Recombinant humanized type iv collagen and expression vector and genetically engineered bacteria thereof

Abstract

The application discloses a kind of recombinant humanized IV type collagen and its expression vector and genetically engineered bacteria.The application obtains 3 segments with potential high activity functional peptide segment by screening, which is designed 10 times tandem repeat respectively to constitute recombinant humanized IV type collagen JYC401, JYC402 and JYC403, and after codon optimization, it is amplified and cloned into pPIC9K vector, linearized after enzyme cutting, and transformed into Pichia pastoris cell, and through G418 resistance gradient screening high copy recombinants to obtain the Pichia pastoris genetically engineered bacteria capable of expressing recombinant humanized IV type collagen JYC401, JYC402 and JYC403.The recombinant humanized IV type collagen JYC401, JYC402 and JYC403 of the application have excellent functions of promoting cell adhesion, cell proliferation and migration, and are suitable for preparing skin repair promoting, skin resistance improving skin care products or medical devices and other products.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to a recombinant humanized type IV collagen, its expression vector, and genetically engineered bacteria. Background Technology

[0002] Collagen is an important structural protein, a major component of the extracellular matrix, widely distributed in the human body and playing a variety of vital biological functions. To date, 28 types of collagen have been discovered. A typical characteristic of the collagen family is its triple helix structure composed of three α-chains. Due to its excellent biological and mechanical properties, collagen has found wide application in fields such as medicine, aesthetics, skincare, and biomaterials.

[0003] The basement membrane, present in most human tissues, is a thin, dense layer of the extracellular matrix, primarily serving a structural support function, such as helping cells attach and anchor to underlying tissues. It also acts as a barrier, preventing cancer cells from invading deeper tissues. Furthermore, it plays a crucial regulatory role in cellular behaviors such as proliferation, differentiation, adhesion, and migration. The components of the basement membrane include type IV collagen, type VII collagen, type XVII collagen, laminin, globin, and nestin. Type IV collagen accounts for approximately 50% of the basement membrane components, forming a network structure that constitutes the core structural framework of the basement membrane and is closely related to its biological functions.

[0004] Type IV collagen is a non-fibrillary collagen with six genetically distinct α chains, α1(IV)-α6(IV), forming three different heterotrimers: α1α1α2, α3α4α5, and α5α5α6. α1(IV)2α2(IV) is the predominant isoform, ubiquitous in the basement membrane of all epithelial tissues. The different α chains share similar domain structures. Each α chain is approximately 400 nm long and can be divided into three domains: a short N-terminal non-collagenous 7S domain (26 kDa, approximately 28 nm in length), a long intermediate triple-helical collagen domain (120 kDa, approximately 320 nm in length or approximately 1400 amino acids), and a C-terminal globular non-collagenous domain NC1 (25 kDa, approximately 52 nm in length or approximately 230 amino acids). Uniquely, the central helical portion of the α-chain, containing 1400 Gly-XY sequences, is interrupted by approximately 20 short non-collagenous regions, thus providing the molecule with flexibility and consequently conferring basement membrane flexibility. The 7S domain is named for its sedimentation properties during centrifugation after proteolytic digestion of type IV collagen. Both the 7S and NC1 domains participate in the formation of the type IV collagen network. The triple-helix collagen domain includes a CB3 region, which studies have shown can bind to cytokines, thereby playing a role in promoting cell adhesion and other functions; therefore, it is widely used in the structural and functional research of type IV collagen. Summary of the Invention

[0005] This invention screens the CB3 domain, which binds to cytokines, and other triple-helix collagen domains in the α1 chain of human type IV collagen to obtain three potentially highly active functional peptides. These peptides are then tandemly repeated 10 times to form recombinant humanized type IV collagen JYC401, JYC402, and JYC403. After codon optimization, these peptides are amplified and cloned into the pPIC9K vector, linearized by enzyme digestion, and transformed into Pichia pastoris cells. High-copy recombinants are selected using the G418 resistance gradient to obtain high-copy Pichia pastoris genetically engineered strains that can express recombinant humanized type IV collagen JYC401, JYC402, and JYC403, respectively.

[0006] The technical solution of the present invention is as follows:

[0007] A functional short peptide, whose sequence is 100% identical to that of human type IV collagen and has potential activity in binding to cytokines, has the amino acid sequence shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

[0008] Recombinant humanized type IV collagen, namely JYC401, JYC402 or JYC403, is composed of 10 tandemly repeated amino acid sequences as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3, and their amino acid sequences are shown in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

[0009] The genes encoding the above-mentioned recombinant humanized type IV collagen JYC401, JYC402 or JYC403 have nucleotide sequences as shown in SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9.

