CN118530476B

CN118530476B - A preparation method and application of type II recombinant human collagen hydrogel

Info

Publication number: CN118530476B
Application number: CN202410478623.3A
Authority: CN
Inventors: 阮仁全; 彭玮; 赵莹; 张海中石
Original assignee: Meierjian Shenzhen Biological Technology Co ltd
Current assignee: Meierjian Shenzhen Biological Technology Co ltd
Priority date: 2024-04-19
Filing date: 2024-04-19
Publication date: 2025-01-28
Anticipated expiration: 2044-04-19
Also published as: CN118530476A

Abstract

The present invention provides a method for preparing a type II recombinant human collagen hydrogel, which belongs to the field of biomaterial technology, and includes: preparing a recombinant type II collagen solution, and then adding a visible light initiator to obtain a mixed solution; exposing the mixed solution to visible light to obtain a photo-crosslinked hydrogel; soaking the photo-crosslinked hydrogel in an EDC buffer for condensation reaction to obtain a type II recombinant human collagen hydrogel; wherein, in the recombinant type II collagen solution, the recombinant type II collagen has an amino acid sequence as described in SEQ ID NO.1. This method uses recombinant human type II collagen as a raw material, and forms a hydrogel by visible light irradiation. Compared with the existing RHC cornea preparation process, the reaction speed is more controllable, and the difficulty of subsequent cornea tissue engineering processing is reduced. The present invention also provides an application of a type II recombinant human collagen hydrogel in the preparation of an artificial cornea.

Description

Preparation method and application of II-type recombinant human collagen hydrogel

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a preparation method and application of II-type recombinant human collagen hydrogel.

Background

The human cornea is the transparent front of the eye, whose main function is to focus the incoming light rays onto the retina, thus achieving visual perception. The cornea consists of several distinct layers, including the outer epithelial layer, the stroma, which consists of collagenous extracellular matrix (ECM) and interconnected keratocytes, and the innermost endothelial layer. Although the cornea is not vascular, its nerve supply is abundant. However, once damaged or affected by disease, it may result in loss of permanent transparency, surface deformation, and even corneal blindness. It is estimated that approximately one thousand or more people worldwide suffer from blindness due to damaged or diseased cornea. In the most severe cases, transplantation of corneal tissue from a human donor is the only viable treatment. However, high quality, safe corneal organisations are severely deficient worldwide, particularly in third world and developing countries. Therefore, a cornea substitute is being actively explored.

Although a variety of total synthetic materials have been developed as cornea substitutes, synthetic materials have proven to be far less biocompatible than natural materials. Numerous studies have demonstrated that corneal stroma constructed from porcine or bovine collagen has significant biological advantages. Not only is this matrix excellent in biocompatibility, but also animal-derived collagen is abundant and low in cost, and thus has been one of the first choice in many biomaterial applications. However, most of the proteins extracted from animals are not highly purified, resulting in poor stability between batches, possibly eliciting immune responses, and carrying the risk of infectious agents. The amino acid sequence of recombinant collagen (RHC) is identical to that of natural human collagen and the level of hydroxylation is comparable, so that RHC exhibits similar properties and stability to natural human collagen, and it is possible to reduce the immune response associated with the use of extracted collagen in animals using RHC. In addition, RHC may reduce the risk of transmission of animal-related infectious agents (particularly viruses or prions, etc.), and significantly reduce the batch-to-batch variability typically present in animal-derived collagen extraction (e.g., recombinant human type III collagen (RHCIII) has been found to be a homogeneous, non-heterologous protein selected to minimize the risk of allergic reactions to animal-derived collagen or transmission of zoonotic diseases). Therefore, the development of RHCs as a source of biomedical applications would offer an important prospect for providing safe, predictable and chemically defined collagen.

Currently, there have been several studies on the use of recombinant RHC in the preparation of cornea. However, most of the existing researches use EDC/NHS condensation reaction to glue RHC into hydrogel, and the reaction speed of the glue-bonding mode is very high, so that certain challenges are brought to the later cornea tissue engineering processing.

Disclosure of Invention

In order to solve the problem of high operation difficulty of the process for preparing the cornea by using the RHC, the invention provides a preparation method of the II-type recombinant human collagen hydrogel, which takes the recombinant human II-type collagen as a raw material, and forms the hydrogel through visible light irradiation.

The invention also provides application of the II-type recombinant human collagen hydrogel in preparing artificial cornea.

The invention is realized by the following technical scheme:

The invention provides a preparation method of II-type recombinant human collagen hydrogel, which comprises the following steps:

preparing a recombinant type II collagen solution, and then adding a visible light initiator to obtain a mixed solution;

Exposing the mixed solution to visible light for irradiation to obtain photocrosslinked hydrogel;

Soaking the photocrosslinked hydrogel in EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) buffer solution for condensation reaction to obtain type II recombinant human collagen hydrogel;

Wherein, in the recombinant type II collagen solution, the recombinant type II collagen (named Recombinant Human Collagen Type II and abbreviated rhCOL A1) has an amino acid sequence as shown in SEQ ID NO. 1.

