CN116514956A

CN116514956A - Recombinant humanized III type collagen and application thereof

Info

Publication number: CN116514956A
Application number: CN202310057682.9A
Authority: CN
Inventors: 马永; 赵百学; 高慧
Original assignee: Jiangsu Jingsen Biomedical New Material Technology Co ltd
Current assignee: Jiangsu Jingsen Biomedical New Material Technology Co ltd
Priority date: 2022-01-28
Filing date: 2023-01-16
Publication date: 2023-08-01

Abstract

The invention relates to recombinant humanized III type collagen, and a preparation method and application thereof. At present, the production of macromolecular humanized collagen or fully human collagen is expected to be realized in the industry, but the production is limited by the existing recombinant expression technology, and the production is a point which is difficult to completely break through. The recombinant humanized III type collagen of the invention effectively screens the amino acid sequence based on the chain amino acid sequence of human III type collagen alpha 1 (COL 3A 1), not only remains the core functional sequence of the collagen, but also has a molecular weight closer to that of natural collagen; meanwhile, the coding gene is optimized, so that the high-efficiency secretion expression of the recombinant collagen is realized, and the yield is high. Experiments prove that the collagen has higher safety, and the hemostatic sponge and the composite bone repair material prepared by the collagen have greatly improved hemostatic function and bone repair function. Therefore, the collagen can meet the industrial production and application of various products such as hemostatic sponge, bone repair material, medical or medical filling material, cosmetics and the like.

Description

Recombinant humanized III type collagen and application thereof

Technical Field

The invention belongs to the field of bioengineering and biological materials, and particularly relates to recombinant humanized III-type collagen, a preparation method and application thereof.

Background

Collagen is a family of proteins with the most abundant content in animals, and the bioactivity of the collagen enables the collagen to participate in cell migration, differentiation and reproduction, so that connective tissue has mechanical strength, and in addition, the collagen can promote cell growth and has the properties of hemostasis, biocompatibility, biodegradability and the like. Based on the characteristics, the collagen has wide application in the medical fields of burn, wound, cornea diseases, wound surface hemostasis, drug delivery, slow release technology and the like.

It has been found that more than 30 collagen chain encoding genes can form more than 16 collagen molecules, and the common types are I, II, III, V and XI. The type I collagen has the highest content in animals and accounts for 80-90% of the total collagen content, and has the strongest effect on the body functions, so that the type I collagen has the greatest clinical application. Type I collagen is mainly present in adult skin, tendons and bone tissues, and type III collagen is mainly present in infant skin or vascular intima, intestinal tract, and adult cartilage, vitreous body, intervertebral disc, etc. Among them, type III collagen (CLO 3 A1) has a strong synthesis ability and is capable of differentiating stem cells derived from bone marrow into fibroblasts that aid repair in wound repair. Therefore, in biomedical engineering, the type III collagen has wider application in the aspects of repairing tissue wounds, supporting cell reorganization, improving skin state, improving skin elasticity and tenderness, and the like.

The traditional collagen production process mainly extracts from animal tissues by an acid-base hydrolysis method, but the product obtained by the process has complex components and poor purity, and is extremely easy to lose part of biological activity in the extraction process, clinically shows rejection reaction and has extremely great potential virus hazard. In addition, the possibility of natural extraction is also limited due to the low level of type III collagen in animal tissue.

In recent years, with the development of DNA recombination technology, researchers have selected various host cells (such as E.coli, yeast, insects, mammals, etc.) in order to make full use of the excellent properties of collagen, and have desired to produce recombinant human collagen with high safety, high reproducibility, and stable quality. The escherichia coli expression system has the characteristics of high expression quantity, but has the defects of heat source generation, difficult purification of inclusion body expression, no post-translational modification of a prokaryotic system, low biological activity of a product and the like; however, the expression systems such as mammalian cells and insect cells have the post-translational modification function, but the disadvantages of long production cycle, large culture difficulty, high cost and low expression level limit the application. The pichia pastoris expression system is taken as a microbial system, has the functions of high product expression quantity, easy purification and post-translational modification, and has very outstanding advantages in expressing collagen. The molecular weight of the collagen subunit is generally about 120kDa, which is far greater than that of the common bioactive protein, and the amino acid sequence of the collagen subunit contains a large number of Gly-X-Y (X, Y represents amino acid, wherein Y is mainly proline) repeats, so that the recombinant collagen or the like obtained by a biological fermentation method by means of a genetic engineering technology has great difficulty in the length and the expression level of an expression product.

