the experiment is recombinant humanized type V collagen JYC501. The results showed that recombinant humanized type V collagen JYC501 has a firming effect. Compared with the normal control group, the relative expression of eln1 gene was upregulated in the 10, 100, and 1000ppm concentration groups of recombinant humanized type V collagen JYC501, and the gene expression upregulation rates were 33%, 62%, and 73%, respectively. The effect increased with the increase of concentration.

[0061] Example 7 Detection of the expression of type I collagen α1 chain gene in zebrafish promoted by recombinant humanized type V collagen JYC501

[0062] 1. Test method:

[0063] According to the standard "Standard Operating Procedure for Zebrafish Anti-wrinkle Efficacy Evaluation Experiment", the effect of recombinant humanized type V collagen JYC501 on promoting the expression of type I collagen α1 chain gene in zebrafish was detected to study its anti-wrinkle effect.

[0064] Skin growth, repair, nutrition, elasticity and tension are all related to collagen. Its loss will reduce the smoothness of the skin and produce wrinkles. In tetrapods, type I collagen is a trimer, mainly composed of two α1 chains and one α2 chain, encoded by col1a1a and col1a2 genes, respectively, and performs collagen-related biological functions in connective tissue and bone. There are three type I collagen genes in zebrafish, encoding col1a1a, col1a1b and col1a2 for α1(I), α2(I) and α3(I) chains, respectively. Therefore, by detecting the relative expression of col1a1a or (and) col1a1b or (and) col1a2 genes, it can be indicated whether the sample has anti-wrinkle effect.

[0065] 2. Experimental Procedure

[0066] (1) Zebrafish were randomly selected and placed in a 6-well plate, with 30 zebrafish in each well.

[0067] (2) Water-soluble administration samples and a normal control group were set up at the same time, with a volume of 3 mL per well. Three biological replicates were performed.

[0068] (3) Incubate at 28°C in the dark for 24 h.

[0069] (4) Total RNA was extracted from zebrafish in each experimental group, cDNA was synthesized, and the gene expression of β-actin and target genes was detected by q-PCR.

[0070] (5) Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene.

[0071] 3. Test results

[0072] Table 2

[0073]

[0074] Table 2 and Fig.10 The recombinant type V collagen JYC501 in the experiment is recombinant humanized type V collagen JYC501. The results showed that recombinant humanized type V collagen JYC501 has anti-wrinkle effect. Compared with the normal control group, the relative expression of col1a1a gene was upregulated in the 10, 100, and 1000ppm concentration groups of recombinant humanized type V collagen JYC501, and the gene expression upregulation rates were 8%, 25%, and 92%, respectively. The efficacy increased with the increase of concentration.

[0075] Example 8 Detection of the inhibition of inflammatory factor TNF-α gene expression in zebrafish by recombinant humanized type V collagen JYC501

[0076] 1. Test method:

[0077] According to the standard "Standard Operating Procedure for Zebrafish Soothing Efficacy Evaluation Experiment", the effect of recombinant humanized type V collagen JYC501 in inhibiting the expression of the inflammatory factor TNF-α gene in zebrafish was tested to study its soothing effect.

[0078] Zebrafish tail fin regeneration is divided into three processes: wound healing, blastema formation, and regeneration outcome, with blastema formation being the core. In this process, immune cells play an indispensable protective role in clearing dead cells, preventing microbial invasion, and promoting regeneration of damaged tissues. Neutrophils and macrophages are the two main immune system cells involved in this process. Neutrophils are the first immune cells to arrive at the site of injury. TNF-α is the earliest and most important inflammatory cytokine that appears during the inflammatory response and can activate neutrophils. Therefore, the relative expression of the zebrafish TNF-α gene can be detected to evaluate whether the sample has a soothing effect.

[0079] 2. Experimental Procedure

[0080] (1) The zebrafish tail fin was removed with a scalpel to establish a zebrafish tail fin injury inflammation model.

[0081] (2) Transfer the model zebrafish to a 6-well plate, with 30 zebrafish per well.

[0082] (3) Water-soluble administration samples, normal control group and model control group were set up at the same time, and the volume of each well was 3 mL.

[0083] (4) Incubate at 28°C in the dark for 5 h.

[0084] (5) Total RNA was extracted from zebrafish in each experimental group, cDNA was synthesized, and the gene expression of β-actin and target genes was detected by q-PCR.

[0085] (6) Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene.

[0086] 3. Test results

[0087] Table 3

[0088]

[0089] Table 3 and Fig.11 The recombinant type V collagen JYC501 in the experiment is recombinant humanized type V collagen JYC501. The results showed that recombinant humanized type V collagen JYC501 has a soothing effect. Compared with the model control group, the relative expression of TNF-α gene in the recombinant humanized type V collagen JYC501 10, 100, and 1000ppm concentration groups were all downregulated, with the gene expression downregulation rates being 5%, 35%, and 63%, respectively. The efficacy increased with increasing concentration.

Claims

1. A functional short peptide, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. Recombinant humanized type V collagen JYC501, characterized in that: It is composed of 10 tandemly repeated functional short peptides according to claim 1, and its amino acid sequence is shown in SEQ ID No.

2.

3. The gene encoding the recombinant humanized type V collagen JYC501 according to claim 2, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

3.

4. A recombinant expression vector, characterized in that: It is a recombinant expression vector pPIC9K-JYC501 containing the gene encoding the recombinant humanized type V collagen JYC501 as claimed in claim 3.

5. The method for constructing the recombinant expression vector according to claim 4, characterized in that: The specific steps are as follows: The gene sequence encoding the recombinant humanized type V collagen JYC501 described in claim 3 is amplified by high-fidelity PCR, and the target fragment after gel recovery and purification is seamlessly cloned into the downstream of the α-mating factor secretion signal peptide of the pPIC9K empty vector, and enriched after colony PCR and sequencing verification to obtain the recombinant expression vector pPIC9K-JYC501.

6. The genetically engineered Pichia pastoris for producing the recombinant humanized type V collagen JYC501 according to claim 2, characterized in that: It contains the recombinant expression vector pPIC9K-JYC501 described in claim 4.

7. The method for constructing the genetically engineered Pichia pastoris according to claim 6, characterized in that: Here are the steps: The recombinant expression vector pPIC9K-JYC501 was linearized by restriction digestion and then transformed into Pichia pastoris GS115 competent cells, positive transformants were selected, and high-copy recombinants were screened through G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria that produce recombinant humanized type V collagen JYC501.

8. The method for preparing the recombinant humanized type V collagen JYC501 according to claim 2, characterized in that: Here are the steps: The genetically engineered Pichia pastoris described in claim 6 is inoculated into a BMMY induction medium, induced to express with methanol, and the fermentation supernatant is purified to obtain a highly pure recombinant humanized type V collagen JYC501.

9. A composition or product, characterized in that Comprising the recombinant humanized type V collagen JYC501 as described in claim 2.

10. The composition or product according to claim 9, characterized in that The composition is a skin care product or a pharmaceutical composition, and the product is a medical device, a biomaterial or a tissue engineering product.

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