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

Abstract

The present invention discloses a recombinant humanized type I collagen protein and its expression vector and genetically engineered bacteria. The present invention screens and combines the functional domains of human type I collagen protein that bind to cell integrins and fibronectin, or the carboxyl terminal peptide to form recombinant humanized type I collagen proteins JYC103 and JYC104, and amplifies and clones them into the pPIC9K vector after codon optimization, and transforms them into Pichia pastoris cells after enzyme linearization, and screens high-copy recombinants through G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria that can express recombinant humanized type I collagen proteins JYC103 and JYC104. The recombinant humanized type I collagen proteins JYC103 and JYC104 of the present invention exhibit excellent functions of promoting cell adhesion, cell proliferation and migration, can be used to promote the repair of tissues such as skin, enhance the resistance of the skin, and are suitable for preparing products such as skin care or medical devices.

Description

Technical Field

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

[0002] Type I collagen is the most abundant component in dermal connective tissue, accounting for more than 80% of the skin. Type I collagen molecules are long and tough right-handed triple helical structures, and can spontaneously assemble into a supramolecular advanced structure - collagen fibers, which are widely distributed in the extracellular matrix.

[0003] The gene of type I procollagen is about 40kbp, and the monomeric α peptide chain contains 1464 amino acid residues. The type I procollagen polypeptide chain is translated and synthesized by ribosomes on the rough endoplasmic reticulum. A signal peptide sequence consisting of 22 amino acids located at the amino terminal will be recognized by the signal peptide receptor on the endoplasmic reticulum membrane, and then the signal peptide will be cut and removed by the signal peptidase in the cavity. Before being secreted into the extracellular space, type I procollagen needs to undergo a series of post-translational modifications in the endoplasmic reticulum, such as the hydroxylation of proline and lysine. Subsequently, under the action of the N-terminal and C-terminal propeptides, nucleation begins from the C-terminus, and type I procollagen with the correct triple helix structure is formed by the action of chaperone proteins and enzymes in the endoplasmic reticulum and enters the Golgi apparatus, and is finally secreted into the extracellular space in the form of secretory vesicles. In the extracellular matrix, type I procollagen will be enzymatically cleaved by specific proteases to remove the N-propeptide and C-propeptide, thereby forming mature type I collagen containing the terminal peptide and triple helix region. The hydrophobic and electrostatic interactions between type I collagen monomers help the monomers form a 1 / 4 embedded structure, which then aggregates into a five-chain fiber structure, and forms a higher-level fiber structure based on this. Type I collagen fibers with advanced fiber structures have different orientations in different tissues. For example, in tendons, type I collagen fibers are arranged in parallel to form fiber bundles, while in skin, they form a complex mesh structure with random orientations. Among them, the telopeptide plays a key role in the formation of intermolecular cross-links of fibers. These cross-linked structures are mainly Schiff base structures formed by the condensation of the aldehyde groups of lysine residues on adjacent telopeptides, which give collagen fibers certain physical and mechanical properties.

[0004] There are also many key functional domains involved in life activities in type I collagen, such as the classic GER / GEK integrin binding site, GVMGFP discoidin domain receptor binding motif, etc. Among them, the functional domain of type I collagen binding to fibronectin plays an irreplaceable role in the formation of fibronectin fibers. Studies have shown that mutation of the fibronectin binding site in type I collagen will lead to the formation of short fibronectin fibers. Fibronectin is an important component of the extracellular matrix and is widely involved in cell migration, adhesion, proliferation, hemostasis and tissue repair. It can also mobilize monocytes to remove harmful substances from damaged tissues and has a function similar to that of growth factors.

[0005] With the rapid development of synthetic biology, artificial design and construction of biological systems has become the mainstream trend of biology today, and the de novo design of gene element modules is the foundation of synthetic biology. Based on this concept, combined with the rational transformation strategy of protein engineering, the rational design of recombinant humanized type I collagen with high biological performance and its efficient expression in heterologous hosts will greatly contribute to the application of type I collagen in high-end skin care products, medical beauty and tissue engineering. Pichia pastoris is the most common eukaryotic expression system after Escherichia coli. Thousands of proteins have been successfully expressed in the Pichia pastoris system, and it has also been recognized as a GRAS (Generally Recognized As Safe) microorganism by the US FDA. In addition, Pichia pastoris also has a systematic gene editing system and a complete protein secretion expression mechanism, and is a heterologous host widely used in the fields of food and medicine. Therefore, the secretion expression of recombinant humanized type I collagen by Pichia pastoris will greatly promote the development of type I collagen in the fields of beauty and skin care, medical devices and tissue engineering. Summary of the invention

[0006] The present invention screens and combines functional domains or carboxyl terminal peptides in human type I collagen that bind to cell integrins and fibronectin to form recombinant humanized type I collagen JYC103 and JYC104, amplifies and clones them into a pPIC9K vector after codon optimization, linearizes them after enzyme digestion, and transforms them into Pichia pastoris cells. High-copy recombinants are screened through a G418 resistance gradient to obtain high-copy Pichia pastoris genetic engineering strains that can express recombinant humanized type I collagen JYC103 and JYC104.