[0010] A recombinant expression vector is a recombinant expression vector containing the gene encoding recombinant humanized type IV collagen JYC401, JYC402, or JYC403. In a specific embodiment of the present invention, the recombinant expression vectors pPIC9K-JYC401, pPIC9K-JYC402, or pPIC9K-JYC403 containing the gene encoding recombinant humanized type IV collagen JYC401, JYC402, or JYC403 are used as examples.

[0011] The specific steps for constructing the above-mentioned recombinant expression vector are as follows:

[0012] The gene sequences encoding recombinant humanized type IV collagen JYC401, JYC402, or JYC403, as shown in SEQ ID No. 7, SEQ ID No. 8, or SEQ ID No. 9, were amplified by high-fidelity PCR. The target fragments, purified by gel recovery, were seamlessly cloned downstream of the α-mating factor secretion signal peptide of the empty pPIC9K vector. After verification by colony PCR and sequencing, the fragments were enriched to obtain the recombinant expression vectors pPIC9K-JYC401, pPIC9K-JYC402, or pPIC9K-JYC403.

[0013] The Pichia pastoris genetically engineered strains that produce the aforementioned recombinant humanized type IV collagen JYC401, JYC402, or JYC403 contain the aforementioned recombinant expression vectors. In a specific embodiment of the present invention, an example is taken containing the aforementioned recombinant expression vectors pPIC9K-JYC401, pPIC9K-JYC402, or pPIC9K-JYC403.

[0014] The construction method of the above-mentioned Pichia pastoris genetically engineered strain is as follows:

[0015] The recombinant expression vector was linearized by enzyme digestion and transformed into Pichia pastoris GS115 competent cells. Positive transformants were selected, and high-copy recombinants were screened using the G418 resistance gradient to obtain Pichia pastoris genetically engineered strains that produce recombinant humanized type IV collagen JYC401, JYC402, or JYC403.

[0016] In a specific embodiment of the present invention, the method for constructing the above-mentioned Pichia pastoris genetically engineered strain comprises the following steps:

[0017] The recombinant expression vectors pPIC9K-JYC401, pPIC9K-JYC402, or pPIC9K-JYC403 were linearized by enzyme digestion and transformed into Pichia pastoris GS115 competent cells. Positive transformants were selected, and high-copy recombinants were screened using the G418 resistance gradient to obtain Pichia pastoris genetically engineered strains that produce recombinant humanized type IV collagen JYC401, JYC402, or JYC403.

[0018] The preparation methods for the above-mentioned recombinant humanized type IV collagen JYC401, JYC402, or JYC403 are as follows:

[0019] The Pichia pastoris genetically engineered strains that produce recombinant humanized type IV collagen JYC401, JYC402, or JYC403 were inoculated into BMMY induction medium, induced to express with methanol, and the fermentation supernatant was purified to obtain high-purity recombinant humanized type IV collagen JYC401, JYC402, or JYC403.

[0020] A composition comprising one or more of the above-mentioned recombinant humanized type IV collagen JYC401, JYC402 or JYC403, wherein the composition includes, but is not limited to, skin care products, pharmaceutical compositions, etc.

[0021] An article comprising one or more of the above-mentioned recombinant humanized type IV collagen JYC401, JYC402 or JYC403, the article including but not limited to medical devices, biomaterials, tissue engineering products, etc.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] This invention screened the CB3 domain, which binds to cytokines, and other triple-helix collagen domains in the α1 chain of human type IV collagen to obtain three potentially highly active short peptide sequences. These sequences were then tandemly repeated 10 times to obtain recombinant humanized type IV collagen JYC401, JYC402, and JYC403, which promote cell adhesion, proliferation, and migration. Cell experiments showed that recombinant humanized type IV collagen JYC401, JYC402, and JYC403 all exhibited excellent efficacy in promoting the adhesion, proliferation, and migration of human fibroblasts and epidermal cells, with JYC401 and JYC402 showing superior efficacy compared to JYC403. Furthermore, this invention optimizes the coding gene sequences of recombinant humanized type IV collagen JYC401, JYC402, and JYC403 according to the codon preference of Pichia pastoris, constructs recombinant expression vectors for each, and transforms them into Pichia pastoris strains. High-copy recombinants are selected using the G418 resistance gradient to obtain high-copy Pichia pastoris genetically engineered strains capable of producing recombinant humanized type IV collagen JYC401, JYC402, and JYC403, respectively, thus achieving large-scale production of recombinant humanized type IV collagen JYC401, JYC402, and JYC403. The recombinant humanized type IV collagen JYC401, JYC402, and JYC403 of this invention can be used to promote the repair of skin and other tissues, enhance skin resistance, and are suitable for the preparation of skincare or medical device products. Attached Figure Description

[0024] Figure 1 Schematic diagram of plasmid structures expressing recombinant humanized type IV collagen JYC401(a), JYC402(b), and JYC403(c).