Further, the visible light initiator comprises riboflavin and sodium persulfate.

Further, the preparation of the recombinant type II collagen solution, and then adding a visible light initiator to obtain a mixed solution, specifically comprising:

preparing a recombinant type II collagen solution by using water or normal saline, and then adding riboflavin and sodium persulfate, or adding the riboflavin solution and the sodium persulfate solution to obtain a mixed solution;

the concentration of the recombinant type II collagen solution is 50-500 mg/mL, the concentration of the riboflavin solution is 0.5-5 mmol/L, and the concentration of the sodium persulfate solution is 10-40 mmol/L;

The volume ratio of the recombinant type II collagen solution to the riboflavin solution to the sodium persulfate solution is 100 (1-10): 2-10.

Further, the exposing the mixed solution to visible light to obtain the photo-crosslinked hydrogel specifically comprises:

and exposing the mixed solution to visible light with the wavelength of 400-760 nm for irradiation for 5-30 min to obtain the photo-crosslinked hydrogel.

Further, the photo-crosslinking hydrogel is soaked in EDC buffer solution for condensation reaction, and the type II recombinant human collagen hydrogel is obtained, which comprises the following specific steps:

Soaking the photocrosslinked hydrogel in EDC buffer for 5-60 min to perform condensation reaction to obtain type II recombinant human collagen hydrogel;

wherein the concentration of EDC in the EDC buffer solution is 0.5% -2% (w/v).

Further, the photo-crosslinking hydrogel is soaked in EDC buffer solution for 5-60 min to perform condensation reaction, so as to obtain the type II recombinant human collagen hydrogel, which specifically comprises the following steps:

And soaking the photocrosslinked hydrogel in EDC buffer for 5-60 min to perform condensation reaction, and washing after the condensation reaction is finished to remove redundant visible light initiator and EDC to obtain the type II recombinant human collagen hydrogel.

Based on the same inventive concept, the invention provides another preparation method of type II recombinant human collagen hydrogel, which comprises the following steps:

preparing a recombinant type II collagen solution, and then adding a visible light initiator and EDC mother liquor to obtain a reaction solution;

Exposing the reaction liquid to visible light for irradiation to obtain type II recombinant human collagen hydrogel;

Wherein, in the recombinant type II collagen solution, the recombinant type II collagen has an amino acid sequence as shown in SEQ ID NO. 1;

the photoinitiator is riboflavin and sodium persulfate;

or the photoinitiator is riboflavin solution and sodium persulfate solution.

Further, the reaction solution is exposed to visible light for irradiation to obtain the type II recombinant human collagen hydrogel, which specifically comprises the following steps:

And exposing the reaction liquid to visible light with the wavelength of 400-760 nm for irradiation for 5-60 min, and then washing to obtain the type II recombinant human collagen hydrogel.

Based on the same inventive concept, the invention provides a type II recombinant human collagen hydrogel, which is prepared by the preparation method of the type II recombinant human collagen hydrogel;

based on the same inventive concept, the invention provides an application of type II recombinant human collagen hydrogel in preparing artificial cornea.

Based on the same inventive concept, the invention provides an artificial cornea, and the preparation material of the artificial cornea comprises the type II recombinant human collagen hydrogel.

Based on the same inventive concept, the invention also provides recombinant type II collagen, which has an amino acid sequence shown in SEQ ID NO. 1.

Based on the same inventive concept, the invention also provides an application of the recombinant type II collagen in preparing type II recombinant human collagen hydrogel or artificial cornea.

One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:

1. The invention relates to a preparation method of II-type recombinant human collagen hydrogel, which takes recombinant II-type collagen rhCOL A1 as a raw material, takes riboflavin and sodium persulfate as visible light initiators, forms photo-linked hydrogel through visible light irradiation, and then is soaked in EDC buffer solution to perform condensation reaction to realize secondary crosslinking, the II-type recombinant human collagen hydrogel obtained through secondary crosslinking has excellent stability and toughness, and can be applied to preparation of artificial cornea.

2. The invention relates to a preparation method of II-type recombinant human collagen hydrogel, which takes recombinant II-type collagen rhCOL A1 as a raw material, adds visible light initiator riboflavin and sodium persulfate, and simultaneously adds EDC buffer solution, and forms II-type recombinant human collagen hydrogel by visible light irradiation, wherein rhCOL A1 is in photo-crosslinking with riboflavin and sodium persulfate, and simultaneously rhCOL A1 is in condensation reaction with EDC, so that the stability and toughness of the prepared II-type recombinant human collagen hydrogel can be enhanced.