[0007] The technical solution of the present invention is as follows:

[0008] Recombinant humanized type I collagen JYC103 or JYC104, wherein the recombinant humanized type I collagen JYC103 comprises a functional domain for human type I collagen to bind to cell integrins and fibronectin, and its amino acid sequence is as shown in SEQ ID No.1; the recombinant humanized type I collagen JYC104 comprises a functional domain for human type I collagen to bind to cell integrins and fibronectin as shown in SEQ ID No.1 and a carboxyl terminal peptide as shown in SEQ ID No.3, and its amino acid sequence is as shown in SEQ ID No.4.

[0009] The gene encoding the above-mentioned recombinant humanized type I collagen JYC103 or JYC104, the nucleotide sequence of the gene encoding the above-mentioned recombinant humanized type I collagen JYC103 is shown as SEQ ID No.5, and the nucleotide sequence of the gene encoding the above-mentioned recombinant humanized type I collagen JYC104 is shown as SEQ ID No.8.

[0010] A recombinant expression vector, which is a recombinant expression vector pPIC9K-JYC103 or pPIC9K-JYC104 containing a gene encoding recombinant humanized type I collagen JYC103 as shown in SEQ ID No.5 or a gene encoding recombinant humanized type I collagen JYC104 as shown in SEQ ID No.8.

[0011] The method for constructing the above-mentioned recombinant expression vector comprises the following specific steps:

[0012] The gene sequence encoding the recombinant humanized type I collagen JYC103 shown in SEQ ID No.5 or the gene sequence encoding the recombinant humanized type I collagen JYC104 shown in SEQ ID No.8 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, and enriched after colony PCR and sequencing verification to obtain the recombinant expression vector pPIC9K-JYC103 or pPIC9K-JYC104.

[0013] The genetically engineered Pichia pastoris bacteria for producing the above-mentioned recombinant humanized type I collagen JYC103 or JYC104 contains the above-mentioned recombinant expression vector pPIC9K-JYC103 or pPIC9K-JYC104.

[0014] The construction method of the above-mentioned Pichia pastoris genetically engineered bacteria comprises the following steps:

[0015] The recombinant expression vector pPIC9K-JYC103 or pPIC9K-JYC104 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 I collagen JYC103 or JYC104.

[0016] The preparation method of the above-mentioned recombinant humanized type I collagen JYC103 or JYC104 comprises the following steps:

[0017] The genetically engineered Pichia pastoris producing the recombinant humanized type I collagen JYC103 or JYC104 is inoculated into BMMY induction medium, induced to express with methanol, and the fermentation supernatant is purified to obtain high-purity recombinant humanized type I collagen JYC103 or JYC104.

[0018] A composition comprising the above-mentioned recombinant humanized type I collagen JYC103 or JYC104, wherein the composition includes but is not limited to skin care products, pharmaceutical compositions, etc.

[0019] A product comprising the above-mentioned recombinant humanized type I collagen JYC103 or JYC104, the product including but not limited to medical devices, biomaterials, tissue engineering products, etc.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The present invention obtains a recombinant humanized type I collagen JYC103 having the function of promoting fibronectin fiber formation, cell adhesion, cell proliferation and migration by screening the sequence of human type I collagen, and further rationally combines it with the carboxyl terminal peptide on this basis to obtain a recombinant humanized type I collagen JYC104 that can further enhance the proliferation, migration and adhesion of epidermal cells and fibroblasts. Cell experiments have found that the effect of recombinant humanized type I collagen JYC104 is significantly better than that of recombinant humanized type I collagen JYC102 containing only the integrin binding domain, and recombinant humanized type I collagen JYC103 containing the integrin and fibronectin binding domains. In addition, the present invention optimizes the coding gene sequences of recombinant humanized type I collagen JYC103 and JYC104 according to the codon preference of Pichia pastoris, constructs a recombinant expression vector, and transfers it into a Pichia pastoris strain, and screens high-copy recombinants through a G418 resistance gradient to obtain high-copy Pichia pastoris genetic engineering bacteria capable of producing recombinant humanized type I collagen JYC103 and JYC104, thereby achieving large-scale production of recombinant humanized type I collagen JYC103 and JYC104. The recombinant humanized type I collagen JYC103 or JYC104 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

[0022] Figure 1 Schematic diagram of the plasmids expressing recombinant humanized type I collagen JYC103 and JYC104.

