Recombinant human type III collagen and its application

Abstract

The present invention belongs to the field of cosmetics technology, specifically relating to a recombinant humanized type III collagen and its applications. The amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID No. 1. This recombinant humanized type III collagen exhibits high hydrophilicity and cell adhesion. Furthermore, this recombinant humanized type III collagen is constructed using a yeast expression system, enabling large-scale production.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to a recombinant humanized type III collagen and its application. Background Art

[0002] Collagen is the most abundant protein family in the extracellular matrix and the primary component of animal connective tissue. It is also the most abundant and widely distributed functional protein in mammals, accounting for approximately 25% to 30% of total protein. It performs numerous biological functions, including transmitting growth factors and cytokines, maintaining tissue structural integrity, and influencing cell adhesion and proliferation. Among the numerous collagen subtypes, type III collagen is the predominant collagen found in human skin, fascia, and tendons, primarily distributed in the dermis. Its fine structure and excellent repair properties make it crucial for skin elasticity and scar healing. Its reduction contributes to skin aging. Type III collagen levels begin to decline after birth. Normal infant skin accounts for 80% of type III collagen. With growth and development, this level of type III collagen continues to decrease, leading to its nickname, "baby collagen." It is crucial for supporting the epidermis, and its deficiency in mature skin is a major contributor to skin sagging.

[0003] Traditionally, collagen has been extracted from terrestrial animal connective tissue and aquatic product processing byproducts through methods such as hot water extraction, acid-base hydrolysis, and enzymatic hydrolysis. However, the isolation and purification of animal-derived collagen is complex, and monomer separation is difficult. It may also carry viruses, posing a safety hazard. Furthermore, the collagen obtained through extraction has lost its original biological activity, making it unsuitable for biomedical applications. Genetic engineering technology, however, offers advantages over extraction methods, such as high product purity, stability, and safety, and is currently a hot topic of research.

[0004] Recombinant collagen genetic engineering technology involves different expression systems such as Escherichia coli, yeast, insect cells, mammalian cells, and transgenic crops. Expression systems such as mammalian cells and insect cells are costly and time-consuming, making it difficult to meet industrialization needs. In contrast, the production of recombinant collagen by microbial fermentation is low-cost, has a short cycle, is easier to culture, and is more amenable to commercial production. However, bacterial expression systems generally have shortcomings such as: the production of pyrogens makes the expression product difficult to apply clinically; the target protein is expressed in the form of inclusion bodies, making product purification difficult; the post-translational processing and modification system of the prokaryotic expression system is imperfect, the biological activity of the expression product is low, and endotoxins are difficult to remove. Public patent CN115948441A uses an Escherichia coli system for expression. The research process mentions the need to use a low temperature of 16°C to increase the expression of soluble protein, but low-temperature culture is not conducive to large-scale production.

[0005] The interaction between cells and the matrix is ​​based on cell adhesion. Integrins can bind to components of the extracellular matrix, such as collagen, to attach cells to the matrix. After cell adhesion is established, the intracellular force generated by the cytoskeleton is transmitted to the matrix through the adhesion focal points, resulting in a certain amount of stress within the matrix. The intracellular force has a direct regulatory effect on gene expression.

[0006] Therefore, it is very necessary to provide a recombinant protein that has cell adhesion function and can be scaled up for production. Summary of the Invention

[0007] To address the above technical issues, the present invention provides a recombinant humanized type III collagen with high hydrophilicity and cell adhesion. Furthermore, the recombinant humanized type III collagen is constructed using a yeast expression system and can be produced and translated on a large scale.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a recombinant humanized type III collagen, the amino acid sequence of which is shown in SEQ ID No. 1.

[0010] As a preferred embodiment of the present invention, the recombinant humanized type III collagen sequence comprises four tandemly repeated GPRGDK sequences, seven GXXGER integrin sites, and four GXXGEN integrin sites.

[0011] In a second aspect, the present invention provides a recombinant vector, which is obtained by connecting the gene encoding the recombinant humanized type III collagen to an expression plasmid.

[0012] As a preferred embodiment of the present invention, the expression plasmid is any one of pPICZαA, pPIC9, pPIC9K, pHIL-S1 and pYAM75P vectors.

[0013] In a third aspect, the present invention provides an engineered strain, which is constructed according to the following steps:

[0014] An expression vector containing a prolyl 4-hydroxylase encoding gene was constructed, transformed into competent cells, and cultured to obtain a proline hydroxylase expressing strain; the nucleotide sequence of the prolyl 4-hydroxylase encoding gene is shown in SEQ ID No. 4;

[0015] The recombinant vector is transformed into competent cells of the proline hydroxylase expression strain, cultured, and screened to obtain the engineered strain.

[0016] As a preferred embodiment of the present invention, the competent cells are competent cells of any one of the Pichia pastoris X33, GS115, SMD1168, KM71 and KM71H strains.

[0017] As a preferred embodiment of the present invention, the prolyl 4-hydroxylase is derived from the Chlorella virus PBCV-1.