Therefore, in the existing report of recombinant human (human-like) III collagen, a part of fragments of natural human III collagen or a part of fragments of human III collagen are spliced with other types of fragments of human collagen to carry out recombinant expression. For example, chinese patent CN111363029a discloses a method for expressing recombinant human type III collagen mature peptide by pichia pastoris, wherein the expressed collagen is only 498 amino acids. And as disclosed in Chinese patent CN103122027B, the expressed protein is 257-501 amino acids spliced peptide composed of human collagen III peptide and human collagen II peptide. Further, as disclosed in chinese patent CN103725623a, a pichia pastoris engineering bacterium for secretion expression of human type III collagen alpha chain protein, and a construction method and application thereof are disclosed, wherein although it is stated that human type III collagen mature peptide expressed with 1069 amino acids can be secreted, no evidence is provided for detailed expression, and it is well known that the commonly used escherichia coli and yeast expression system does not contain proline hydroxylase, and thus collagen directly expressed by yeast is not possible to have hydroxyproline without introducing external, e.g., co-expression of proline hydroxylase (e.g., P4H), and thus, calculation of collagen expression amount by hydroxyproline content is not possible in the patent, and the expression amount is only 1g/L, which is still low for industrial production. On the other hand, the humanized collagen with a large molecular weight has the following advantages compared with the small molecular weight humanized collagen or human-like collagen: (1) The molecular weight of the micromolecular humanized collagen or the human-like collagen is small, and the number of site groups capable of providing crosslinking reaction is small, so that the macromolecule humanized collagen is easier to crosslink and fix under the same crosslinking condition. The prepared biological material has a longer upper degradation time limit (the time can be adjusted by a crosslinking process). Especially for the biological material application scene of in vivo repair, the long degradation time can possibly act along with the repair of the wound, and the micromolecular collagen biological material is likely to be completely degraded and absorbed when the wound is not repaired, so that the material disintegration and the wound repair cannot be synchronous. (2) The small molecular humanized collagen or artificial designed human-like collagen is more similar to the degradation product gelatin of macromolecular collagen in terms of molecular weight, and the molecular integrity is lost to a great extent, so that the collagen activity property exerted by the macromolecular collagen in organisms is weakened or lost to a great extent. Thus, a greater molecular weight and structure that is closer to natural collagen would be of greater advantage.

At present, the production of macromolecular humanized collagen or fully human collagen is expected to be realized in the industry, but the production is limited by the existing recombinant expression technology, and the production is a technical difficulty which is difficult to completely break through.

The recombinant humanized III collagen of the invention effectively screens the amino acid sequence based on the chain amino acid sequence of human III collagen alpha 1 (COL 3A 1), not only remains the core functional sequence of the collagen, but also has a molecular weight closer to that of natural collagen; meanwhile, the coding gene is optimized to realize the efficient secretion expression of the recombinant collagen and improve the yield.

Disclosure of Invention

The invention aims to provide recombinant humanized collagen expressed by using pichia pastoris, which has a molecular weight close to that of natural human protein, and has high expression level and excellent biological functions.

One of the purposes of the invention is to provide a recombinant humanized type III collagen which is secreted and expressed by pichia pastoris, and the amino acid sequence is shown as SEQ ID NO. 4.

Preferably, the recombinant humanized III type collagen has a coding gene shown in SEQ ID NO. 5.

Another object of the present invention is to provide a method for preparing recombinant humanized type III collagen, comprising the steps of

(1) Cloning the gene sequence into an expression vector, converting pichia pastoris engineering bacteria, and screening to obtain engineering bacteria capable of efficiently expressing recombinant humanized collagen;

(2) Fermenting and culturing the genetically engineered bacteria, and inducing and expressing recombinant humanized collagen to obtain a fermentation broth containing target protein;

(3) Purifying the fermentation broth to obtain the recombinant humanized III-type collagen.

Preferably, the expression vector is pGAPZ alpha A, and the Pichia pastoris engineering bacteria are Pichia pastoris GS115.

Another object of the present invention is to provide the use of recombinant humanized type III collagen in the preparation of hemostatic sponges, bone repair materials, medical or medical filler materials, cosmetics.

Another object of the present invention is to provide a recombinant humanized type III collagen hemostatic sponge comprising the above recombinant humanized type III collagen.

