Recombinant humanized IV-type collagen as well as expression vector and genetically engineered bacterium thereof

Abstract

The invention discloses a recombinant humanized IV-type collagen as well as an expression vector and a genetic engineering bacterium thereof. The preparation method comprises the following steps: screening to obtain three functional peptide fragments with potential high activity, designing the functional peptide fragments for 10 times of tandem repetition to respectively form recombinant humanized IV-type collagen JYC401, JYC402 and JYC403, amplifying and cloning the recombinant humanized IV-type collagen JYC401, JYC402 and JYC403 into a pPIC9K vector after codon optimization, transforming the recombinant humanized IV-type collagen JYC401, JYC402 and JYC403 into pichia pastoris cells after enzyme digestion linearization, and screening to obtain the recombinant humanized IV-type collagen. And high-copy recombinants are screened through G418 resistance gradient, so that the pichia pastoris genetically engineered bacteria capable of expressing the recombinant humanized IV-type collagen JYC401, JYC402 and JYC403 are obtained. The recombinant humanized IV type collagen JYC401, JYC402 and JYC403 disclosed by the invention have excellent functions of promoting cell adhesion, cell proliferation and migration, and are suitable for preparing products such as skin care products or medical instruments for promoting repair of tissues such as skin and improving skin resistance.

Description

Technical Field

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

[0002] Collagen is an important structural protein that is a major component of the extracellular matrix. It is widely distributed in the human body and plays a variety of important biological functions. So far, 28 types of collagen have been discovered. The typical feature of the collagen family is that it has a triple helix structure composed of three α chains. Due to its good biological and mechanical properties, collagen has been widely used in medical treatment, medical beauty, skin care, biomaterials and other fields.

[0003] The basement membrane exists in most human tissues. It is a thin, dense layer in the extracellular matrix that mainly plays a structural support role, such as helping cells attach and anchor to the underlying tissue. It also acts as a barrier to prevent cancer cells from invading deep tissues. At the same time, it also has an important regulatory role in cell behaviors such as proliferation, differentiation, adhesion or migration. The components of the basement membrane include type IV collagen, type VII collagen, type XVII collagen, laminin, beaded proteoglycan, entactin, etc. Among them, type IV collagen accounts for about 50% of the basement membrane components. It is interwoven into a mesh structure, forming the core structural skeleton of the basement membrane, and is also closely related to the biological function of the basement membrane.

[0004] Type IV collagen is a non-fibrillar collagen with six genetically different α chains, including α1(IV)-α6(IV), forming three different heterotrimers: α1α1α2, α3α4α5, and α5α5α6, of which α1(IV)2α2(IV) is the main subtype and is ubiquitous in the basement membranes of all epithelial tissues. Different α chains have similar domain structures. Each α chain is about 400 nm long and can be divided into three domains: a short N-terminal non-collagenous 7S domain (26 kDa, about 28 nm in length), a long middle triple helical collagen domain (120 kDa, about 320 nm in length or about 1400 amino acids), and a C-terminal globular non-collagenous domain NC1 (25 kDa, about 52 nm in length or about 230 amino acids). Uniquely, the central helical portion of the α chain, which contains 1400 Gly-XY sequences, is interrupted by approximately 20 short non-collagenous regions, providing flexibility to the molecule and, in turn, to the basement membrane. The 7S domain is named for its sedimentation properties during centrifugation following proteolytic digestion of type IV collagen. Both the 7S and NC1 domains participate in the formation of the type IV collagen network. The triple helical collagen domain contains a CB3 region, and studies have shown that this region can bind to cell integrins, thereby promoting cell adhesion and other functions. Therefore, it is widely used in the structural and functional studies of type IV collagen. Summary of the invention

[0005] The present invention obtains three functional peptide segments with potential high activity by screening the CB3 functional domain and other triple helical collagen domains in the α1 chain of human type IV collagen, and repeats them in series 10 times to form recombinant humanized type IV collagen JYC401, JYC402 and JYC403, 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, so as to obtain high-copy Pichia pastoris genetic engineering strains that can respectively express the recombinant humanized type IV collagen JYC401, JYC402 and JYC403.

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

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

[0008] The recombinant humanized type IV collagen is JYC401, JYC402 or JYC403, which is composed of 10 tandemly repeated functional short peptides as shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3, and its amino acid sequence is shown in SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6.

[0009] The nucleotide sequence of the gene encoding the above-mentioned recombinant humanized type IV collagen JYC401, JYC402 or JYC403 is shown in SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9.

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

[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 IV collagen JYC401, JYC402 or JYC403 shown in SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 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-JYC401, pPIC9K-JYC402 or pPIC9K-JYC403.

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

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

[0015] The above 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 by G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria that produce recombinant humanized type IV collagen JYC401, JYC402 or JYC403.

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

[0017] The above-mentioned recombinant expression vector pPIC9K-JYC401, pPIC9K-JYC402 or pPIC9K-JYC403 is linearized by restriction 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 bacteria that produce recombinant humanized type IV collagen JYC401, JYC402 or JYC403.