[0018] In a fourth aspect, the present invention provides a hydroxyproline-modified recombinant humanized type III collagen, which is obtained by fermenting and culturing the engineered strain and inducing protein expression.

[0019] As a preferred embodiment of the present invention, the fermentation culture of the engineered strain is carried out at 25° C. to 30° C., pH=4.5 to 6.5, and dissolved oxygen of 20% to 60%.

[0020] As a preferred embodiment of the present invention, after inducing protein expression, the supernatant containing the fermentation culture fluid is sequentially concentrated by ultrafiltration and purified by chromatography to obtain the hydroxyproline-modified recombinant humanized type III collagen.

[0021] In a fifth aspect, the present invention provides a use of the recombinant humanized type III collagen, the recombinant vector or the engineered strain or the hydroxyproline-modified recombinant humanized type III collagen in preparing a cell adhesive product.

[0022] As a preferred embodiment of the present invention, the product is any one of the following (1) to (3):

[0023] (1) Functional cosmetics that improve cell adhesion;

[0024] (2) Medical cosmetics;

[0025] (3) Medical dressings.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1. The recombinant humanized type III collagen provided by the present invention selects the hydrophilic segment in the triple helical region of the natural human type III collagen α1 chain protein sequence, and the selected segment contains the RGD sequence, and the sequence containing RGD is GPRGDK, and the GPRGDK sequence is repeated in tandem so that the final recombinant humanized type III collagen sequence contains quadruple RGD; in addition, the hydrophilic segment in the triple helical region of the natural human type III collagen α1 chain protein sequence selected by the present invention also contains 7 GXXGER integrin sites and 4 GXXGEN integrin sites. The presence of the integrin sites and the GPRGDK sequence can improve the overall hydrophilic properties of the recombinant humanized type III collagen and achieve the effect of enhancing cell adhesion.

[0028] 2. The hydroxyproline-modified recombinant humanized type III collagen provided by the present invention utilizes the co-expression of the proline hydroxylase gene of Chlorella and Paramecium virus 1 to compensate for the lack of proline hydroxylase in the host cells, allowing proline to be smoothly hydroxylated and the single amino acid chain to successfully form a triple helical structure, thereby improving the triple helical conformation stability and thermal stability of the hydroxyproline-modified recombinant humanized type III collagen at body temperature.

[0029] 3. The present invention uses the Pichia pastoris expression system to co-transduce prolyl 4-hydroxylase to perform proline hydroxylation modification on the recombinantly expressed collagen. The hydroxylated and glycosylated collagen is encapsulated in vesicles and delivered to the Golgi apparatus. The three α chains are entangled with the C-terminus as an anchor point to form a structurally stable triple-helical collagen that is secreted outside the cell. The high secretory expression of the hydroxyproline-modified recombinant humanized type III collagen makes the collection and purification operations simpler, and avoids the impurity proteins, endotoxins, and peptidoglycans brought about by the bacterial lysis process, making it have application potential in tissue engineering, clinical medicine, food industry, packaging materials, cosmetics, medical cosmetology, biomaterials, and medical devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the proline hydroxylase expression plasmid map;

[0031] Figure 2 This is the recombinant human type III collagen expression plasmid map;

[0032] Figure 3 The expression of A085 was screened by Western blot; 1 to 10 represent different clone strains; M is a protein marker, which is 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, and 15 kDa from top to bottom;

[0033] Figure 4 The expression of hydroxyproline-modified recombinant humanized type III collagen was screened by SDS-PAGE. 1 to 12 represent different cloned strains. M is a protein marker, which is 180 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, and 25 kDa from top to bottom.

[0034] Figure 5 The figure shows the fermentation expression of hydroxyproline-modified recombinant humanized type III collagen detected by SDS-PAGE. M is a protein marker, which is 100 kDa, 70 kDa, 55 kDa, 40 kDa, and 35 kDa from top to bottom.

[0035] Figure 6 This is the SDS-PAGE detection of hydroxyproline-modified recombinant humanized type III collagen purified protein; the left side is the protein marker, from top to bottom are

[0036] 180kD, 130kD, 100kD, 70kD, 55kD, 45kD, 35kD;

[0037] Figure 7 It is the hydrophobicity analysis of hydroxyproline-modified recombinant humanized type III collagen;

[0038] Figure 8 The molecular structure of hydroxyproline-modified recombinant humanized type III collagen was detected by Fourier transform infrared spectroscopy;

[0039] Figure 9 Circular dichroism was used to detect the secondary structure of hydroxyproline-modified recombinant humanized type III collagen;

[0040] Figure 10 Cell staining to detect the cell adhesion promoted by hydroxyproline-modified recombinant humanized type III collagen; Scale bar: 250 μm; A, control group; B, commercially available sample; C, HYCol.III quadruple RGD;

[0041] Figure 11 Cell adhesion test results. * indicates significant difference between the hydroxyproline-modified recombinant humanized type III collagen sample group and the blank control group; * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. # indicates significant difference between the commercially available competitor recombinant humanized type III collagen group and the hydroxyproline-modified recombinant humanized type III collagen sample group; # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001. DETAILED DESCRIPTION

[0042] The technical solutions of the present invention are described below with reference to the following embodiments; however, the present invention is not limited to the following embodiments.