[0023] Figure 2The electrophoresis diagram of plasmids expressing recombinant humanized type I collagen JYC103 and JYC104, wherein lane 1: linearized fragment of pPIC9K empty plasmid; lane 2: linearized fragment of pPIC9K+recombinant humanized type I collagen JYC103 plasmid; lane 3: linearized fragment of pPIC9K+recombinant humanized type I collagen JYC104 plasmid.

[0024] Figure 3 This 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 I collagen JYC103 and JYC104, wherein lanes 1 to 5: SDS-PAGE of the supernatant of the high-copy strain JYC104; lanes 6 to 9: SDS-PAGE of the supernatant of the high-copy strain JYC103.

[0025] Figure 4 The effects of recombinant humanized type I collagen JYC103, JYC104 and JYC102 on the proliferation of epidermal cells and fibroblasts.

[0026] Figure 5 The effects of recombinant humanized type I collagen JYC103, JYC104 and JYC102 on the migration of epidermal cells and fibroblasts.

[0027] Figure 6 The effects of recombinant humanized type I collagen JYC103, JYC104 and JYC102 on the adhesion of epidermal cells and fibroblasts. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] Example 1: Design and construction of high-copy Pichia pastoris genetically engineered bacteria of recombinant humanized type I collagen JYC103, JYC104 and JYC102

[0031] (1) Bioinformatics and protein structure prediction database tools (NCBI, AlphaFold) were used to screen the functional domains that can bind to cell integrins and fibronectin from human type I collagen. The amino acid sequence of the domain is shown in SEQ ID No. 1, and the domain is named as recombinant humanized type I collagen JYC103. The domain that only contains the functional domain that binds to cell integrins is shown in SEQ ID No. 2, and the domain is named as recombinant humanized type I collagen JYC102.

[0032] (2) The carboxyl-terminal end peptide of the amino acid sequence shown in SEQ ID No. 3 is conjugated to the carboxyl-terminal end of the functional domain that binds to cell integrin and fibronectin in human type I collagen in (1), and the resulting amino acid sequence is shown in SEQ ID No. 4, which is named recombinant humanized type I collagen JYC104.

[0033] (3) The amino acid sequences of the recombinant humanized type I collagen JYC103, JYC104 and JYC102 obtained in steps (1) and (2) are codon-optimized according to the codon preference of Pichia pastoris, so as to obtain the optimized nucleotide sequences of the recombinant humanized type I collagen JYC103, JYC104 and JYC102 as SEQ ID No. 5, SEQ ID No. 8 and SEQ ID No. 6, respectively.

[0034] (4) The nucleotide sequence in step (3) was entrusted to Shanghai Qingke Biotechnology Co., Ltd. for gene synthesis. The recombinant humanized type I collagen JYC103 gene sequence shown in SEQ ID No. 5 was amplified using primer P1 (nucleotide sequence as shown in SEQ ID No. 9) and primer P2 (nucleotide sequence as shown in SEQ ID No. 10). The recombinant humanized type I collagen JYC104 gene sequence shown in SEQ ID No. 8 was amplified using primer P3 (nucleotide sequence as shown in SEQ ID No. 11) and primer P4 (nucleotide sequence as shown in SEQ ID No. 12). The recombinant humanized type I collagen JYC102 gene sequence shown in SEQ ID No. 6 was amplified using primer P5 (nucleotide sequence as shown in SEQ ID No. 13) and primer P2 (nucleotide sequence as shown in SEQ ID No. 10). The high-fidelity PCR amplification product was purified by nucleic acid gel purification and then purified by Gibson PCR. The assembly technology was seamlessly cloned into the downstream of the α-mating factor secretion signal peptide of the pPIC9K plasmid, and the recombinant plasmid was enriched after verification by colony PCR and sequencing (such as Figure 1 and 2The plasmid was purified by gel recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) and then electroporated into Pichia pastoris GS115 competent cells. The electroporated cells were activated and cultured with fresh YPD medium for 2-3 hours. Then, the cells were plated into YNB selection medium and placed in an incubator at 30°C for inversion culture for 2-3 days to grow transformants. First, the transformant monoclones were verified by colony PCR. The verified transformants were plated onto screening plates containing 1g / L, 2g / L, 3g / L and 4g / L G418, respectively. After 2-4 days of culture, the transformants with good growth on the high-concentration G418 plates were selected for purification and re-screening. The genome of the re-screened transformants was extracted and verified to be correct before preservation in glycerol.