[0018] The preparation method of the above-mentioned recombinant humanized type IV collagen JYC401, JYC402 or JYC403 comprises the following steps:

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

[0020] A composition comprising at least one 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] A product comprising at least one of the above-mentioned recombinant humanized type IV collagen JYC401, JYC402 or JYC403, wherein the product includes but is not limited to medical devices, biomaterials, tissue engineering products, etc.

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

[0023] The present invention obtains three short functional peptide sequences with potential high activity by screening the CB3 functional domain and other triple helical collagen domains in the α1 chain of human type IV collagen, and repeats them in series 10 times to obtain recombinant humanized type IV collagen JYC401, JYC402 and JYC403 with the function of promoting cell adhesion, proliferation and migration. Cell experiments have found that recombinant humanized type IV collagen JYC401, JYC402 and JYC403 all show excellent effects of promoting the adhesion, proliferation and migration of human fibroblasts and epidermal cells, and the effects of recombinant humanized type IV collagen JYC401 and JYC402 are better than those of JYC403. In addition, the present 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 respectively, and transfers them into Pichia pastoris strains respectively, and selects high-copy recombinants through G418 resistance gradient to obtain high-copy Pichia pastoris genetic engineering bacteria capable of producing recombinant humanized type IV collagen JYC401, JYC402 and JYC403 respectively, and realizes the large-scale production of recombinant humanized type IV collagen JYC401, JYC402 and JYC403. The recombinant humanized type IV collagen JYC401, JYC402 and JYC403 of the present invention can be used to promote the repair of tissues such as skin, improve the resistance of skin, and are suitable for preparing products such as skin care or medical devices. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 The electrophoresis diagram of plasmids expressing recombinant humanized type IV collagen JYC401, JYC402 and JYC403, wherein (a) Lane 1: pPIC9K empty plasmid linearized fragment; Lane 2: pPIC9K+recombinant humanized type IV collagen JYC401 plasmid linearized fragment; (b) Lane 1: pPIC9K empty plasmid linearized fragment; Lane 2: pPIC9K+recombinant humanized type IV collagen JYC402 plasmid linearized fragment; (c) Lane 1: pPIC9K empty plasmid linearized fragment; Lane 2: pPIC9K+recombinant humanized type IV collagen JYC403 plasmid linearized fragment.

[0026] Figure 3The present invention 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 IV collagen JYC401, JYC402 and JYC403, wherein (a) Lanes 2-3: SDS-PAGE of the supernatant of the high copy strain JYC401, Lane 1: the fermentation supernatant of the blank control strain; (b) Lanes 1-8: SDS-PAGE of the supernatant of the high copy strain JYC402, Lane 9: the fermentation supernatant of the blank control strain; (c) Lanes 2-4: SDS-PAGE of the supernatant of the high copy strain JYC403, Lane 1: the fermentation supernatant of the blank control strain.

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

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

[0029] Figure 6 This figure shows the effects of recombinant humanized type IV collagen JYC401, JYC402 and JYC403 on the adhesion of epidermal cells and fibroblasts. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] Example 1: Design and construction of high-copy Pichia pastoris genetic engineering bacteria of recombinant humanized type IV collagen JYC401, JYC402 and JYC403

[0033] (1) Bioinformatics and protein structure prediction database tools (NCBI, AlphaFold) were used to screen functional short peptides that can bind to cell integrins from the α1 chain of human type IV collagen. The amino acid sequences of these short peptides are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively. After 10 tandem repetitions, recombinant humanized type IV collagen JYC401, JYC402, and JYC403 were formed, respectively. The amino acid sequences of these short peptides are shown in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively.

[0034] (2) The amino acid sequences of the recombinant humanized type IV collagen JYC401, JYC402 and JYC403 obtained in step (1) are codon-optimized according to the codon preference of Pichia pastoris to obtain the optimized nucleotide sequences of the 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 sequence in step (2) was entrusted to Shanghai Qingke Biotechnology Co., Ltd. for gene synthesis, and the gene sequence of the recombinant humanized type IV collagen JYC401 shown in SEQ ID No.7 was amplified using primers P1 (nucleotide sequence as shown in SEQ ID No.10) and primers P2 (nucleotide sequence as shown in SEQ ID No.11), the gene sequence of the recombinant humanized type IV collagen JYC402 shown in SEQ ID No.8 was amplified using primers P3 (nucleotide sequence as shown in SEQ ID No.12) and primers P4 (nucleotide sequence as shown in SEQ ID No.13), and the gene sequence of the recombinant humanized type IV collagen JYC402 shown in SEQ ID No.8 was amplified using primers P5 (nucleotide sequence as shown in SEQ ID No.14) and primers P6 (nucleotide sequence as shown in SEQ ID No.15). The gene sequence of the recombinant humanized type IV collagen JYC403 shown in No.9 was obtained. 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 the 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 in Figure 1 As shown, the plasmid electrophoresis diagram is as follows Figure 2As shown. Then, the recombinant plasmids pPIC9K-JYC401, pPIC9K-JYC402 and pPIC9K-JYC403 were 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 plasmids were purified using a 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, 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 single clone was verified by colony PCR, and the verified correct transformants were spread on 1g / L, 2g / L, 3g / L and 4g / L On the G418 screening plate, after 2-4 days of culture, the transformants that grew well on the high-concentration G418 plate were picked for purification and re-screening verification. After the genome of the re-screened transformants was extracted and verified to be correct, high-copy Pichia pastoris genetically engineered strains expressing recombinant humanized type IV collagen JYC401, JYC402 and JYC403 were obtained and preserved in glycerol.