[0043] The experimental methods and detection methods in the following embodiments are all conventional methods unless otherwise specified; the agents and materials are all commercially available unless otherwise specified; the indicator data are all based on conventional measurement methods unless otherwise specified.

[0044] The culture medium and components involved in the following examples are as follows:

[0045] The culture medium was prepared with depurified water and sterilized at 121°C for 15 to 30 minutes.

[0046] YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose. Add 20 g / L agar powder to prepare YPD solid medium. The solvent is water.

[0047] BMGY medium: peptone 20 g / L, yeast powder 10 g / L, glycerol 20 g / L, 50 mmol / L potassium phosphate buffer solution, 10% volume fraction of 10× YNB yeast basic nitrogen source mother solution, biotin 4×10 4 g / L, the solvent is water.

[0048] BMMY medium: peptone 20 g / L, yeast powder 10 g / L, methanol 10 g / L, 50 mmol / L potassium phosphate buffer solution, 10% volume fraction of 10×YNB yeast basic nitrogen source mother solution, biotin 4×10 4 g / L, the solvent is water.

[0049] MD plate: glucose 20g / L, agar powder 20g / L, 10% volume fraction of 10×YNB yeast basic nitrogen source stock solution, biotin 4×10 4 g / L, the solvent is water.

[0050] Example 1:

[0051] Construction of yeast expression cells expressing proline hydroxylase

[0052] 1. Construction of proline hydroxylase expression vector

[0053] The target gene Flag-A085R-DNAsequence carried by the plasmid is synthesized by reverse translation of the amino acid sequence of prolyl 4-hydroxylase A085R of Chlorella virus PBCV-1 and then optimization of Pichia pastoris codons. The synthesized gene fragment is connected to the expression vector pPICZB through the EcoRI+NotI double restriction site to obtain the proline hydroxylase expression vector. This process was entrusted to General Bio (Anhui) Co., Ltd. The nucleotide sequence of the target gene Flag-A085R-DNAsequence is shown in SEQ ID No.4. The schematic diagram of the construction of the proline hydroxylase expression plasmid pPICZB-Flag-A085R is shown in Figure 1 shown.

[0054] The amino acid sequence of prolyl 4-hydroxylase A085R, namely the Flag-A085R sequence, consists of 216 amino acids. The specific sequence is shown in SEQ ID No. 3.

[0055] SEQ ID No.3:

[0056] MDYKDDDDKEGFETSDRPGVCDGKYYEKIDGFLSDIECDVLINAAIKKGLIKSEVGGATENDPIKLDPKSRNSEQTWFMPGEHEVIDKIQKKTREFLNSKKHCIDKYNFEDVQVARYKPGQYYYYHHYDGDDDCDDACPKDQRLATLMVYLKAPEEGGGGETDFPTLTKKIKPKKGTSIFFWVADPTRKLYKETLHAGLPVKSGEKIIANQWIRAVK

[0057] SEQ ID No.4:

[0058] GATTACAAGGATGATGATGATAAGGAAGGTTTCGAAACTTCTGATAGAC

[0059] CAGGTGTTTGTGATGGTAAACTACGAAAAGATTGATGGTTTCTCTGTCTGA

[0060] TATTGAATGTGATGTTTTGATTAACGCTGCTATTAAGAAAGGTTTGATTAAGT

[0061] CTGAAGTCGGTGGTGCTACTGAAAATGATCCAATTAAGTTGGACCCTAAATC

[0062] TCGTAACTCTGAACAAACTTGGTTCATGCCTGGTGAACATGAAGTTATTGAT

[0063] AAAATTCAGAAGAAGACCAGAGAATTTTTGAACTCTAAAAAGCATTGCATC

[0064] GATAAATACAACTTCGAAGATGTTTCAAGTTGCTAGATACAAACCTGGTCCAAT

[0065] ATTATTATCACCATTACGATGGTGACGATTGTGATGATGCTTGTCCCAAAGGAT

[0066] CAAAGATTGGCTACTTTGATGGTTTACTTGAAAGCTCCAGAAGAAGGTGGT

[0067] GGTGGTGAAACTGATTTCCCAACTTTGAAGACTAAGATTAAGCCAAAGAAG

[0068] GGTACTTCTATTTTCTTTTGGGTTGCTGATCCAGTTACTAGAAAGTTGTACAA

[0069] AGAAACTTTGCATGCTGGTTTGCCAGTTAAATCTGGTGAAAAGATTATTGCT

[0070] AACCAATGGATTAGAGCTGTTAAATAA

[0071] 2. Preparation of GS115 yeast competent cells

[0072] Streak the GS115 yeast strain onto a YPD plate and culture at 30°C for 5 days until a single colony grows.