Preferably, the recombinant humanized type III collagen hemostatic sponge is prepared according to the following method:

(1) Adding recombinant III type humanized collagen into an acidic solution to prepare a collagen solution;

(2) And freeze-drying the collagen solution, and then crosslinking to obtain the collagen.

Preferably, the acidic solution may be acetic acid or dilute hydrochloric acid. More preferably, the acidic solution is acetic acid,

preferably, the crosslinking is vacuum thermal crosslinking or chemical crosslinking. More preferably, the crosslinking is vacuum thermal crosslinking.

(1) Adding 1-3% (wt/vol) of the recombinant III type humanized collagen into 1-3% (vol/vol) acetic acid solution, and magnetically stirring at normal temperature until the protein is completely dissolved to obtain a collagen solution;

(2) And (3) injecting the collagen solution into a proper mold, freeze-drying, and performing vacuum thermal crosslinking in a vacuum drying box of the freeze-dried sample to obtain the collagen hemostatic sponge sample.

Another object of the invention is the use of a hemostatic sponge for the hemostasis of wound surfaces in surgery, obstetrics and gynecology, orthopedics, stomatology and the like.

The hemostatic sponge is mainly obtained by directly freeze-drying a collagen solution, other raw materials are not added, so that the applicant tries to add other raw materials (such as hydroxyapatite) on the basis of the hemostatic sponge to prepare a composite material, and surprisingly, compared with natural collagen, the hydroxyapatite powder in the collagen solution of the invention is greatly improved in dispersion, is more beneficial to simulating the structure of the natural bone, and the actual repairing effect of the bone repairing composite material prepared by the process is far higher than that of the natural collagen or other recombinant collagens. The collagen of the invention also has more advantages in the preparation of composite materials such as bone repair and the like.

Accordingly, another object of the present invention is to provide a humanized type III collagen-hydroxyapatite composite bone repair material containing the above recombinant humanized type III collagen.

Preferably, the bone repair material further comprises hydroxyapatite.

Preferably, the bone repair material is prepared as follows:

(1) Adding recombinant humanized III type collagen into an acidic solution to prepare a collagen solution;

(2) Respectively adding hydroxyapatite powder into the collagen solution to prepare hydroxyapatite-containing slurry;

(3) And freeze-drying the slurry, and then crosslinking to obtain the modified starch.

Preferably, the preparation method of the humanized collagen-hydroxyapatite composite bone repair material specifically comprises the following steps:

(2) Adding 1-5% (wt/vol) of 200nm hydroxyapatite powder into the collagen solution, and stirring in an ice water bath to prepare hydroxyapatite-containing slurry;

(3) And respectively injecting the slurry into a proper mold, freeze-drying, and performing vacuum thermal crosslinking in a vacuum drying box of the freeze-dried sample to obtain the collagen-hydroxyapatite composite bone repair material sample.

Another object of the present invention is to provide a recombinant humanized type III collagen-hydroxyapatite composite bone repair material for filling and repairing defects of a tooth (jaw) bone; use of the composition in filling repair of bone defects, bone nonunions, delayed bone healing or nonunions caused by various reasons.

Compared with the prior art, the invention has the remarkable advantages that: (1) The designed human-like collagen gene is based on the amino acid sequence of natural type III human collagen, residues 154-1232 are obtained by screening, and the core functional sequence of the collagen is reserved, so that the molecular weight is closer to that of the natural collagen, the secretory expression of the recombinant collagen is realized, the yield is increased, the purification is easy, and the recombinant collagen has excellent biological functions; (2) The pGAPZ alpha A vector can realize high-level expression of the recombinant humanized collagen gene in pichia pastoris, methanol is not needed to induce in the fermentation process, the fermentation period is short, the cost is low, and the production process is relatively safe; (3) The secretory expression of the recombinant humanized collagen can be realized by inducing the pichia pastoris engineering bacteria, the secretory expression is beneficial to the separation and purification of the target protein, and the production period and the production cost can be reduced; (4) The target protein with the purity of more than 97 percent can be obtained by only one-step salting-out crude purification and one-step hydrophobic chromatography purification, and the method has the advantages of simple operation, short production period and remarkable economic benefit. (5) Compared with the hemostatic sponge prepared from the type III human collagen sold in the market, the hemostatic sponge prepared from the recombinant humanized type III collagen has better hemostatic effect. (6) Compared with the bone repair material prepared from the commercially available type III human collagen or natural type I collagen, the bone repair material prepared from the recombinant humanized type III collagen has the function of better promoting the growth of osteoblasts.