3. The invention relates to a type II recombinant human collagen hydrogel, which takes recombinant type II collagen rhCOL A1 as a raw material, has an amino acid sequence which is highly similar to that of natural human collagen, presents similar characteristics and stability to those of natural human collagen, has high biocompatibility, provides safe and effective material assurance for cornea preparation, has rhCOL A1 which additionally comprises HHHHHHGGGGSEDLYFQ sequences at the N-terminal compared with the natural human collagen or type II recombinant human collagen RHCII, and has more excellent biocompatibility and higher light transmittance compared with RHCII compared with rhCOL A1, and is more suitable for preparing artificial cornea.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a scanning electron microscope image of a type II recombinant human collagen hydrogel according to the present invention.

FIG. 2 shows the result of SDS-PAGE electrophoresis of recombinant type II collagen genetically engineered bacteria.

FIG. 3 shows the tensile properties of the recombinant human collagen type II hydrogels of the present invention.

FIG. 4 shows the results of a biocompatibility test for the type II recombinant human collagen hydrogels of the present invention and a control group.

FIG. 5 is a diagram showing the appearance of a type II recombinant human collagen hydrogel according to the present invention and a control group.

FIG. 6 is a graph showing the stability test of type II recombinant human collagen hydrogels prepared by different crosslinking methods according to the present invention.

FIG. 7 shows the results of light transmittance test of type II recombinant human collagen hydrogels according to the present invention.

FIG. 8 shows the expansion ratio test results of the type II recombinant human collagen hydrogel according to the present invention.

Fig. 9 is a diagram of an artificial cornea made in accordance with the present invention.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.

The following will describe the preparation method of a type II recombinant human collagen hydrogel according to the present application in detail by referring to examples and experimental data.

Example 1

The preparation method of the recombinant type II collagen comprises the following steps:

1. Recombinant type II collagen expression vector construction

According to the amino acid sequence of the recombinant II-type collagen shown in SEQ ID NO.1, designing a corresponding nucleotide sequence (namely a coding sequence of rhCOL A1) according to the codon preference of pichia pastoris, adding a Xho I restriction enzyme cutting site CTCGAG at the 5 'end of the nucleotide sequence for subsequent molecular operation, adding a stop codon and a Not I restriction enzyme cutting site GCGGCCGC at the 3' end, designing to obtain a nucleotide sequence COL-1, and carrying out total gene synthesis on the sequence COL-1.

The synthesized sequence COL-1 is connected to a pichia pastoris vector pPIC9K through double enzyme digestion of Xho I and Not I, and a recombinant type II collagen expression vector pPIC9K-rhCOL A1 is obtained.

2. Construction of genetically engineered bacteria

The recombinant type II collagen expression strain is constructed by linearizing a vector pPIC9K-rhCOL A1 by Sal I endonuclease, respectively introducing the linearized vector pPIC9K-rhCOL A1 into competent cells of pichia pastoris host bacteria GS115 through electric conversion, screening and converting a positive strain by G418 antibiotics, identifying the expression condition of the positive strain by shaking, and selecting the optimal strain.

And (3) identifying the expression condition by shaking the bottle, and selecting the optimal high-expression recombinant strain as recombinant type II collagen gene engineering bacteria, wherein the SDS-PAGE electrophoresis result of the expression product of the engineering bacteria is shown in figure 2. As can be seen from FIG. 2, the color development position of the target protein band is about Marker 100kDa, and in order to further verify the accuracy of the sequence, the protein sequence is identified by GC/MS mass spectrometry to be consistent with the theoretical protein sequence. Meets the theory expectation.

3. Fermentation of recombinant type II collagen

Fermenting the recombinant type II collagen rhCOL A1 genetic engineering bacteria screened in the step 2, taking an inorganic salt BSM culture medium as a base, controlling the pH to 5.0 and the temperature to 28.0 ℃, controlling the dissolved oxygen to be 30%, starting methanol induction when the wet weight of thalli in a fermentation tank (100L) reaches 200-220 mg/mL, placing fermentation liquor in the tank after fermentation induction is performed for 40-50 hours, and completing fermentation production, wherein the yield of the formed recombinant type II collagen is about 2g/L. The preparation of recombinant collagen by using the biosynthesis method has short period, high yield and considerable economic benefit.

4. Purification of recombinant type II collagen and removal of endotoxins

Centrifuging the fermentation liquor obtained in the step 3 to collect fermentation supernatant, ultrafiltering, desalting and concentrating the fermentation supernatant by using a hollow fiber ultrafiltration system with a molecular weight cutoff of 30KD, collecting the retentate, and finally performing column hanging gradient elution and purification on the recombinant type II collagen in the retentate by using a weak cation exchange chromatographic column. Wherein the loading buffer solution is 20mM phosphate buffer solution (pH 7.5), the eluting phase adopts 20mM phosphate buffer solution containing 1M NaCl, pH7.5, and the NaCl concentration in gradient elution is automatically regulated by an instrument within the range of 0.1-1.0M.