[0073] Pick a single clone to 50 mL YPD culture medium and culture in a constant temperature shaking incubator at 30°C and 300 rpm for 2 days; transfer to 100 mL YPD culture medium and culture at 30°C and 300 rpm for 2 days, and measure the OD 600 =1.3, centrifuge at 1500g, 4°C for 5 min to collect the strains, add 250 mL of pre-cooled sterile water to resuspend the strains to wash the strains; centrifuge at 1500g, 4°C for 5 min to collect the strains, add 50 mL of pre-cooled sterile water to resuspend the strains to wash the strains; centrifuge at 1500g, 4°C for 5 min to collect the strains.

[0074] Subsequently, 10 mL of pre-cooled 1 M sorbitol was added to resuspend the bacteria, and the suspension was gently pipetted; the bacteria were collected by centrifugation at 1500 g and 4 °C for 5 min, and 300 μL of pre-cooled 1 M sorbitol was added to resuspend the bacteria, and the suspension was gently pipetted to a final volume of about 500 μL. Aliquot 100 μL into each tube and place on ice for electroporation.

[0075] 3. Construction of proline hydroxylase expression strain

[0076] Take 50ug of freeze-dried pPICZB-Flag-A085R plasmid powder, add sterile water to dissolve it into 100ng / μL, use SacⅠ restriction endonuclease, digest it with agarose gel electrophoresis, and then save it for electroporation.

[0077] Remove the electroporation cuvette from the 75% ethanol container and place it on ice for a few minutes. Add 10 μg of the linearized pPICZB-Flag-A085R plasmid to 80 μL of GS115 competent yeast cells and gently pipette to mix. Transfer the GS115 competent cells and plasmid mixture to the electroporation cuvette and place it on ice for 5 minutes.

[0078] Place the cuvette in an electroporator at 2 kV for electroporation. Immediately add 1 mL of 1 M pre-cooled sorbitol and mix thoroughly by pipetting. Place the mixture in a 1.5 mL EP tube and incubate at 30°C for 1 h.

[0079] Take 100 μL of the bacterial solution obtained from the above incubation and spread it on a YPD plate containing 0.1 mg / mL bleomycin and culture it at 30°C for 5 days; select clones with better growth and then streak them onto YPD plates containing 1 mg / mL and 2 mg / mL bleomycin respectively, and culture them at 30°C for 5 days; try to select clones with better growth on the YPD plate with 2 mg / mL bleomycin for shake flask induced expression.

[0080] Single clones were selected and cultured in 50 mL of BMGY medium at 30°C and 250 rpm for 24 h. One mL of the bacterial culture was transferred to 50 mL of BMMY medium. The amount of bacterial culture added was calculated based on the OD value of each clone to ensure a consistent initial OD for each clone. The culture was then incubated at 30°C and 250 rpm. Methanol was added every 24 h to a final concentration of 0.5%. Expression was analyzed by SDS-PAGE at 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h of induction. After 72 h of induction, the supernatant was collected at 8000 rpm for 10 min at 4°C. Western blot analysis of the Falg tag was performed to determine protein expression.

[0081] 4. Western Blot Detection of Proline Hydroxylase Expression

[0082] Positive clone samples from the small-scale induced expression screening were collected, randomly numbered, and added with 5× loading buffer, and heated in a water bath at 100°C for 10 minutes; 10 μL of sample was loaded and subjected to 15% polyacrylamide gel electrophoresis at 120V for 1.5 hours; after the electrophoresis, the concentrated gel portion was cut off, the gel was removed and transferred to the membrane at a current of 400mA for 120 minutes; the membrane was placed in 5% skim milk powder for overnight blocking at 4°C; the primary antibody containing Flag-HRP was diluted with 5% skim milk powder at a dilution ratio of 1:10,000, and the membrane was incubated in the primary antibody dilution solution for 120 minutes. The membrane was removed and washed three times with PBST for 10 minutes each time, and then developed and photographed.

[0083] Figure 3The results of immunoblotting for expression identification are shown in Figure 1. Figure 3 ,Depend on Figure 3 It can be seen that different clone strains 2, 3, 7, 8, and 10 all express the gene, among which clone strain 10 has the best expression, which is the yeast expression cell expressing proline hydroxylase obtained by screening and is marked as GS115-A085R yeast.

[0084] Example 2

[0085] 1. Recombinant humanized type III collagen sequence selection and plasmid construction

[0086] The hydrophilic region of the triple helical region of the native human type III collagen α1 chain protein sequence was obtained using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). This region is located between 168aa and 1196aa within the triple helical region. This hydrophilic region contains the RGD sequence, which is GPRGDK. The GPRGDK sequence was tandemly repeated, resulting in a collagen sequence containing quadruple RGD. Furthermore, the hydrophilic region of the triple helical region of the selected native human type III collagen α1 chain protein sequence also contains seven GXXGER integrin sites and four GXXGEN integrin sites. The presence of these integrin sites and the RGD peptide sequence can enhance the overall hydrophilicity, expression efficiency, and cell adhesion of the recombinant humanized type III collagen. The resulting recombinant humanized type III collagen sequence is shown in SEQ ID No. 1.