Drawings

FIG. 1 is a diagram of linearized nucleic acid electrophoresis of recombinant humanized collagen A expression vectors.

FIG. 2 is a diagram of nucleic acid electrophoresis of recombinant humanized collagen B (pre-optimization) expression vector linearization.

FIG. 3 is a diagram of nucleic acid electrophoresis of recombinant humanized collagen B (optimized) expression vector linearization.

FIG. 4 is a diagram of nucleic acid electrophoresis of recombinant humanized collagen C expression vector linearization.

FIG. 5 is a SDS-PAGE analysis of recombinant humanized collagen A induced expression. Wherein lane 1 is GS115 competent induced expression supernatant (negative control); lane 2 is recombinant humanized collagen a.

FIG. 6 is a SDS-PAGE analysis of recombinant humanized collagen B (pre-optimization) induced expression. Wherein lane 1 is GS115 competent induced expression supernatant (negative control); lane 2 is recombinant humanized collagen B (before optimization).

FIG. 7 is a SDS-PAGE analysis of recombinant humanized collagen B (after optimization) induced expression. Lane 1 is recombinant humanized collagen B (before optimization); lane 2 is recombinant humanized collagen B (after optimization).

FIG. 8 is a SDS-PAGE analysis of recombinant humanized collagen C induced expression. Wherein lane 1 is GS115 competent induced expression supernatant (negative control); lane 2 is recombinant humanized collagen C.

FIG. 9 is a SDS-PAGE examination of recombinant humanized collagen fermentation broth.

FIG. 10 is a SDS-PAGE examination of crude purification of recombinant humanized collagen.

FIG. 11 is a chromatographic purification chromatogram of recombinant humanized collagen.

FIG. 12 is a SDS-PAGE examination of purified recombinant humanized collagen

Fig. 13 shows an in vitro hemostatic model evaluation-related device and blood elevation. The first photograph on the left is an evaluation device formulated with reference to industry standard YY1477.5-2020, and the three photographs on the right are photographs of the location of the blood entering the syringe with the test sample.

FIG. 14 is a schematic view of a hemostatic sponge implantation site

FIG. 15 20G05 VS "Ai Wei stop" implant site musculature microscopy results

FIG. 16 is a schematic diagram of the site of application of a rabbit stimulation or intradermal reaction test, 1-head; 2-a test site; 3-control site; 4-dehaired back area; 5-tail

FIG. 17 results of acute toxicity test on collagen sponge ICR mice

FIG. 18 microstructure of 20G05-HA bone repair material

FIG. 19G291-HA bone repair material microstructure

FIG. 20 microstructure of COL-HA bone repair material

FIG. 21 20G05-HA seeded cell cultures 2, 5, 7d

FIG. 22 19G291-HA seeded cell culture 2d

FIG. 23COL-HA inoculated cell cultures 2, 5, 7d

FIG. 24 is a schematic view of the application site of stimulation or intradermal reaction of 20G05-HA bone repair material

FIG. 25 results of acute toxicity test of ICR mice with 20G05-HA bone repair material

FIG. 26 surface and lateral cell growth of sample 7d inoculated with 20G05-HA-1 and MC3T3-E1 cell culture

FIG. 27 growth of cells on the surface and side of samples of 20G05-HA-2 seeded MC3T3-E1 cell culture 7d

FIG. 28 growth of cells on the surface and side of samples of 20G05-HA-3 seeded MC3T3-E1 cell culture 7d

FIG. 29 surface and lateral cell growth of 20G05-HA-1 seeded MC3T3-E1 cell culture 14d sample

FIG. 30 growth of cells on the surface and side of sample 14d seeded with MC3T3-E1 cells 20G05-HA-2

FIG. 31 surface and lateral cell growth of sample 14d inoculated with 20G05-HA-3 MC3T3-E1 cell culture

Detailed Description

The present invention will be described in further detail with reference to the following examples and the accompanying drawings.

