Recombinant type XVII collagen and its expression strain

Abstract

The present invention discloses a recombinant type XVII collagen and its expression strain. The recombinant type XVII collagen sequence is obtained by combining the functional domains that bind to cell integrins in human type XVII collagen, which are respectively located in the collagen domain and non-collagen domain of human type XVII collagen, and contain 2 non-collagen domains and 4 collagen domains in total. After codon optimization, it is transformed into Pichia pastoris, and the high-copy Pichia pastoris genetic engineering bacteria obtained by G418 resistance gradient screening can stably express recombinant type XVII collagen. The recombinant type XVII collagen expressed by the present invention has excellent cell proliferation, migration and adhesion performance of promoting human hair follicle stem cells and human fibroblasts, and can be used for the repair of skin and hair follicle tissues, and enhance the resistance of skin and hair follicles.

Description

Technical Field

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

[0002] Collagen XVII, also known as BP180, is a type II transmembrane protein located at the junction of the dermis and epidermis. As a key structural component of hemidesmosomes (a multiprotein complex located in the dermis-epidermal basement membrane region that mediates the adhesion of keratinocytes to the underlying membrane), it plays an important role in maintaining the tight connection between the basement membrane and the extracellular matrix. There are two homotrimeric forms of type XVII collagen in the human body, but both are encoded by the COL17α1 gene, namely the full-length transmembrane protein (1497 amino acids) and the truncated soluble extracellular domain (also known as LAD-1, 1009 amino acids). Among them, the extracellular domain mainly mediates the binding with laminin and type IV collagen, and is an important part involved in maintaining the stability of the basement membrane. There are 15 collagen domains and 16 non-collagen domains in the extracellular domain of type XVII collagen, and these collagen domains and non-collagen domains jointly participate in the interaction with the extracellular matrix. Studies have shown that the expression of type XVII collagen can help inhibit the aging of hair follicle stem cells, thereby delaying hair loss. In addition, in terms of anti-aging, studies have found that type XVII collagen can promote the removal of senescent cells, thereby delaying the aging of the body. Since the content of type XVII collagen in humans and animals is extremely low, the traditional collagen separation and extraction process obviously cannot meet the market demand. The development of an efficient green manufacturing platform for mass production of recombinant type XVII collagen is of great significance to promoting the application of rare collagen in daily chemicals, skin care, health care and medical fields.

[0003] With the rapid development of synthetic biology, artificial design and construction of biological systems has become the mainstream trend in biology today, and 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 type XVII collagen with high biological performance and its efficient expression in heterologous hosts will greatly contribute to the application of type XVII collagen in industrial production.

[0004] 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 now widely used in the food and pharmaceutical fields. Therefore, the secretion expression of recombinant type XVII collagen by Pichia pastoris will greatly promote the development of type XVII collagen in the fields of beauty and skin care, medical devices and tissue engineering. Summary of the invention

[0005] The invention provides a recombinant type XVII collagen and an expression strain thereof.

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

[0007] The recombinant type XVII collagen described in the present invention involves 5 basic functional domains, including the amino acid sequences shown in SEQ No. 1, SEQ No. 2, SEQ No. 3, SEQ No. 4 and SEQ No. 5, and is composed of 2 non-collagenous domains and 4 collagenous domains.

[0008] The recombinant type XVII collagen described in the present invention is a recombinant type XVII collagen 1 whose amino acid sequence is formed by sequentially combining the amino acid sequences of SEQ No.1, SEQ No.2, SEQ No.3 and SEQ No.4, as shown in SEQ No.6, or a recombinant type XVII collagen 2 whose amino acid sequence is formed by sequentially combining the amino acid sequences of SEQ No.1, SEQ No.2, SEQ No.3, SEQ No.4 and SEQ No.5, as shown in SEQ No.7.

[0009] According to the codon preference of Pichia pastoris, the present invention performs codon optimization on five basic functional domains, and obtains nucleotide sequences corresponding to SEQ No.1, SEQ No.2, SEQ No.3, SEQ No.4 and SEQ No.5, which are SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12, respectively.

[0010] The present invention performs codon optimization on recombinant type XVII collagen according to the codon preference of Pichia pastoris, and the nucleotide sequence of the obtained recombinant type XVII collagen 1 is shown as SEQ ID No.13, and the nucleotide sequence of the recombinant type XVII collagen 2 is shown as SEQ ID No.14.