[0087] SEQ ID No. 1:

[0088] GAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGPKGNDGAPGKNGERGGP GGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGAPGQNGEPGGKGERGAPGEKGEGGPPGPPG GSGPAGPPGPQGVKGERGSPGGPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGEPGRDGNPGSDGLPGRDGSPGG KGDRGENGSPGAPGAPGHPGPPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGPRGDKGPRGDKGPRGDK

[0089] The recombinant humanized type III collagen sequence was reverse translated and then optimized for Pichia pastoris codons to obtain the target gene HYCol.III Quadruple RGD. The target gene was then linked into the expression vector pPIC9K through the EcoRI+NotI double restriction sites to obtain the plasmid pPIC9K-HYCol.III Quadruple RGD. This process was commissioned to General Bio (Anhui) Co., Ltd. The construction diagram of the plasmid pPIC9K-HYCol.III Quadruple RGD is shown below. Figure 2 Its nucleotide sequence is shown in SEQ ID No.2.

[0090] SEQ ID No. 2:

[0091]

[0092] 2. Preparation of competent cells of GS115-A085R yeast

[0093] The GS115-A085R yeast strain in Example 1, i.e., clone No. 10, was streaked onto a YPD plate and cultured at 30°C for 5 days until a single colony grew;

[0094] Pick a single clone to 50mL YPD culture medium, culture in a constant temperature shaking incubator at 30℃, 300rpm for 2 days; transfer to 100mL YPD culture medium, culture at 30℃, 300rpm for 2 days, and measure OD 600 =1.3, centrifuge at 1500g, 4°C for 5 min to collect the strains, add 250 mL of pre-cooled sterile water to resuspend the strains to wash the strains; centrifuge at 1500g, 4°C for 5 min to collect the strains, add 50 mL of pre-cooled sterile water to resuspend the strains to wash the strains; centrifuge at 1500g, 4°C for 5 min to collect the strains.

[0095] Subsequently, 10 mL of pre-cooled 1 M sorbitol was added to resuspend the bacteria, and the suspension was gently pipetted; the bacteria were collected by centrifugation at 1500 g and 4 °C for 5 min, and 300 μL of pre-cooled 1 M sorbitol was added to resuspend the bacteria, and the suspension was gently pipetted to a final volume of about 500 μL. Aliquot 100 μL into each tube and place on ice for electroporation.

[0096] 3. Construction of a strain co-expressing recombinant humanized type III collagen and proline hydroxylase

[0097] Take 50ug of freeze-dried pPIC9K-HYCol.III quadruple RGD plasmid powder, add sterile water to dissolve it into 100ng / μL, use SalⅠ restriction endonuclease, digest it with agarose gel electrophoresis, and then save it for electroporation.

[0098] The pPIC9K-HYCol.III quadruple RGD plasmid after enzyme digestion was electroporated into GS115-A085R yeast competent cells, spread onto MD plates, and cultured at 30°C until a single colony grew;

[0099] Single colonies grown on MD plates were streaked onto YPD plates containing 0.5 mg / mL G418 antibiotics and cultured at 30°C for 5 days; clones with better growth were selected and streaked onto YPD plates containing 2 mg / mL and 4 mg / mL G418 antibiotics, respectively, and cultured at 30°C for 5 days; clones with better growth on YPD plates containing 4 mg / mL G418 were selected for induced expression in shake flasks; single colonies were picked and cultured overnight in BMGY medium. After 72 hours of induced expression in BMMY medium, the expression supernatant was collected at 8000 rpm, 10 min, and 4°C.

[0100] The results of SDS-PAGE detection of small-scale expression identification are as follows Figure 4 .Depend on Figure 4 It can be seen that different clone strains all express the protein, among which clone strain No. 8 has the best expression, which is the strain co-expressing recombinant humanized type III collagen and proline hydroxylase obtained by screening; the screened monoclonal strains are randomly numbered.

[0101] Example 3

[0102] Fermentation culture of hydroxyproline-modified recombinant humanized type III collagen

[0103] The recombinant humanized type III collagen and proline hydroxylase co-expressing strain in Example 2 was inoculated with 10% of the inoculum into a fermenter containing sterilized batch fermentation medium; the fermentation parameters were set as fermentation temperature of 25°C, pH of 5.5, and dissolved oxygen DO of 35%; samples were taken regularly after the start of fermentation to measure OD 600 The wet weight of the cells was determined. Once the glycerol in the base feed was consumed and dissolved oxygen had risen, the glycerol feed solution was fed at a rate of approximately 40 mL / hour of working volume. This stage ended when the wet weight reached 220 g / L. Samples were taken at the beginning of this stage, 5 hours after the feed, and at the end of the feed, and the parameters were recorded and the wet weight was determined. After the glycerol feed phase ended and the DO rapidly increased, a starvation treatment was performed for 90 minutes. The temperature was lowered to 25°C and the dissolved oxygen was controlled at 35%. The methanol induction phase then began. After the starvation treatment was terminated, methanol feed was resumed. The feed rate was approximately 50 mL / hour / L in the first hour of induction; approximately 100 mL / hour / L in the second hour; approximately 150 mL / hour / L in the third hour; and 200 mL / hour / L in the fourth hour. After 4 hours of induction, the feed rate was increased to 300 mL / hour / L until the end of fermentation. From the 0th hour of induction, samples were taken every 8 hours and when the tank was put out, and the parameters were recorded and the wet weight was measured. After 112 hours of induction, the tank was taken out for treatment, and the supernatant was collected by centrifugation. The results of the samples taken at different time periods were run on gels for testing. The results are as follows Figure 5 As shown, hydroxyproline-modified recombinant humanized type III collagen was obtained and labeled as HYCol.III quadruple RGD.