Collecting eluent containing recombinant type II collagen, regulating the salt ion concentration of the eluent to 150mM, removing endotoxin by using a Q-type strong anion exchange chromatographic column (the trapped liquid is directly put on the column, the endotoxin is combined with the chromatographic column and protein passes through), desalting and concentrating the passing liquid by using a hollow fiber ultrafiltration system with the molecular weight cutoff of 30KD, collecting the trapped liquid, and finally, freeze-drying the trapped liquid to obtain rhCOL A1 protein with the purity of more than or equal to 95% and the endotoxin of less than 0.5 EU/mg.

RhCOL2A1 obtained based on biosynthesis has single component, high purity and high safety.

Example 2

The preparation method (step-by-step crosslinking) of the type II recombinant human collagen hydrogel comprises the following steps:

(1) Preparing a recombinant type II collagen solution with the concentration of 50mg/mL by using physiological saline, preparing riboflavin with the concentration of 5mMol/L and sodium sorbate with the concentration of 10mMol/L by using ultrapure water, and preparing an EDC solution with the concentration of 0.5% -2% (w/v) by using MES buffer solution (pH=5-7) to obtain the EDC buffer solution for standby.

(2) And adding 20ul of riboflavin solution and 20ul of sodium sorbate solution (the molar ratio of the riboflavin to the sodium sorbate is 1:2) into 1mL of recombinant type II collagen solution, and irradiating for 5-30 min under 400-760 nm of visible light to form the photo-crosslinked hydrogel.

(3) And (3) soaking the photo-crosslinked hydrogel in EDC buffer solution for 5-60 min, and performing condensation reaction on rhCOL A1 to perform secondary crosslinking.

(4) And (3) washing the secondary gel product with normal saline to remove redundant visible light initiator and EDC, thereby obtaining the type II recombinant human collagen hydrogel.

The scanning electron microscope image of the type II recombinant human collagen hydrogel prepared by the embodiment is shown in figure 1, and as can be seen from figure 1, the type II recombinant human collagen hydrogel prepared by the invention has uniformly distributed pores, which is beneficial to cell survival.

Example 3

The preparation method (step-by-step crosslinking) of the type II recombinant human collagen hydrogel is provided in the embodiment.

The only difference between the preparation method of this example and example 2 is that step (2) is different, step (2) of this example is as follows:

(2) And adding 20ul of riboflavin solution and 50ul of sodium sorbate solution (the molar ratio of the riboflavin to the sodium sorbate is 1:5) into 2mL of recombinant type II collagen solution, and irradiating for 5-30 min under 400-760 nm of visible light to form the photo-crosslinked hydrogel.

Example 4

The preparation method (single-step crosslinking) of the type II recombinant human collagen hydrogel comprises the following steps:

(1) Preparing a recombinant type II collagen solution with the concentration of 500mg/mL by using physiological saline, preparing riboflavin with the concentration of 5mMol/L and sodium salt peroxide with the concentration of 20mMol/L by using ultrapure water, and preparing EDC mother solution with the concentration of 10% (w/v) by using MES buffer solution (pH=5-7) for later use.

(2) Adding 300ul of riboflavin solution, 150ul of sodium sorbate solution (molar ratio of riboflavin to sodium sorbate is 1:2) and 30ul of EDC mother liquor into 3mL of recombinant type II collagen solution, and irradiating for 5-60 min under 400-760 nm of visible light;

(3) And (3) washing the crosslinked product obtained in the step (2) in normal saline to remove redundant visible light initiator and EDC, thereby obtaining the type II recombinant human collagen hydrogel.

Example 5

The preparation method (single-step crosslinking) of the type II recombinant human collagen hydrogel is provided in the embodiment.

The only difference between the preparation method of this example and example 4 is that step (2) is different, step (2) of this example is as follows:

(2) 20ul of riboflavin solution, 25ul of sodium sorbate solution (molar ratio of riboflavin to sodium sorbate is 1:5) and 30ul of EDC mother solution are added into 1mL of recombinant type II collagen solution, and the mixture is irradiated for 5-60 min under 400-760 nm of visible light.

Example 6

The embodiment relates to a preparation method of II-type recombinant human collagen hydrogel.

Compared with the preparation method of the embodiment 2, the only difference of the embodiment is that the step (3) is omitted, and the photo-linked hydrogel is directly washed in normal saline to obtain the type II recombinant human collagen hydrogel.

Example 7

The only difference in this example compared to the preparation method of example 2 is that in step (1), recombinant type II collagen rhCOL A1 is replaced with recombinant type II human collagen RHCII.

The amino acid sequence of the type II recombinant human collagen RHCII is shown as SEQ ID NO. 2.

Example 8

The only difference in this example compared to the preparation method of example 2 is that in step (1), recombinant type II collagen rhCOL A1 was replaced with silk fibroin (available from silk biotechnology Co., ltd. In Su.).