[0025] Figure 2 Electrophoresis images of plasmids expressing recombinant humanized type IV collagen JYC401, JYC402, and JYC403, where (a) lane 1: linearized fragment of pPIC9K empty vector plasmid; lane 2: linearized fragment of pPIC9K+ recombinant humanized type IV collagen JYC401 plasmid; (b) lane 1: linearized fragment of pPIC9K empty vector plasmid; lane 2: linearized fragment of pPIC9K+ recombinant humanized type IV collagen JYC402 plasmid; (c) lane 1: linearized fragment of pPIC9K empty vector plasmid; lane 2: linearized fragment of pPIC9K+ recombinant humanized type IV collagen JYC403 plasmid.

[0026] Figure 3SDS-PAGE images of the supernatant of Pichia pastoris genetically engineered strains (induced with methanol for 72 hours) for the production of recombinant humanized type IV collagen JYC401, JYC402 and JYC403. In Figure (a), lanes 2-3: SDS-PAGE of supernatant of high copy strain JYC401, lane 1: fermentation supernatant of blank control strain; (b) lanes 1-8: SDS-PAGE of supernatant of high copy strain JYC402, lane 9: fermentation supernatant of blank control strain; (c) lanes 2-4: SDS-PAGE of supernatant of high copy strain JYC403, lane 1: fermentation supernatant of blank control strain.

[0027] Figure 4 Figure showing the effects of recombinant humanized type IV collagen JYC401, JYC402, and JYC403 on the proliferation of epidermal cells and fibroblasts.

[0028] Figure 5 Figure showing the effects of recombinant humanized type IV collagen JYC401, JYC402, and JYC403 on the migration of epidermal cells and fibroblasts.

[0029] Figure 6 Figure showing the effects of recombinant humanized type IV collagen JYC401, JYC402, and JYC403 on the adhesion of epidermal cells and fibroblasts. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below through preferred embodiments of the present invention. However, the following embodiments do not limit the scope of protection of the present invention.

[0031] In the embodiments of the present invention, unless otherwise described, conventional molecular biology experimental methods were used. The processes involved in the embodiments, such as PCR, enzyme digestion, seamless cloning, and codon optimization, are all understandable and easily implemented by those skilled in the art based on the product manual or basic knowledge in the field, and therefore will not be described in detail.

[0032] Example 1: Design and construction of high-copy Pichia pastoris genetically engineered strains containing recombinant humanized type IV collagen JYC401, JYC402, and JYC403.

[0033] (1) Using bioinformatics and protein structure prediction database tools (NCBI, AlphaFold), functional short peptides that can bind to cytokines were screened from the α1 chain of human type IV collagen. Their amino acid sequences are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively. After being repeated 10 times in tandem, they constituted recombinant humanized type IV collagen JYC401, JYC402 and JYC403, respectively. Their amino acid sequences are shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6.

[0034] (2) The amino acid sequences of the recombinant humanized type IV collagen JYC401, JYC402 and JYC403 obtained in step (1) were codon optimized according to the codon preference of Pichia pastoris to obtain the optimized nucleotide sequences of recombinant humanized type IV collagen JYC401, JYC402 and JYC403, as shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, respectively.