[0104] Example 4:

[0105] Purification of Hydroxyproline-Modified Recombinant Humanized Type III Collagen

[0106] The supernatant of the fermentation broth containing the hydroxyproline-modified humanized type III collagen obtained in Example 3 was subjected to ultrafiltration and concentration to replace the liquid. The specific steps are as follows:

[0107] ① Hollow fiber column pretreatment: Rinse with 3 L of injection water, then 2 L of 0.5 M NaOH, and circulate for 30 minutes. Drain the liquid in the tubing. Finally, rinse the hollow fiber column with 20 L of 10 mM PB buffer until the pH and Cd at the permeate and reflux ends are consistent with those of the buffer.

[0108] ② Microfiltration clarification: Use PB buffer to rinse until the pH and Cd of the permeate end are consistent with those of the buffer solution. Connect the pipeline to the centrifugal supernatant for microfiltration clarification. The flow rate of the microfiltration process is 7L / min, and the transmembrane pressure is ≤1Bar. When the interception volume is less than 5% of the total volume, add PB solution with an equal volume of interception volume to wash 3 times. The microfiltration clarification is completed and the permeate end sample is collected throughout the process.

[0109] ③ Ultrafiltration concentration: Use PB buffer to flush until the pH and Cd at the reflux end are consistent with those of the buffer solution. Connect the pipeline to the microfiltration clarified sample and concentrate it about 5 times. After the concentration is completed, use equal volume continuous liquid exchange to replace the concentrate with 10mM PB buffer until the sample conductivity reaches 4ms / cm. After the liquid exchange is completed, purification is carried out.

[0110] ④ Column purification: Purification by cationic filler SP Sepharose FF and elution with different salt concentrations. The column buffer is pH 5.0, 20mM citric acid buffer, and the elution buffer is pH 5.0, 20mM citric acid buffer plus 800mM sodium chloride. The eluted sample is again ultrafiltered and concentrated to remove salt, and the pH is adjusted.

[0111] 5.0, 8000rpm high-speed centrifugation for 20min, and then anion QFF chromatography was performed to collect the flow-through, which was the pure protein sample. Finally, it was dried and collected and stored. SDS-PAGE detection of hydroxyproline-modified recombinant humanized type III collagen purified protein Figure 6 shown.

[0112] Example 5

[0113] Identification and Analysis of Hydroxyproline-Modified Recombinant Humanized Type III Collagen

[0114] 1. Protein identification and LC-MSMS modification site identification

[0115] Sample preparation: Dissolve the sample in 500 μl of 0.1% TFA, shake and mix, centrifuge for about 5 min, aspirate the supernatant into an ultrafiltration tube, centrifuge for 15 min, freeze-dry, and re-dissolve in 30 μl of 0.1% TFA. Desalt by ultrafiltration at 10 kd and perform mass spectrometry detection.

[0116] Peptide enzymatic digestion: Add 40 μL of trypsin / chymotrypsin / pepsin buffer to each sample and incubate at 37°C for 18 h.

[0117] Chromatographic separation: Liquid A was a 0.1% (volume concentration) formic acid in water, and Liquid B was a 0.1% (volume concentration) formic acid in acetonitrile. The acetonitrile concentration in Liquid B was 84%. The HPLC column was a 0.15 mm x 150 mm RP-C18 column equilibrated with 95% Liquid A. Samples were loaded into Zorbax 300SB-C18 peptide traps via an autosampler and separated on the column. The HPLC gradient settings were as follows: from 0 to 50 minutes, a linear gradient of 4% to 50% for Liquid B; from 50 to 54 minutes, a linear gradient of 50% to 100% for Liquid B; and from 54 to 60 minutes, Liquid B was maintained at 100%.

[0118] Mass spectrometry identification: The enzymatic digestion products were separated by capillary high-performance liquid chromatography and analyzed by mass spectrometry on a Q Exactive HF-X mass spectrometer for 60 minutes. Detection was positive ion. The mass-to-charge ratios of the peptides and their fragments were collected using the following method: 10 fragment spectra were acquired after each full scan.

[0119] Data analysis: The original mass spectrometry test files were searched against the corresponding database using the software MaxQuant 1.5.5.1 to obtain protein identification and quantitative analysis results.