EXAMPLE 1 construction of Pichia pastoris expression System containing collagen III Gene

(1) Construction of recombinant expression vectors

1) Gene design

According to the protein resource database UniProt (website: http:// www.uniprot.org /) published human III type collagen alpha 1 chain amino acid sequence (P02461-1), the core functional amino acid of the collagen is reserved, and the optimal design of the gene is carried out on the amino acid sequence aiming at the codon preference of pichia pastoris, wherein the main design scheme is as follows: (1) Removing residues 1-153 of the N-terminal propeptide and residues 1233-1466 of the C-terminal propeptide, and reserving 154-1232 amino acids; (2) Removing residues 1-153 of the N-terminal propeptide, and reserving 154-1466 amino acids; (3) selecting 154 th to 615 th amino acids. Then EcoRI cleavage sites were introduced at the 5' ends thereof, respectively, the 3' ends carried NotI cleavage sites, and stop codons were introduced before the 3' NotI cleavage sites. All genes were codon optimized for pichia pastoris expression system, and then related gene sequences were synthesized by the Nanjing Jinsri biotechnology limited company, and the corresponding amino acid sequences and nucleotide sequences were shown in the following table, respectively:

TABLE 1 design of different human type III collagen sequences

2) Ligation transformation

The pGAPZ alpha A expression vector and each COL3A1 gene are respectively subjected to double enzyme digestion by EcoRI and NotI, enzyme digestion is carried out for 4 hours at 37 ℃, pGAPZ alpha A vector skeleton and COL3A1 gene are respectively recovered by gel digestion, and the pGAPZ alpha A vector skeleton and the COL3A1 gene are connected by a connection system: 2. Mu.l of vector fragment, 6. Mu.l of target gene, 1. Mu.l of T4 DNA ligase Buffer, and 1h of enzyme ligation at 22 ℃. Transforming the ligation product into an E.coli TOP10 cloning host, and coating an LB plate of 25ug/ml bleomycin; and (3) selecting the monoclonal antibodies to 2ml of sterile tubes containing 300 μl of LB liquid medium, culturing for 4 hours at 37 ℃ and 220rpm, and respectively taking 1 μl of bacterial liquid for PCR identification to obtain positive monoclonal antibodies which are recombinant escherichia coli containing target genes. Respectively inoculating positive monoclone into 200ml liquid LB culture medium, culturing at 37 deg.C and 220rpm overnight, respectively extracting plasmid by using Tiangen endotoless plasmid large extraction kit, so as to obtain a large quantity of recombinant expression plasmid for transforming Pichia pastoris.

(2) Transformation of Pichia pastoris

The recombinant expression plasmids were linearized using avril endonuclease (fig. 1-4) in a system of: 3 μl of AvrII enzyme, 100 μg of recombinant expression plasmid, 100 μl of 10×Cutsmart Buffer, and ddH ₂ O is added for 1ml, and enzyme cutting is carried out for 8-12 h at 37 ℃; firstly, treating a linearization system at 65 ℃ for 5min to inactivate enzymes, then adding 900 μl of isopropanol, standing at-20 ℃ for 10min, centrifuging at 14000rpm at 4 ℃ to collect precipitate, washing twice with 70% ethanol, drying in a super clean bench, adding 80 μl of ddH ₂ O, namely obtaining a linearization product; the linearized product was then transformed into Pichia pastoris GS115 competent, and recombinant yeast strains were obtained on YPD plates containing 0.1mg/ml bleomycin. Monoclonal antibodies were picked from the electrotransformed plates and replica plated onto YPD plates containing different concentrations of bleomycin, thereby obtaining multicopy recombinant yeast strains with an anti-bleomycin concentration of 2mg/ml, respectively.

(3) Expression identification

Inoculating the multicopy yeast strain and GS115 in 5ml BMGY culture medium (GS 115 competence is used as negative control and induction is carried out at the same time), culturing for 3 days at 30 ℃ and 200rpm, supplementing 1% glycerol every 24 hours, collecting induction expression supernatant for SDS-PAGE detection (figures 5-8), and displaying that 4 fragments designed according to the SDS-PAGE detection result can effectively express recombinant humanized collagen, but the expression quantity of each protein has obvious difference, and the results are shown in the following table:

TABLE 2 different designs of collagen expression

According to the molecular weight and the expression quantity of the expressed protein, the strain for expressing the recombinant humanized collagen B (after optimization) is finally determined to be used as a genetic engineering strain for expressing the human-like collagen, and the subsequent invention description is carried out by taking the strain as an engineering strain, so that the expression of other fragments does not obtain ideal effects.

EXAMPLE 2 Induction of expression of recombinant humanized collagen

Fermenting and culturing the genetically engineered bacteria to obtain fermentation liquor containing recombinant humanized collagen.