The type II recombinant human collagen hydrogels prepared in example 2 and example 6 were subjected to tensile mechanical property test using a universal material tester (68FM 300,Instron,USA). By normalizing the length, width and thickness of each sample (specific length: 15.5mm, width: 5mm, thickness: 1.5 mm), a tensile force with a slope of 24mN/min was applied to the sample, which force was stopped immediately upon breaking of the hydrogel fiber tube sample and the data was recorded, receiving strain and stress from the displacement and load data, respectively. To improve the accuracy and versatility of the test, each set of tests was performed at least 3 times. The test results are shown in FIG. 3. As can be seen from fig. 3, the hydrogel prepared in example 2 has higher tensile properties.

The type II recombinant human collagen hydrogels prepared in example 2 and example 7 were used as raw materials, human cornea fibroblasts were mixed with the hydrogels prepared in example 2 and example 7, respectively, the final cell concentration was 2×10 ⁶ cells/mL, the absorbance values of 1d and 3d were measured according to the MTT method, and the cell proliferation rates were obtained, and the results are shown in FIG. 4. From FIG. 4, it is understood that the cell proliferation rate of example 7 is higher, indicating that the biocompatibility of the hydrogel prepared in example 7 is better.

The type II recombinant human collagen hydrogels prepared in example 2, example 7, and example 8 were tested for compressibility, light transmittance, and expansion ratio.

The light transmittance was measured as follows:

samples were placed in 96-well plates and absorbance of the hydrogels was measured with a microplate reader (TECAN) at wavelengths between 400nm and 750 nm. Here, the absorbance of the PBS was used as a control, and the absorbance of the sample was converted into the corresponding light transmittance after subtracting the absorbance of the control from the absorbance of the sample.

The expansion ratio was measured as follows:

After immersing the samples in PBS at 37℃for 24 hours, 6 samples were taken per group, surface moisture was absorbed, the weight of the samples was measured (Wwet), and recorded. The samples were then freeze dried for 24 hours and recorded by weighing (Wdry). The calculation formula is that the expansion rate (%) = [ (Wwet-Wdry)/Wdry ]. Times.100%.

The compression performance test is as follows:

First, the hydrogel samples were cut into standard pieces of the same size and the surface was ensured to be flat. Then, a digital compression tester is prepared and appropriate parameters such as compression rate and range are set. The sample was compressed by gradually applying pressure under standard temperature and humidity conditions, and pressure and deformation data were recorded.

Test results are shown in table 1, fig. 7, and fig. 8. In Table 1, samples 1,2 and 3 were taken from type II recombinant human collagen hydrogels prepared in examples 2, 7 and 8, respectively. The physical diagrams of samples 1,2 and 3 are shown in the left, middle and right of fig. 5, respectively.

Table 1 compression properties and light transmittance test results of samples 1,2, and 3

As can be seen from table 1, fig. 7 and fig. 8, the type II recombinant human collagen hydrogel prepared in example 2 has more excellent light transmittance, significantly higher compressive strength than the hydrogel prepared in example 8, and lower expansion rate than the hydrogels prepared in examples 7 and 8. Compared with the existing hydrogel, the II-type recombinant human collagen hydrogel prepared by the invention has more excellent compression performance, expansion rate and light transmittance, and is more suitable for preparing artificial cornea.

The type II recombinant human collagen hydrogels prepared in examples 2 to 5 were subjected to stability test (test method: hydrogels prepared in examples 2, 3, 4, 5 were placed in 3ml of DMEM medium, respectively, and the DMEM medium was replaced every 2, 3 days. At different time points (1 d, 3d, 7 d), samples (n=3) were randomly extracted from each group, and the results were recorded by photographing) as shown in FIG. 6. As can be seen from fig. 6, the hydrogel prepared in example 3 had the best stability, followed by the hydrogels prepared in examples 5 and 4.

The nucleotide and amino acid sequences involved in the invention are specifically as follows:

SEQ ID NO.1

HHHHHHGGGGSEDLYFQGPMGPMGPRGPPGPAGAPGPQGFQGNPGEPGEPGVSGPMGPRGPPGPPGKPGDDGEAGKPGKAGERGPPGPQGARGFPGTPGLPGVKGHRGYPGLDGAKGEAGAPGVKGESGSPGENGSPGPMGPRGLPGERGRTGPAGAAGARGNDGQPGPAGPPGPVGPAGGPGFPGAPGAKGEAGPTGARGPEGAQGPRGEPGTPGSPGPAGASGNPGTDGIPGAKGSAGAPGIAGAPGFPGPRGPPGPQGATGPLGPKGQTGEPGIAGFKGEQGPKGEPGPAGPQGAPGPAGEEGKRGARGEPGGVGPIGPPGERGAPGNRGFPGQDGLAGPKGAPGERGPSGLAGPKGANGDPGRPGEPGLPGARGLTGRPGDAGPQGKVGPSGAPGEDGRPGPPGPQGARGQPGVMGFPGPKGANGEPGKAGEKGLPGAPGLRGLPGKDGETGAAGPPGPAGPAGERGEQGAPGPSGFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLPGTPGTDGPKGASGPAGPPGAQGPPGLQGMPGERGAAGIAGPKGDRGDVGEKGPEGAPGKDGGRGLTGPIGPPGPAGANGEKGEVGPPGPAGSAGARGAPGERGETGPPGPAGFAGPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPTGVTGPKGARGAQGPPGATGFPGAAGRVGPPGSNGNPGPPGPPGPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQGLAGQRGIVGLPGQRGERGFPGLPGPSGEPGKQGAPGASGDRGPPGPVGPPGLTGPAGEPGREGSPGADGPPGRDGAAGVKGDRGETGAVGAPGAPGPPGSPGPAGPTGKQGDRGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGLQGLPGPPGPSGDQGASGPAGPSGPRGPPGPVGPSGKDGANGIPGPIGPPGPRGRSGETGPAGPPGNPGPPGPPGPPGPGIDMSAFAGLGPREKGPDPLQYMRAD