[0120] The results of hydroxyproline modification site identification showed that the selected fragment had a total of 459 amino acids, of which 53 prolines were modified by prolyl 4-hydroxylase, i.e., a modification rate of 11.5%. The hydroxyproline content of human collagen is 13%. Therefore, the hydroxyproline-modified recombinant humanized type III collagen constructed by the present invention can achieve more than 88% hydroxyproline modification homology with humans.

[0121] 2. Molecular weight detection

[0122] Liquid Phase Conditions: Separation was performed using an ultra-high performance liquid chromatography system. Liquid A consisted of 0.1% FA in water, and Liquid B consisted of 0.1% FA in acetonitrile. The column was equilibrated with Liquid A. The sample was loaded via an autosampler and separated on the column at a flow rate of 0.3 ml / min, a detection wavelength of 280 nm, and a column temperature of 80°C.

[0123] The relevant liquid phase gradients are shown in Table 1.

[0124] Table 1 Molecular weight detection liquid phase gradient

[0125] Retention time (min) Flow rate (ml / min) A％ B％ 1.00 0.300 98.0 2.0 7.00 0.300 10.0 90.0 7.50 0.300 10.0 90.0 7.60 0.300 98.0 2.0 8.00 0.300 98.0 2.0

[0126] Mass spectrometry conditions: Samples were analyzed using a Triple TOF 4600 mass spectrometer. Analysis time: 8 min, detection mode: positive ion, parent ion scan range: 105 m / z to 50,000 m / z.

[0127] Data analysis: The raw data were processed by PeakView 2.2 software.

[0128] High-resolution mass spectrometry determined that the hydroxyproline-modified recombinant humanized type III collagen HYCol.III quadruple RGD showed an obvious signal peak in the range of 41454.3 Da, that is, the molecular weight of the hydroxyproline-modified recombinant humanized type III collagen HYCol.III quadruple RGD was 41.5 KD.

[0129] 3. Hydrophobicity analysis

[0130] The HYCol.III quadruple RGD sequence and the native human type III procollagen sequence were analyzed for hydrophobicity using a website. The protein property analysis results indicate the overall average hydrophilicity of the protein. Positive values ​​indicate greater hydrophobicity, while negative values ​​indicate greater hydrophilicity. Amphipathic amino acids are primarily represented between 0.5 and -0.5. Further analysis of protein hydrophilicity can be performed using the online analysis tool Protscale (https: / / web.expasy.org / protscale / ).

[0131] The results of amino acid hydrophobicity analysis of hydroxyproline modified recombinant humanized type III collagen HYCol.III quadruple RGD are as follows Figure 7 As shown, overall, hydrophilic amino acids are slightly more than hydrophobic ones, and the hydroxyproline-modified recombinant humanized type III collagen HYCol.III quadruple RGD is hydrophilic due to the presence of multiple hydrophilic amino acids in the selected fragment.

[0132] Example 6

[0133] Higher-order structural analysis of hydroxyproline-modified recombinant humanized type III collagen

[0134] 1. Protein detection by Fourier transform infrared spectroscopy

[0135] Take about 10 mg of hydroxyproline-modified recombinant humanized type III collagen HYCol.III quadruple RGD and mix it with the prepared potassium bromide to make a transparent sample slice. Scan it with Fourier transform infrared spectrometer at room temperature and record the wavelength at 4000 cm -1 ~500cm -1 Spectrum of the interval.

[0136] FT-IR results of hydroxyproline modified recombinant humanized type III collagen freeze-dried powder are as follows Figure 8 As shown, it contains all the characteristic peaks of collagen, that is, the peak is higher than 2500cm -1 The amide A band and amide B band have peaks at 1200 cm -1 ~1700cm -1 Amide I, II, and III bands.

[0137] 2. Circular dichroism detection of proteins

[0138] Take an appropriate amount of hydroxyproline-modified recombinant humanized type III collagen HYCol.III quadruple RGD sample to prepare a 0.1 mg / mL aqueous solution, place it in a sample cell with an optical path of 1 mm, and perform scanning analysis at room temperature with a scanning wavenumber of 190 nm to 250 nm. Figure 9 The protein showed a clear negative absorption peak near 198 nm, a weaker positive absorption peak at 223 nm, and a crossover point at 219 nm, showing the typical characteristics of the collagen triple helix structure.

[0139] Example 7

[0140] Biological activity analysis of hydroxyproline-modified recombinant humanized type III collagen

[0141] Cell adhesion biological efficacy test

[0142] A 24-well plate was coated with 500 μL of a sample solution of hydroxyproline-modified recombinant humanized type III collagen and a commercially available recombinant humanized type III collagen solution. A blank control group consisted of 500 μL of PBS solution. The commercially available recombinant humanized type III collagen solution was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd. and is designated recombinant human collagen SFH. After incubating each well in a 37°C, 5% CO2 incubator for 4 hours, the excess coating solution was removed from the wells, and 500 μL of a 1% BSA-PBS solution was added. The wells were then incubated for another hour at 37°C, 5% CO2. After removing the liquid from the wells, the wells were washed three times with PBS, and the wash solution was discarded for later use. Human skin fibroblasts (HFB) were seeded into 24-well plates at 10,000 cells / well and incubated in a 37°C, 5% CO2 incubator for 2 hours. The supernatant was aspirated and washed once with PBS. The plates were stained with Hoechst 33342 staining solution, photographed, and the cell adhesion rate was calculated. Six replicates were made for each group, and the average value was calculated.