[0011] Furthermore, the present invention constructs a strain expressing recombinant type XVII collagen, which synthesizes the nucleotide sequence of recombinant type XVII collagen 1 or recombinant type XVII collagen 2, then amplifies and seamlessly clones it into the pPIC9K empty vector, enriches the recombinant plasmid after colony PCR and sequencing verification, linearizes it after enzyme digestion, and transforms it into Pichia pastoris, and obtains the Pichia pastoris genetically engineered bacteria expressing recombinant type XVII collagen 1 or recombinant type XVII collagen 2 through G418 resistance gradient screening.

[0012] Furthermore, the present invention provides a method for expressing the above-mentioned recombinant type XVII collagen, specifically: inoculating Pichia pastoris genetically engineered bacteria expressing recombinant type XVII collagen 1 or recombinant type XVII collagen 2 into BMMY culture medium, inducing expression with methanol at 30°C for 72 to 120 hours, collecting the supernatant, and purifying to obtain recombinant type XVII collagen 1 or recombinant type XVII collagen 2.

[0013] The present invention screens the basic functional domains that bind to cell integrins in human type XVII collagen, and specially combines the above basic functional domains to form a recombinant type XVII collagen sequence, and amplifies and clones it into the pPIC9K vector after codon optimization, and transforms it into Pichia pastoris cells after enzyme linearization, and screens high-copy recombinants through G418 resistance gradient to obtain a high-copy Pichia pastoris genetic engineering strain that can express recombinant type XVII collagen. The recombinant type XVII collagen 1 and 2 of the present invention can significantly enhance the proliferation, migration and adhesion capabilities of human fibroblasts and human hair follicle stem cells, and the effect of recombinant type XVII collagen 2 is better than that of recombinant type XVII collagen 1. The recombinant type XVII collagen of the present invention can be used for the repair of skin and hair follicle tissues, enhance the resistance of skin and hair follicles, and is suitable for preparing products for use in hair care, skin care and related medical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the recombinant plasmid containing recombinant type XVII collagen 1.

[0015] Figure 2 Schematic diagram of the recombinant plasmid containing recombinant type XVII collagen 2.

[0016] Figure 3 It is the nucleic acid electrophoresis diagram of each plasmid, wherein lane 1: pPIC9K empty vector plasmid linearized fragment; lane 2: pPIC9K+recombinant type XVII collagen 1 plasmid linearized fragment; lane 3: pPIC9K+recombinant type XVII collagen 2 plasmid linearized fragment.

[0017] Figure 4It is the SDS-PAGE diagram of the culture supernatant of high-copy Pichia genetically engineered bacteria XVII.1 and XVII.2 (methanol induced for 72 hours), wherein lanes 1 and 2: SDS-PAGE of the supernatant of high-copy strain XVII.2, lanes 3 and 4: SDS-PAGE of the supernatant of high-copy strain XVII.1.

[0018] Figure 5 The figure shows the effects of different concentrations of recombinant XVII human collagen 1, 2 and fetal bovine serum (BSA) on the proliferation of human hair follicle stem cells.

[0019] Figure 6 The figure shows the effects of different concentrations of recombinant XVII human collagen 1, 2 and BSA on the proliferation of human fibroblasts.

[0020] Figure 7 This graph shows the effects of different concentrations of recombinant XVII human collagen 1, 2 and BSA on the migration rate of human hair follicle stem cells.

[0021] Figure 8 This graph shows the effects of different concentrations of recombinant XVII human collagen 1, 2 and BSA on the migration rate of human fibroblasts.

[0022] Fig. 9 This graph shows the effects of different concentrations of recombinant XVII human collagen 1, 2 and BSA on the relative adhesion of human hair follicle stem cells.

[0023] Fig.10 This graph shows the effects of different concentrations of recombinant XVII human collagen 1, 2 and BSA on the relative adhesion of human fibroblasts. DETAILED DESCRIPTION

[0024] The present invention is further described below by specific examples and drawings, but the present invention is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product specifications.