SEQ ID NO.2

GPMGPMGPRGPPGPAGAPGPQGFQGNPGEPGEPGVSGPMGPRGPPGPPGKPGDDGEAGKPGKAGERGPPGPQGARGFPGTPGLPGVKGHRGYPGLDGAKGEAGAPGVKGESGSPGENGSPGPMGPRGLPGERGRTGPAGAAGARGNDGQPGPAGPPGPVGPAGGPGFPGAPGAKGEAGPTGARGPEGAQGPRGEPGTPGSPGPAGASGNPGTDGIPGAKGSAGAPGIAGAPGFPGPRGPPGPQGATGPLGPKGQTGEPGIAGFKGEQGPKGEPGPAGPQGAPGPAGEEGKRGARGEPGGVGPIGPPGERGAPGNRGFPGQDGLAGPKGAPGERGPSGLAGPKGANGDPGRPGEPGLPGARGLTGRPGDAGPQGKVGPSGAPGEDGRPGPPGPQGARGQPGVMGFPGPKGANGEPGKAGEKGLPGAPGLRGLPGKDGETGAAGPPGPAGPAGERGEQGAPGPSGFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLPGTPGTDGPKGASGPAGPPGAQGPPGLQGMPGERGAAGIAGPKGDRGDVGEKGPEGAPGKDGGRGLTGPIGPPGPAGANGEKGEVGPPGPAGSAGARGAPGERGETGPPGPAGFAGPPGADGQPGAKGEQGEAGQKGDAGAPGPQGPSGAPGPQGPTGVTGPKGARGAQGPPGATGFPGAAGRVGPPGSNGNPGPPGPPGPSGKDGPKGARGDSGPPGRAGEPGLQGPAGPPGEKGEPGDDGPSGAEGPPGPQGLAGQRGIVGLPGQRGERGFPGLPGPSGEPGKQGAPGASGDRGPPGPVGPPGLTGPAGEPGREGSPGADGPPGRDGAAGVKGDRGETGAVGAPGAPGPPGSPGPAGPTGKQGDRGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGLQGLPGPPGPSGDQGASGPAGPSGPRGPPGPVGPSGKDGANGIPGPIGPPGPRGRSGETGPAGPPGNPGPPGPPGPPGPGIDMSAFAGLGPREKGPDPLQYMRAD

sequence COL-1 in example 1:

ggcggcggcggcagcgaagatctgtattttcagggcccgatgggcccgatgggcccgcgc

ggcccgccgggcccggcgggcgcgccgggcccgcagggctttcagggcaacccgggcgaa

ccgggcgaaccgggcgtgagcggcccgatgggcccgcgcggcccgccgggcccgccgggc

aaaccgggcgatgatggcgaagcgggcaaaccgggcaaagcgggcgaacgcggcccgccg

ggcccgcagggcgcgcgcggctttccgggcaccccgggcctgccgggcgtgaaaggccat

cgcggctatccgggcctggatggcgcgaaaggcgaagcgggcgcgccgggcgtgaaaggc

gaaagcggcagcccgggcgaaaacggcagcccgggcccgatgggcccgcgcggcctgccg

ggcgaacgcggccgcaccggcccggcgggcgcggcgggcgcgcgcggcaacgatggccag

ccgggcccggcgggcccgccgggcccggtgggcccggcgggcggcccgggctttccgggc

gcgccgggcgcgaaaggcgaagcgggcccgaccggcgcgcgcggcccggaaggcgcgcag

ggcccgcgcggcgaaccgggcaccccgggcagcccgggcccggcgggcgcgagcggcaac

ccgggcaccgatggcattccgggcgcgaaaggcagcgcgggcgcgccgggcattgcgggc

gcgccgggctttccgggcccgcgcggcccgccgggcccgcagggcgcgaccggcccgctg

ggcccgaaaggccagaccggcgaaccgggcattgcgggctttaaaggcgaacagggcccg

aaaggcgaaccgggcccggcgggcccgcagggcgcgccgggcccggcgggcgaagaaggc

aaacgcggcgcgcgcggcgaaccgggcggcgtgggcccgattggcccgccgggcgaacgc

ggcgcgccgggcaaccgcggctttccgggccaggatggcctggcgggcccgaaaggcgcg

ccgggcgaacgcggcccgagcggcctggcgggcccgaaaggcgcgaacggcgatccgggc

cgcccgggcgaaccgggcctgccgggcgcgcgcggcctgaccggccgcccgggcgatgcg

ggcccgcagggcaaagtgggcccgagcggcgcgccgggcgaagatggccgcccgggcccg

ccgggcccgcagggcgcgcgcggccagccgggcgtgatgggctttccgggcccgaaaggc

gcgaacggcgaaccgggcaaagcgggcgaaaaaggcctgccgggcgcgccgggcctgcgc

ggcctgccgggcaaagatggcgaaaccggcgcggcgggcccgccgggcccggcgggcccg

gcgggcgaacgcggcgaacagggcgcgccgggcccgagcggctttcagggcctgccgggc

ccgccgggcccgccgggcgaaggcggcaaaccgggcgatcagggcgtgccgggcgaagcg

ggcgcgccgggcctggtgggcccgcgcggcgaacgcggctttccgggcgaacgcggcagc

ccgggcgcgcagggcctgcagggcccgcgcggcctgccgggcaccccgggcaccgatggc

ccgaaaggcgcgagcggcccggcgggcccgccgggcgcgcagggcccgccgggcctgcag

ggcatgccgggcgaacgcggcgcggcgggcattgcgggcccgaaaggcgatcgcggcgat

gtgggcgaaaaaggcccggaaggcgcgccgggcaaagatggcggccgcggcctgaccggc

ccgattggcccgccgggcccggcgggcgcgaacggcgaaaaaggcgaagtgggcccgccg

ggcccggcgggcagcgcgggcgcgcgcggcgcgccgggcgaacgcggcgaaaccggcccg

ccgggcccggcgggctttgcgggcccgccgggcgcggatggccagccgggcgcgaaaggc

gaacagggcgaagcgggccagaaaggcgatgcggg

Nucleotide sequence (II) (nucleotide sequence of recombinant type II collagen )：ggcccgatgggcccgatgggcccgcgcggcccgccgggcccggcgggcgcgccgggcccgcagggctttcagggcaacccgggcgaaccgggcgaaccgggcgtgagcggcccgatgggcccgcgcggcccgccgggcccgccgggcaaaccgggcgatgatggcgaagcgggcaaaccgggcaaagcgggcgaacgcggcccgccgggcccgcagggcgcgcgcggctttccgggcaccccgggcctgccgggcgtgaaaggccatcgcggctatccgggcctggatggcgcgaaaggcgaagcgggcgcgccgggcgtgaaaggcgaaagcggcagcccgggcgaaaacggcagcccgggcccgatgggcccgcgcggcctgccgggcgaacgcggccgcaccggcccggcgggcgcggcgggcgcgcgcggcaacgatggccagccgggcccggcgggcccgccgggcccggtgggc