The culture medium used for fermentation is:

(1) Seed medium (YPD): 20g/L of peptone, 10g/L of yeast powder and 20g/L of glucose;

(2) Fermentation medium (BSM): phosphoric acid 26.7ml/L, caSO ₄ ·2H ₂ O 0.93g/L，K ₂ SO ₄ 18.2g/L，MgSO ₄ ·2H ₂ 14.9g/L of O, 4.13g/L of KOH, 40g/L of glycerol and 4.0ml/L of PTM.

PTM1：CuSO ₄ ·5H ₂ O 6.0g/L，KI 0.088g/L，MnSO ₄ ·2H ₂ O 3.0g/L，Na ₂ MOO ₄ ·2H ₂ O 0.2g/L，H ₃ BO ₃ 0.02g/L，CoCl ₂ ·6H ₂ O 0.5g/L，ZnCl ₂ 20.0g/L，FeSO ₄ ·7H ₂ O65.0 g/L, biotin 0.2g/L, concentrated H ₂ SO ₄ 5.0ml, and stored at 4 ℃.

(3) Feed medium: 50% glycerol (W/V), each 1L contains 12ml PTM1 trace elements.

The fermentation and induction expression processes of the recombinant humanized collagen engineering bacteria are as follows:

firstly, inoculating engineering bacteria into YPD liquid culture medium for culture, wherein the culture temperature is 30 ℃. When OD is ₆₀₀ When the fermentation time reaches 4-8, the fermentation seed can be used as fermentation seed. Setting parameters of fermentation tank at 30deg.C, rotation speed of 200-700rpm, and ventilation rate of 4L/min (automatic adjustment of fermentation tank) Festival), dissolved oxygen is kept above 30%, and ammonia water is used for adjusting pH to 5.0. And then the fermentation seed liquid is put into a sterile fermentation tank to start fermentation culture. And after the carbon source is exhausted, the feeding culture medium is fed, and when the wet weight of the thalli reaches 220g/L, the fermentation is finished, and the fermentation liquid is collected centrifugally.

SDS-PAGE showed that the target protein was expressed in a yield of about 10g/L as shown in FIG. 9.

The invention utilizes pichia pastoris to produce the recombinant humanized collagen, does not need to add methanol in the fermentation process, saves the production cost, simplifies the operation flow, has short fermentation period, and simultaneously ensures the high-level expression of the recombinant humanized collagen.

EXAMPLE 3 purification of recombinant humanized collagen

Purifying the fermentation liquor to obtain recombinant humanized collagen, wherein the purification steps are as follows:

(1) Adding ammonium sulfate to the fermentation broth at a concentration of 160g/L, and centrifuging at 8000rpm to collect salting-out precipitate, wherein the salting-out precipitate is redissolved with purified water (FIG. 10);

(2) Adding 0.8M ammonium sulfate into the salting-out precipitation re-dissolved product, regulating the pH to 7.4, filtering by a filter membrane, performing chromatographic purification by using a hydrophobic chromatography column, wherein the purification binding buffer is 10mM sodium dihydrogen phosphate, 0.8M ammonium sulfate, pH7.4, and the elution buffer is 10mM sodium dihydrogen phosphate, pH7.4, collecting elution peaks in the purification process (figure 11), and detecting the SDS-PAGE result shows that the purity of the recombinant humanized collagen can reach more than 97 percent after the purification (figure 12).

(3) And freeze-drying to obtain the recombinant humanized collagen pure product.

The purification method only uses one-step salting-out and one-step hydrophobic chromatography purification, does not need liquid exchange in the purification process, has simple operation and short production period, can reach more than 97% of final purity, and can meet the development requirement of biological materials.

Example 4 safety experiment of collagen raw materials

In order to verify whether the recombinant humanized collagen 20G05 expressed by the present invention meets the biosafety standard requirements, the following safety evaluation was performed, using native bovine type I collagen COL (purchased from Hebei Coolisen Biotechnology Co., ltd.) as a control.