[0025] In the following examples, Pichia pastoris GS115 was used as the starting strain. The expression vectors of recombinant type XVII collagen 1 and 2 used pPIC9K as the integration vector, and the nucleotide sequences of SEQ ID No. 13 and SEQ ID No. 14 synthesized by Shanghai Qingke Biotechnology Co., Ltd. were PCR amplified and purified, and seamlessly cloned into pPIC9K.

[0026] Example 1

[0027] Construction of high-copy Pichia pastoris genetic engineering bacteria:

[0028] (1) Bioinformatics and protein structure prediction tools (NCBI, Uniprot, AlphaFold) were used to screen five basic functional domains (amino acid sequences as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5) that can bind to cellular integrins from human type XVII collagen, including two non-collagenous domains in the extracellular domains and four collagenous domains in the extracellular domains.

[0029] (2) The five basic functional domains are arranged in the order of 1-2-3-4 to obtain a recombinant type XVII collagen 1 with an amino acid sequence as shown in SEQ ID No. 6, and the five basic functional domains are arranged in the order of 1-2-3-4-5 to obtain a recombinant type XVII collagen 2 with an amino acid sequence as shown in SEQ ID No. 7.

[0030] (3) The amino acid sequences of the two recombinant type XVII collagens were codon-optimized according to the codon preference of Pichia pastoris to obtain the optimized nucleotide sequence of recombinant type XVII collagen 1, as shown in SEQ ID No. 13, and the nucleotide sequence of recombinant type XVII collagen 2, as shown in SEQ ID No. 14.

[0031] (4) The nucleotide sequences of recombinant type XVII collagen 1 and 2 were entrusted to Shanghai Qingke Biotechnology Co., Ltd. for gene synthesis. Primer P1 (nucleotide sequence as shown in SEQ ID No. 15) and primer P2 (nucleotide sequence as shown in SEQ ID No. 16) were used to amplify the target sequence of SEQ ID No. 13, and primer P1 (nucleotide sequence as shown in SEQ ID No. 15) and primer P3 (nucleotide sequence as shown in SEQ ID No. 17) were used to amplify the target sequence of SEQ ID No. 14. After high-fidelity PCR amplification and nucleic acid gel purification, the high-fidelity PCR amplification was seamlessly cloned into the downstream of the α factor secretion signal peptide of the pPIC9K plasmid using the Gibson assembly technology. After verification by colony PCR and sequencing, the recombinant plasmid 1 (structural schematic diagram as shown in Figure 1 2 (structural diagram as shown) and Figure 2As shown), the recombinant plasmids 1 and 2 were linearized by SalI and then electro-transformed into Pichia pastoris competent cells, and the electro-transformed cells were activated and cultured with 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. After transformants grew out, the transformant monoclones were first verified by colony PCR, and the verified correct transformants were respectively spread on screening plates containing 1g / L, 2g / L, 3g / L and 4g / L G418. After 2-4 days of culture, the transformants that grew well on the high-concentration G418 plates were picked for purification and re-screening verification. The genome of the re-screened transformants was extracted and verified to be correct, and then glycerol seeds were preserved to obtain high-copy Pichia pastoris genetic engineering strain XVII.1 expressing recombinant type XVII collagen 1 and high-copy Pichia pastoris genetic engineering strain XVII.2 expressing recombinant type XVII collagen 2. Figure 3 It is the nucleic acid electrophoresis diagram of each plasmid, wherein lane 1: pPIC9K empty vector plasmid linearized fragment; lane 2: pPIC9K+recombinant type XVII collagen 1 plasmid linearized fragment; lane 3: pPIC9K+recombinant type XVII collagen 2 plasmid linearized fragment.

[0032] Example 2

[0033] Fermentation of high-copy Pichia pastoris genetically engineered strains and expression of recombinant type XVII collagen 1 and 2:

[0034] High-copy Pichia genetically engineered strains XVII.1 and XVII.2 were 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 remainder was water, and the percentages were weight percentages), cultured for 16-20 hours, and then the initial OD 600 The inoculum size of 1 was 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, and the percentages were mass percentages) for cultivation, and 300 μL of filtered and sterilized methanol was added every 24 hours. After 72 hours, 2 mL of the sample was centrifuged to collect the supernatant, and 500 μL of the supernatant was concentrated in an ultrafiltration tube with a size of 10 kDa to obtain about 50 μL of concentrated solution. 24 μL of the concentrated solution and 6 μL of 5× loading buffer were mixed evenly and placed in boiling water for treatment for 10-15 minutes. After centrifugation for 15 minutes, 10-15 μL of the supernatant was taken and loaded into SDS-PAGE to complete the qualitative analysis of recombinant type XVII collagen 1 and 2. The results of SDS-PAGE detection are shown in Figure 4 . Figure 4 In the figure, lanes 1 and 2 are the supernatant SDS-PAGE of high copy strain XVII.2, and lanes 3 and 4 are the supernatant SDS-PAGE of high copy strain XVII.1. It can be seen from the figure that the present invention obtains high copy Pichia genetically engineered strains XVII.1 and XVII.2 that can successfully express recombinant type XVII collagen 1 and 2.

[0035] Example 3

[0036] 1. Cell Proliferation Assay

[0037] The MTT method was used to detect the ability of recombinant type XVII collagen 1, 2 and BSA to promote cell proliferation using human hair follicle stem cells and human fibroblasts as experimental cells. Figure 5 and 6 As shown, it can be seen that recombinant type XVII collagen 1 and 2 exhibit better ability to promote cell proliferation than BSA, and recombinant type XVII collagen 2 has the highest ability to promote cell proliferation.

[0038] 2. Cell Migration Assay

[0039] The cell migration was determined by the cell scratch method, and human hair follicle stem cells and human fibroblasts were used as experimental cells to detect the ability of recombinant type XVII collagen 1, 2 and BSA to promote cell migration. Figure 7 and 8 As shown, it can be seen that recombinant type XVII collagen 1 and 2 exhibit better ability to promote cell migration than BSA, and recombinant type XVII collagen 2 has the highest ability to promote cell migration.

[0040] 3. Cell Adhesion Assay

[0041] The centrifugation method was used to detect the ability of recombinant type XVII collagen 1, 2 and BSA to promote cell adhesion using human hair follicle stem cells and human fibroblasts as experimental cells. Fig. 9 and 10 As shown, it can be seen that recombinant type XVII collagen 1 and 2 exhibit better ability to promote cell adhesion than BSA, and recombinant type XVII collagen 2 has the highest ability to promote cell adhesion.

[0042] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, 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 protection scope of the present invention.

Claims

1. Recombinant type XVII collagen, characterized in that It is a recombinant type XVII collagen 1 whose amino acid sequence is formed by sequentially combining the amino acid sequences of SEQ No.1, SEQ No.2, SEQ No.3 and SEQ No.4, as shown in SEQ No.6, or a recombinant type XVII collagen 2 whose amino acid sequence is formed by sequentially combining the amino acid sequences of SEQ No.1, SEQ No.2, SEQ No.3, SEQ No.4 and SEQ No.5, as shown in SEQ No.

7.

2. The gene encoding the recombinant type XVII collagen according to claim 1, characterized in that: The nucleotide sequence of the gene encoding recombinant type XVII collagen 1 is shown in SEQ ID No.13, and the nucleotide sequence of the gene encoding recombinant type XVII collagen 2 is shown in SEQ ID No.

14.

3. The strain expressing the recombinant type XVII collagen according to claim 1, characterized in that: The nucleotide sequence of the recombinant type XVII collagen 1 or recombinant type XVII collagen 2 described in claim 1 is synthesized, and then amplified and seamlessly cloned into the pPIC9K empty vector. The recombinant plasmid is enriched after verification by colony PCR and sequencing, and then transformed into Pichia pastoris after enzyme linearization. The Pichia pastoris genetically engineered bacteria expressing recombinant type XVII collagen 1 or recombinant type XVII collagen 2 are obtained by G418 resistance gradient screening.

4. A method for expressing recombinant type XVII collagen, specifically comprising: inoculating the Pichia pastoris genetically engineered bacteria expressing the recombinant type XVII collagen 1 or recombinant type XVII collagen 2 described in claim 1 as described in claim 3 into BMMY culture medium, inducing expression with methanol at 30°C for 72 to 120 hours, collecting the supernatant, and purifying to obtain recombinant type XVII collagen 1 or recombinant type XVII collagen 2.

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