ccggcgggcggcccgggctttccgggcgcgccgggcgcgaaaggcgaagcgggcccgacc

ggcgcgcgcggcccggaaggcgcgcagggcccgcgcggcgaaccgggcaccccgggcagc

ccgggcccggcgggcgcgagcggcaacccgggcaccgatggcattccgggcgcgaaaggc

agcgcgggcgcgccgggcattgcgggcgcgccgggctttccgggcccgcgcggcccgccg

ggcccgcagggcgcgaccggcccgctgggcccgaaaggccagaccggcgaaccgggcatt

gcgggctttaaaggcgaacagggcccgaaaggcgaaccgggcccggcgggcccgcagggc

gcgccgggcccggcgggcgaagaaggcaaacgcggcgcgcgcggcgaaccgggcggcgtg

ggcccgattggcccgccgggcgaacgcggcgcgccgggcaaccgcggctttccgggccag

gatggcctggcgggcccgaaaggcgcgccgggcgaacgcggcccgagcggcctggcgggc

ccgaaaggcgcgaacggcgatccgggccgcccgggcgaaccgggcctgccgggcgcgcgc

ggcctgaccggccgcccgggcgatgcgggcccgcagggcaaagtgggcccgagcggcgcg

ccgggcgaagatggccgcccgggcccgccgggcccgcagggcgcgcgcggccagccgggc

gtgatgggctttccgggcccgaaaggcgcgaacggcgaaccgggcaaagcgggcgaaaaa

ggcctgccgggcgcgccgggcctgcgcggcctgccgggcaaagatggcgaaaccggcgcg

gcgggcccgccgggcccggcgggcccggcgggcgaacgcggcgaacagggcgcgccgggc

ccgagcggctttcagggcctgccgggcccgccgggcccgccgggcgaaggcggcaaaccg

ggcgatcagggcgtgccgggcgaagcgggcgcgccgggcctggtgggcccgcgcggcgaa

cgcggctttccgggcgaacgcggcagcccgggcgcgcagggcctgcagggcccgcgcggc

ctgccgggcaccccgggcaccgatggcccgaaaggcgcgagcggcccggcgggcccgccg

ggcgcgcagggcccgccgggcctgcagggcatgccgggcgaacgcggcgcggcgggcatt

gcgggcccgaaaggcgatcgcggcgatgtgggcgaaaaaggcccggaaggcgcgccgggc

aaagatggcggccgcggcctgaccggcccgattggcccgccgggcccggcgggcgcgaac

ggcgaaaaaggcgaagtgggcccgccgggcccggcgggcagcgcgggcgcgcgcggcgcg

ccgggcgaacgcggcgaaaccggcccgccgggcccggcgggctttgcgggcccgccgggc

gcggatggccagccgggcgcgaaaggcgaacagggcgaagcgggccagaaaggcgatgcg

ggcgcgccgggcccgcagggcccgagcggcgcgcc

In summary, according to the preparation method of the type II recombinant human collagen hydrogel, the type II recombinant collagen rhCOL A1 is used as a raw material, riboflavin and sodium persulfate are used as a visible light initiator, the visible light irradiation is carried out to form the photo-crosslinked hydrogel, and EDC is further used for secondary crosslinking, so that the type II recombinant human collagen hydrogel has excellent stability and toughness, and the EDC can activate the carboxyl part of aspartic acid or glutamic acid residues in collagen to form O-acyl isothiourea, so that the O-acyl isothiourea can easily react with amino groups, and the collagen hydrolysis is prevented. The visible light initiator riboflavin and sodium persulfate adopted by the invention are nontoxic, ultraviolet light curing is not needed, the damage of ultraviolet light to cells in the hydrogel is avoided, the method has greater potential in preparing the transplantable cornea, and compared with the existing cornea preparation process by RHC, the method has slower reaction speed and high controllability, and the subsequent cornea tissue engineering processing difficulty is reduced. FIG. 9 is an artificial cornea prepared from the hydrogel of example 5.

The recombinant II-type collagen rhCOL A1 is used as a raw material, the amino acid sequence of the recombinant II-type collagen rhCOL A1 is highly similar to that of natural human collagen, the recombinant II-type collagen has characteristics and stability similar to those of the natural human collagen, the biocompatibility is high, and safe and effective material assurance is provided for cornea preparation. In addition, compared with RHCII, rhCOL A1 has the advantages that the prepared II-type recombinant human collagen hydrogel has more excellent biocompatibility and higher light transmittance, and is more suitable for preparing artificial cornea.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A method for preparing a type II recombinant human collagen hydrogel, comprising the steps of:

Soaking the photocrosslinked hydrogel in EDC buffer solution for condensation reaction to obtain type II recombinant human collagen hydrogel;

the visible light initiator comprises riboflavin and sodium persulfate.

2. The method for preparing a type II recombinant human collagen hydrogel according to claim 1, wherein the preparing the recombinant type II collagen solution, and then adding a visible light initiator to obtain a mixed solution, comprises the following steps:

wherein the concentration of the recombinant type II collagen solution is 50-500 mg/mL, the concentration of the riboflavin solution is 0.5-5 mmol/L, and the concentration of the sodium persulfate solution is 10-40 mmol/L;

3. The method for preparing a type II recombinant human collagen hydrogel according to claim 1, wherein exposing the mixed solution to visible light to obtain a photo-crosslinked hydrogel comprises:

4. The method for preparing a type II recombinant human collagen hydrogel according to claim 1, wherein the step of immersing the photo-crosslinked hydrogel in EDC buffer solution for condensation reaction to obtain the type II recombinant human collagen hydrogel comprises the following steps:

wherein the concentration of EDC in the EDC buffer solution is 0.5% -2% (w/v).

5. The method for preparing a type II recombinant human collagen hydrogel according to claim 4, wherein the photo-crosslinking hydrogel is soaked in EDC buffer for 5-60 min to perform condensation reaction, so as to obtain the type II recombinant human collagen hydrogel, which specifically comprises:

6. A method for preparing a type II recombinant human collagen hydrogel, comprising the steps of:

the photoinitiator is riboflavin and sodium persulfate;

or the photoinitiator is riboflavin solution and sodium persulfate solution.

7. The method for preparing a type II recombinant human collagen hydrogel according to claim 6, wherein the exposing the reaction solution to visible light to obtain the type II recombinant human collagen hydrogel specifically comprises:

8. A type II recombinant human collagen hydrogel, characterized in that the type II recombinant human collagen hydrogel is prepared by the preparation method of any one of claims 1-5;

or the type II recombinant human collagen hydrogel is prepared by the preparation method of claim 6 or 7.

9. Use of a type II recombinant human collagen hydrogel according to claim 8 in the preparation of an artificial cornea.

10. An artificial cornea prepared from the material comprising a type II recombinant human collagen hydrogel according to claim 8.