[0035] The obtained high-copy Pichia genetic engineering strain was inoculated into 30 mL 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) and cultured for 16-20 hours. 600 The 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 I collagen JYC103 and JYC104. The results of SDS-PAGE detection are shown in Figure 3 .

[0036] from Figure 3 It can be seen that the present invention obtains a high-copy Pichia pastoris genetic engineering strain that can successfully express recombinant humanized type I collagen JYC103 and JYC104.

[0037] Example 2 Detection of the cell proliferation activity of recombinant humanized type I collagen JYC103, JYC104 and JYC102

[0038] The MTT method was used to detect the ability of recombinant humanized type I collagen JYC103, JYC104 and JYC102 to promote cell proliferation using human epidermal cells and human fibroblasts as experimental cells. Figure 4 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 recombinant humanized type I collagen JYC103, JYC104 and JYC102 all exhibit cell proliferation-promoting activity, and recombinant humanized type I collagen JYC104 exhibits the best cell proliferation-promoting activity.

[0039] Example 3 Detection of the cell migration promoting activity of recombinant humanized type I collagen JYC103, JYC104 and JYC102

[0040] The cell scratch method was used to measure cell migration. Human epidermal cells and human fibroblasts were used as experimental cells to detect the ability of recombinant humanized type I collagen JYC103, JYC104 and JYC102 to promote cell migration. Figure 5 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 recombinant humanized type I collagen JYC103, JYC104 and JYC102 all exhibit cell migration-promoting activity, and recombinant humanized type I collagen JYC104 exhibits the best cell migration-promoting activity.

[0041] Example 4 Detection of cell adhesion promoting activity of recombinant humanized type I collagen JYC103, JYC104 and JYC102

[0042] The centrifugation method was used to detect the ability of recombinant humanized type I collagen JYC103, JYC104 and JYC102 to promote cell adhesion using human epidermal cells and human fibroblasts as experimental cells. Figure 6 As shown, the blank group is a control experiment in which no components are added and only cell culture medium is used for culture. It can be seen that recombinant humanized type I collagen JYC103, JYC104 and JYC102 all exhibit cell adhesion promoting activity, and recombinant humanized type I collagen JYC104 exhibits the best cell adhesion promoting activity.

[0043] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as

[0044] The scope of protection of the present invention shall be based on the scope defined by the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Recombinant humanized type I collagen JYC103 or JYC104, characterized in that: The amino acid sequence of the recombinant humanized type I collagen JYC103 is shown in SEQ ID No.1; the amino acid sequence of the recombinant humanized type I collagen JYC104 is shown in SEQ ID No.

4.

2. The gene encoding the recombinant humanized type I collagen JYC103 or JYC104 according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the recombinant humanized type I collagen JYC103 is shown as SEQ ID No.5, and the nucleotide sequence of the gene encoding the recombinant humanized type I collagen JYC104 is shown as SEQ ID No.

8.

3. A recombinant expression vector, characterized in that: It is a recombinant expression vector pPIC9K-JYC103 or pPIC9K-JYC104 containing a gene encoding recombinant humanized type I collagen JYC103 as shown in SEQ ID No.5 or a gene encoding recombinant humanized type I collagen JYC104 as shown in SEQ ID No.

8.

4. The method for constructing a recombinant expression vector according to claim 3, characterized in that: The specific steps are as follows: The gene sequence encoding the recombinant humanized type I collagen JYC103 shown in SEQ ID No.5 or the gene sequence encoding the recombinant humanized type I collagen JYC104 shown in SEQ ID No.8 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, and enriched after colony PCR and sequencing verification to obtain the recombinant expression vector pPIC9K-JYC103 or pPIC9K-JYC104.

5. The genetically engineered Pichia pastoris for producing the recombinant humanized type I collagen JYC103 or JYC104 according to claim 1, characterized in that: It contains the recombinant expression vector pPIC9K-JYC103 or pPIC9K-JYC104 according to claim 3.

6. The method for constructing the genetically engineered Pichia pastoris according to claim 5, characterized in that: Here are the steps: The recombinant expression vector pPIC9K-JYC103 or pPIC9K-JYC104 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 I collagen JYC103 or JYC104.

7. The method for preparing the recombinant humanized type I collagen JYC103 or JYC104 according to claim 1, characterized in that: Here are the steps: The genetically engineered Pichia pastoris of claim 5 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 I collagen JYC103 or JYC104.

8. A composition or product, characterized in that Comprising the recombinant humanized type I collagen JYC103 or JYC104 according to claim 1.

9. The composition or product according to claim 8, characterized in that The composition is a skin care product or a pharmaceutical composition.

10. The composition or product according to claim 8, characterized in that The product is a medical device, a biomaterial or a tissue engineering product.

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