< cytotoxicity detection >

The experimental method is briefly described:

TABLE 3 Table 3

Cell name	L929
		Culture medium	MEM+10% FBS culture medium
Number of plates	5000 holes/well
		Administration mode	Protein solution administration
Co-culture conditions	24h，(37±1)℃
		Detection method	CCK8 method
Determination method	Survival rate > 70%, no cytotoxicity was judged

The two collagen raw materials, 20G05 and COL, were dissolved in a culture medium at a concentration of 1.25mg/mL, and after filtration, an L929 cytotoxicity test was performed to complete the evaluation of cytotoxicity of the collagen raw materials themselves. The experimental results are as follows:

TABLE 4 Table 4

From the above results, it can be seen that 20G05 and COL raw materials were not significantly cytotoxic and were relatively safe for collagen hemostatic sponge preparation. Based on the above, it can be presumed that the recombinant humanized type III collagen of the present invention can be used for the preparation of various products such as bone repair materials, hemostatic sponges, medical or medical implantable filling materials, cosmetics, etc., and has no safety problem.

Example 5 preparation of hemostatic sponge sample

1% (wt/vol) of (1) recombinant type III humanized collagen 20G05, (2) recombinant type III humanized collagen 19G291 (purchased from Jiangshan Huiyuan Biotechnology Co., ltd., or prepared according to China patent CN 102443057B) and (3) natural bovine type I collagen COL (purchased from Hebei Coulossie Biotechnology Co., ltd.) were added to 3% (vol/vol) acetic acid solutions, respectively, and magnetically stirred at normal temperature until the proteins were completely dissolved, and filtered using a filter membrane, to obtain three kinds of collagen solutions.

And respectively injecting the three collagen solutions into 100mm moulds, and freeze-drying in a freeze dryer according to a freeze-drying curve (-20 ℃ for 5 hours and gradually heating to room temperature within 30 hours) to obtain the collagen sponge sample.

And placing the three collagen sponge samples subjected to freeze drying in a vacuum drying oven, and performing vacuum thermal crosslinking at 150 ℃ for 10 hours to obtain collagen hemostatic sponge samples which are respectively marked as 20G05-DHT,19G291-DHT and COL-DHT.

This example is only for illustrating the present invention, but is not intended to limit the scope of the present invention, and the related preparation conditions are not limited to the above conditions, for example: the acetic acid solution can be further replaced by dilute hydrochloric acid, and the vacuum thermal crosslinking can also be replaced by a chemical crosslinking method.

EXAMPLE 6 evaluation of Water absorption of hemostatic sponge

The water absorption is an important physical and chemical performance index of the hemostatic sponge, and the higher water absorption (the industry standard is > 2000%) is a basic condition for realizing the hemostatic function. Three collagen sponges were tested for water absorption, the specific method was as follows:

taking a certain amount of sponge, weighing dry weight of the sponge to be m1, immersing the sponge into purified water, slightly pressing the sponge with tweezers until the sponge is completely soaked, standing the sponge for 2 hours, ensuring that all air is completely removed, ensuring the integrity of a sample, slightly clamping a corner of the sponge with small tweezers after the sample absorbs enough water, taking the sponge out of the water without squeezing the sample, slightly holding the tweezers, draining the sponge on the water surface for 1min, weighing the wet weight of the sample again to be m2, and calculating the water absorption rate according to the pressing method. The test was repeated 5 times and the average was taken. The calculation formula is as follows: water absorption (%) = (m 2-m 1)/m 1 × 100% three samples and control "Ai Wei stop microfibril hemostatic collagen (sponge)" (purchased from bard medical science (Shanghai) limited) were each tested for water absorption, and the results are shown in the following table.

Table 5 water absorption of each sample

Experimental results show that the water absorption rates of the three samples and the reference substance are higher than the water absorption rate requirement of 2000% specified by the industry standard, wherein the water absorption rates of the samples of 20G05-DHT and COL-DHT are higher, and the hemostatic effect is achieved.

EXAMPLE 7 evaluation of in vitro blood coagulation efficacy of hemostatic sponge

The hemostatic function is a main index for evaluating the efficacy of the hemostatic sponge, and the efficacy of the collagen hemostatic sponge is compared by adopting an in vitro coagulation experiment, and the specific method is as follows:

experimental animals: rabbit

Test article: 1) Negative control group: gauze (10 mg/sample); 2) Positive control: ai Wei stop microfibril hemostatic collagen (sponge) (10 mg/sample); 3) Test sample: 2 recombinant collagen hemostatic sponge (10 mg/sample)

The operation steps are as follows: 1) Preparation of anticoagulated rabbit blood: fully and uniformly mixing 15mg/mL EDTA anticoagulant with the extracted fresh rabbit blood according to the proportion of 1:9; 2) Placing a test sample (negative control, positive control and detection sample) in a 50mL centrifuge tube, and preparing a group of empty 50mL centrifuge tubes as a blank control group; 3) Mixing anticoagulated rabbit blood and 0.2M calcium chloride solution according to the proportion of 9:1, and adding 0.5mL of mixed rabbit blood into each centrifuge tube; 4) Placing the centrifuge tube containing rabbit blood in a water bath at 37 ℃ for 5min, and confirming that the sponge is fully contacted with the blood during the period; 5) 40mL of distilled water was added to each centrifuge tube, and the tube was shaken at 37℃and 120rpm/min for 20min (to disrupt the free red blood cells); 6) The supernatant from the centrifuge tube was taken and absorbance was measured at 540 nm.