[0143] The results of the cell adhesion promotion test of hydroxyproline-modified recombinant humanized type III collagen HYCol.III quadruple RGD are as follows Figure 10 、 Figure 11As shown in Table 2, compared with the blank control group, the number of cells adhering to the commercial sample group increased by 69.62%. The number of cells adhering to the hydroxyproline-modified recombinant humanized type III collagen HYCol.III quadruple RGD group significantly increased by 134.18%. Compared with the commercial sample group, the number of cells adhering to the hydroxyproline-modified recombinant humanized type III collagen HYCol.III quadruple RGD group increased by 38.06%.

[0144] Table 2 HYCol.III quadruple RGD cell adhesion promotion efficacy test results

[0145] Grouping Average number of adherent cells Adhesion rate (%) control group 13.167 Commercially available sample set 22.333 69.62 HYCol.III Quadruple RGD 30.833 134.18

[0146] By adding a natural RGD peptide motif, the present invention imparts excellent hydrophilicity and adhesion to recombinant humanized type III collagen, thereby enhancing cell adhesion. Compared with recombinant type III collagen expressed in the yeast Pichia pastoris, the hydroxyproline-modified recombinant humanized type III collagen provided by the present invention exhibits significantly improved cell adhesion. Furthermore, the RGD peptide sequence contained in the recombinant human type III collagen can recognize and bind to integrins, enabling the recombinant human type III collagen to target cells.

[0147] The present invention adopts the Pichia pastoris expression system to prepare recombinant humanized type III collagen, which has the advantages of large-scale industrial production, such as being pyrogen-free, capable of high-density culture, low production cost, and high expression level. In addition, the recombinant humanized type III collagen can be secreted extracellularly, avoiding impurity proteins introduced during the bacterial lysis process. At the same time, the cell wall components do not contain endotoxins and peptidoglycans, thereby reducing pyrogens and making the expression product easy to use in clinical practice.

[0148] The hydroxyproline-modified recombinant humanized type III collagen provided by the present invention forms a triple-helical recombinant collagen with stable conformation and thermal stability at body temperature due to the expression of proline hydroxylase, which enables the hydroxyproline between three single chains to form strong hydrogen bonds.

[0149] The present invention provides a recombinant humanized type III collagen with strong cell adhesion function, and is not limited to the description in the Examples and Comparative Examples. Therefore, all modifications of proline hydroxylation, RGD sequence-enhanced cell adhesion, and hydroxyproline modification to stabilize the triple-helical structure in the recombinant collagen expressed in Pichia pastoris as described in the patent scope of the present invention are included within the scope of the present invention.

Claims

1. A hydroxyproline-modified recombinant humanized type III collagen, characterized in that: It is constructed by following the steps below: An expression vector containing a prolyl 4-hydroxylase encoding gene was constructed, transformed into competent cells, and cultured to obtain a proline hydroxylase expressing strain; the nucleotide sequence of the prolyl 4-hydroxylase encoding gene is shown in SEQ ID No. 4; The recombinant vector is obtained by connecting the coding gene of recombinant humanized type III collagen to the expression plasmid, wherein the amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID No. 1; The recombinant vector is transformed into competent cells of the proline hydroxylase expression strain, cultured, and screened to obtain an engineered strain; the competent cells are competent cells of the GS115 strain; The engineered strain is fermented and cultured and protein expression is induced to obtain the hydroxyproline-modified recombinant humanized type III collagen.

2. The hydroxyproline-modified recombinant humanized type III collagen according to claim 1, characterized in that: The expression plasmid is any one of pPICZαA, pPIC9, pPIC9K, pHILS1 and pYAM75P vectors.

3. The hydroxyproline-modified recombinant humanized type III collagen according to claim 1, characterized in that: The engineering strain fermentation culture is carried out at 25 DEG C to 30 DEG C, pH=4.5 to 6.5, and dissolved oxygen of 20% to 60%.

4. The hydroxyproline-modified recombinant humanized type III collagen according to claim 1, characterized in that: After inducing protein expression, the obtained supernatant containing the fermentation culture fluid is sequentially subjected to ultrafiltration concentration and chromatography purification to obtain the hydroxyproline-modified recombinant humanized type III collagen.

5. Use of the hydroxyproline-modified recombinant humanized type III collagen according to claim 1 in preparing a product for improving cell adhesion.

6. The use according to claim 5, characterized in that The product is any one of the following (1) to (2): (1) Functional cosmetics that improve cell adhesion; (2) Medical dressings.

Images