[0036] (4) The high-copy Pichia pastoris genetically engineered strain obtained in step (3) 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 used to determine the growth rate. 600 The inoculum volume 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 centrifuged to collect the supernatant. 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 minutes. After centrifugation for 15 minutes, 10-15 μL of supernatant was taken and loaded into SDS-PAGE to complete the qualitative analysis of recombinant humanized type IV collagen JYC401, JYC402 and JYC403. The results of SDS-PAGE detection are shown in Figure 3 ,from Figure 3 It can be seen that a high-copy Pichia pastoris genetic engineering strain that can successfully express recombinant humanized type IV collagen JYC401, JYC402 and JYC403 was obtained.

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

[0038] The MTT method 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. 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 10ppm, 100ppm, and 1000ppm of recombinant humanized type IV collagen JYC401, JYC402, and JYC403 all exhibit excellent cell proliferation promoting activity, and as the concentration of recombinant humanized type IV collagen JYC401, JYC402, and JYC403 increases, the promoting effect increases. In addition, compared with recombinant humanized type IV collagen JYC403, recombinant humanized type IV collagen JYC401 and JYC402 exhibit more excellent cell proliferation promoting activity.

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

[0040] The cell migration was determined by the cell scratch method. 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. 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 10ppm, 100ppm and 1000ppm of recombinant humanized type IV collagen JYC401, JYC402 and JYC403 all exhibit excellent cell migration promoting activity, and as the concentration of recombinant humanized type IV collagen JYC401, JYC402 and JYC403 increases, the promoting effect increases. In addition, compared with recombinant humanized type IV collagen JYC403, recombinant humanized type IV collagen JYC401 and JYC402 exhibit better cell migration promoting activity.

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

[0042] The centrifugation method was used to detect the ability of recombinant humanized type IV collagen JYC401, JYC402 and JYC403 to promote cell adhesion using human epidermal cells and human fibroblasts as experimental cells. Figure 6As 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 IV collagen JYC401, JYC402 and JYC403 all exhibit excellent cell adhesion promoting activity, and as the concentration of recombinant humanized type IV collagen JYC401, JYC402 and JYC403 increases, the promoting effect increases. In addition, compared with recombinant humanized type IV collagen JYC403, recombinant humanized type IV collagen JYC401 and JYC402 exhibit better cell adhesion promoting activity.

Claims

1. A functional short peptide, characterized in that: Its amino acid sequence is shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.

3.

2. Recombinant humanized type IV collagen, characterized in that: JYC401, JYC402 or JYC403; the amino acid sequence of the recombinant humanized type IV collagen JYC401 is shown in SEQ ID No.4, the amino acid sequence of the recombinant humanized type IV collagen JYC402 is shown in SEQ ID No.5, and the amino acid sequence of the recombinant humanized type IV collagen JYC403 is shown in SEQ ID No.

6.

3. The gene encoding the recombinant humanized type IV collagen according to claim 2, 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.

4. A recombinant expression vector, characterized in that: A recombinant expression vector containing the gene encoding the recombinant humanized type IV collagen JYC401, JYC402 or JYC403 according to claim 3.

5. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector pPIC9K-JYC401, pPIC9K-JYC402 or pPIC9K-JYC403 containing the gene encoding the recombinant humanized type IV collagen JYC401, JYC402 or JYC403 according to claim 3 is constructed by the following steps: The gene sequence encoding the recombinant humanized type IV collagen JYC401, JYC402 or JYC403 shown in SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 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-JYC401, pPIC9K-JYC402 or pPIC9K-JYC403.

6. The genetically engineered Pichia pastoris for producing the recombinant humanized type IV collagen according to claim 2, characterized in that: Contains the recombinant expression vector according to claim 4 or 5.

7. The method for constructing a genetically engineered Pichia pastoris according to claim 6, characterized in that: Here are the steps: The recombinant expression vector according to claim 4 or 5 is linearized and then 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 bacteria that produce recombinant humanized type IV collagen JYC401, JYC402 or JYC403.

8. The method for preparing recombinant humanized type IV collagen according to claim 2, characterized in that: Here are the steps: The genetically engineered Pichia pastoris producing the recombinant humanized type IV collagen JYC401, JYC402 or JYC403 as claimed in claim 6 is inoculated into a BMMY induction medium, induced to express with methanol, and the fermentation supernatant is purified to obtain high-purity recombinant humanized type IV collagen JYC401, JYC402 or JYC403.

9. A composition or product, characterized in that The invention comprises at least one of the recombinant humanized type IV collagen JYC401, JYC402 or JYC403 according to 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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