[0035] (3) The nucleotide sequences from step (2) were entrusted to Shanghai Qingke Biotechnology Co., Ltd. for gene synthesis. The gene sequence of recombinant humanized type IV collagen JYC401 shown in SEQ ID No. 7 was amplified using primer P1 (nucleotide sequence as shown in SEQ ID No. 10) and primer P2 (nucleotide sequence as shown in SEQ ID No. 11). The gene sequence of recombinant humanized type IV collagen JYC402 shown in SEQ ID No. 8 was amplified using primer P3 (nucleotide sequence as shown in SEQ ID No. 12) and primer P4 (nucleotide sequence as shown in SEQ ID No. 13). The gene sequence of SEQ ID No. 8 was amplified using primer P5 (nucleotide sequence as shown in SEQ ID No. 14) and primer P6 (nucleotide sequence as shown in SEQ ID No. 15). The gene sequence of recombinant humanized type IV collagen JYC403 shown in No. 9 was used. The high-fidelity PCR amplification product was purified by nucleic acid gel electrophoresis and seamlessly cloned downstream of the α-mating factor secretion signal peptide of the pPIC9K plasmid using Gibson assembly technology. After verification by colony PCR and sequencing, the recombinant plasmids pPIC9K-JYC401, pPIC9K-JYC402, and pPIC9K-JYC403 were enriched. The structures of the recombinant plasmids pPIC9K-JYC401, pPIC9K-JYC402, and pPIC9K-JYC403 are shown below. Figure 1 As shown, the plasmid electrophoresis image is as follows: Figure 2As shown. Next, the recombinant plasmids pPIC9K-JYC401, pPIC9K-JYC402, and pPIC9K-JYC403 were digested with SalI restriction endonuclease (purchased from NEB, specific procedures followed the kit instructions) overnight at 37°C. The linearized recombinant expression plasmids were then purified using a gel extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) and electroporated into Pichia pastoris GS115 competent cells. The electroporated cells were activated in fresh YPD medium for 2-3 hours, then plated onto YNB selective medium and incubated upside down at 30°C for 2-3 days to allow transformants to grow. First, single-clone transformations were verified by colony PCR. Verified transformants were plated onto plates containing 1 g / L, 2 g / L, 3 g / L, and 4 g / L of [agar / concentrate / medium]. After culturing on G418 screening plates for 2-4 days, transformants with good growth on high-concentration G418 plates were selected for purification and rescreening verification. After genome extraction and verification of the transformants obtained from the rescreening, high-copy Pichia pastoris genetically engineered strains expressing recombinant humanized type IV collagen JYC401, JYC402 and JYC403 were obtained and preserved with glycerol.

[0036] (4) The high-copy Pichia pastoris genetically engineered strain obtained in step (3) was inoculated into 30 mL of BMGY medium (which consists 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, with the remainder being water), and cultured for 16-20 hours. Then, the initial OD was measured. 600 Inoculation was performed at a rate of 1 in 30 mL of BMMY medium (composed of 20 g / L peptone, 10 g / L yeast extract, 100 mL of 10×YNB, 100 mL of 10×potassium phosphate buffer, pH 6.0, with the remainder being water). Every 24 hours, 300 μL of filtered sterilized methanol was added. After 72 hours, 2 mL of sample was collected by centrifugation, and the supernatant was collected. 500 μL of the supernatant was concentrated in a 10 kDa ultrafiltration tube to obtain approximately 50 μL of concentrate. 24 μL of the concentrate was mixed with 6 μL of 5× loading buffer and treated in boiling water for 10–15 min. After centrifugation for 15 min, 10–15 μL of the supernatant was loaded onto an SDS-PAGE sample for qualitative analysis of recombinant humanized type IV collagen JYC401, JYC402, and JYC403. The SDS-PAGE results are shown below. Figure 3 ,from Figure 3 It is known that high-copy Pichia pastoris genetically engineered strains capable of successfully expressing recombinant humanized type IV collagen JYC401, JYC402 and JYC403 were obtained.

[0037] Example 2: Detection of cell proliferation activity of recombinant humanized type IV collagen JYC401, JYC402 and JYC403

[0038] The MTT assay was used to detect the ability of recombinant humanized type IV collagen JYC401, JYC402, and JYC403 to promote cell proliferation, using human epidermal cells and human fibroblasts as experimental cells, respectively. The results are as follows: Figure 4 As shown in the figure, the blank group was a control experiment where no components were added and only cell culture medium was used. It can be seen that recombinant humanized type IV collagen JYC401, JYC402, and JYC403 at concentrations of 10 ppm, 100 ppm, and 1000 ppm all exhibited excellent cell proliferation-promoting activity, and the promoting effect increased with increasing concentrations of recombinant humanized type IV collagen JYC401, JYC402, and JYC403. Furthermore, compared to recombinant humanized type IV collagen JYC403, recombinant humanized type IV collagen JYC401 and JYC402 showed even better cell proliferation-promoting activity.

[0039] Example 3: Detection of cell migration-promoting activities of recombinant humanized type IV collagen JYC401, JYC402 and JYC403

[0040] Cell migration was measured using the cell scratch assay. Human epidermal cells and human fibroblasts were used as experimental cells to detect the ability of recombinant humanized type IV collagen JYC401, JYC402, and JYC403 to promote cell migration. Results are as follows: Figure 5 As shown in the figure, the blank group was a control experiment where no components were added and only cell culture medium was used. It can be seen that recombinant humanized type IV collagen JYC401, JYC402, and JYC403 at concentrations of 10 ppm, 100 ppm, and 1000 ppm all exhibited excellent cell migration-promoting activities, and the promoting effect increased with increasing concentrations of recombinant humanized type IV collagen JYC401, JYC402, and JYC403. Furthermore, compared to recombinant humanized type IV collagen JYC403, recombinant humanized type IV collagen JYC401 and JYC402 showed even better cell migration-promoting activities.

[0041] Example 4: Detection of cell adhesion-promoting activities of recombinant humanized type IV collagen JYC401, JYC402 and JYC403

[0042] Using centrifugation, human epidermal cells and human fibroblasts were used as experimental cells to detect the ability of recombinant humanized type IV collagen JYC401, JYC402, and JYC403 to promote cell adhesion. The results are as follows: Figure 6As shown in the figure, the blank group was a control experiment where no components were added and only cell culture medium was used. It can be seen that recombinant humanized type IV collagen JYC401, JYC402, and JYC403 at concentrations of 10 ppm, 100 ppm, and 1000 ppm all exhibited excellent cell adhesion-promoting activities, and the promoting effect increased with increasing concentrations of recombinant humanized type IV collagen JYC401, JYC402, and JYC403. Furthermore, compared to recombinant humanized type IV collagen JYC403, recombinant humanized type IV collagen JYC401 and JYC402 showed even better cell adhesion-promoting activities.

Claims

1. Recombinant humanized type IV collagen, characterized in that, The recombinant humanized type IV collagen JYC401, JYC402, or JYC403 is described in SEQ ID No. 4, JYC402 in SEQ ID No. 5, and JYC403 in SEQ ID No.

6.

2. The gene encoding the recombinant humanized type IV collagen as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding recombinant humanized type IV collagen JYC401 is shown in SEQ ID No. 7, the nucleotide sequence of the gene encoding recombinant humanized type IV collagen JYC402 is shown in SEQ ID No. 8, and the nucleotide sequence of the gene encoding recombinant humanized type IV collagen JYC403 is shown in SEQ ID No.

9.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the gene encoding recombinant humanized type IV collagen JYC401, JYC402 or JYC403 as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vectors pPIC9K-JYC401, pPIC9K-JYC402, or pPIC9K-JYC403 containing the gene encoding recombinant humanized type IV collagen JYC401, JYC402, or JYC403 as described in claim 2 are constructed through the following steps: The gene sequences encoding recombinant humanized type IV collagen JYC401, JYC402, or JYC403, as shown in SEQ ID No. 7, SEQ ID No. 8, or SEQ ID No. 9, were amplified by high-fidelity PCR. The target fragments, purified by gel recovery, were seamlessly cloned downstream of the α-mating factor secretion signal peptide of the empty pPIC9K vector. After verification by colony PCR and sequencing, the fragments were enriched to obtain the recombinant expression vectors pPIC9K-JYC401, pPIC9K-JYC402, or pPIC9K-JYC403.

5. A Pichia pastoris genetically engineered strain for producing recombinant humanized type IV collagen as described in claim 1, characterized in that, Contains the recombinant expression vector as described in claim 3 or 4.

6. The method for constructing Pichia pastoris genetically engineered strain according to claim 5, characterized in that, The steps are as follows: The recombinant expression vector described in claim 3 or 4 is linearized by enzyme digestion and transformed into Pichia pastoris GS115 competent cells. Positive transformants are selected, and high-copy recombinants are screened by G418 resistance gradient to obtain Pichia pastoris genetically engineered strains that produce recombinant humanized type IV collagen JYC401, JYC402, or JYC403.

7. The method for preparing recombinant humanized type IV collagen according to claim 1, characterized in that, The steps are as follows: The Pichia pastoris genetically engineered strain for producing recombinant humanized type IV collagen JYC401, JYC402, or JYC403 as described in claim 5 was inoculated into BMMY induction medium, and expressed by methanol induction. The fermentation supernatant was then purified to obtain high-purity recombinant humanized type IV collagen JYC401, JYC402, or JYC403.

8. A composition, characterized in that, It contains one or more of the recombinant humanized type IV collagen JYC401, JYC402 or JYC403 as described in claim 1.

9. An article, characterized in that, It contains one or more of the recombinant humanized type IV collagen JYC401, JYC402 or JYC403 as described in claim 1.

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

11. The article of claim 9, characterized in that, The product in question is a medical device.

12. The article of claim 9, characterized in that, The product in question is a biological material.

13. The article of claim 9, characterized in that, The product in question is a tissue-engineered product.

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