The data processing method comprises the following steps: BCI is an index of coagulation effect and is treated as follows:

BCI (%) = sample absorbance/blank absorbance 100% (smaller BCI value indicates better hemostatic effect of hemostatic material

The results of the in vitro coagulation function test of 2 samples and control "Ai Wei stop microfibril hemostatic collagen (sponge)" are shown in the following table.

TABLE 6

As can be seen from the results of in vitro coagulation experiments, compared with the commercial control of 'Ai Wei stop microfibril hemostatic collagen (sponge)' and other hemostatic sponges prepared from recombinant type III collagen, 20G05-DHT shows obvious efficacy advantages (the higher the BCI value is, the worse the coagulation effect is), so that the hemostatic sponge prepared from 20G05 collagen can realize better hemostatic functions.

Example 8 evaluation of in vitro hemostatic model

Experimental animals: rabbit

Test article: 1) Sample: 20G05 recombinant type III humanized collagen sponge; 2) Control: ai Wei stop microfibril hemostatic collagen (sponge)

Operational steps (established with reference to industry standard YY 1477.5-2020):

1) Setting up an experimental device:

the test device for constructing the in-vitro model of the hemostatic performance mainly comprises a sample clamping device, a negative pressure system, a connecting pipeline and the like, as shown in fig. 13:

Selecting a 1mL syringe sleeve and a 0.3mm injection needle (flat needle), cutting a sample to be tested into wafers with the diameter of 5mm and the thickness of 2mm, implanting each sample into the bottom of the syringe by means of a core rod of the syringe, pushing a rubber fixing piece (a fixing piece with a through hole is manufactured by cutting off the blind end of a rubber piston of the syringe, the uncut smooth end is contacted with the sample) to the upper part of the sample, and connecting the tail end of the syringe with a negative pressure pipeline to complete the construction of the device;

2) Preparation of anticoagulated rabbit blood: fully and uniformly mixing 15mg/mL EDTA anticoagulant with the extracted fresh rabbit blood according to the proportion of 1:9;

3) Cutting a sample to be tested and a reference substance according to the requirements in the step 1, preparing three parallel samples, and filling the cut samples into 1mL syringes for standby according to the method of the step 1;

4) Sequentially performing experiments, mixing 0.9mL of anticoagulated rabbit blood and 0.1mL of 0.2M calcium chloride solution in a 2mL centrifuge tube, placing the centrifuge tube at the bottom of a clamping device, placing the syringe provided with the sample in the step 3 on the clamping device, connecting a negative pressure pipeline, starting constant negative pressure, moving the syringe downwards, enabling an injection needle to enter the rabbit blood, enabling the rabbit blood to ascend at a constant speed, and supplying the sample;

5) The time was counted from the time when the blood contacted the lower surface of the sample, and the rise of blood was observed within 60 seconds. After contacting the sample, the blood can be slowly raised under the effect of the hemostasis effect of the sample, so that the difference of the hemostasis effect can be analyzed by comparing the rising condition of the blood of the sample and the reference.

Experimental results:

1) The rise of blood within 60s is shown in FIG. 13;

2) The 20G05 recombinant III type humanized collagen sponge group is free from rising after blood passes through a test sample, and the test sample is taken out for analysis to find that coagulation occurs in the sample, so that the method is a main reason for preventing the blood from rising; the blood in the reference substance group rises to a significantly higher position within 60 seconds and can still continue to rise, which indicates that the reference substance has limited ability to slow down the rising speed of the blood. Therefore, the comparison result of the rising condition of the blood of the tested sample and the control product shows that the 20G05 recombinant III type humanized collagen sponge has a hemostatic function obviously superior to that of the control product in the in-vitro hemostatic model evaluation, and has obvious advantages in realizing the hemostatic function.

EXAMPLE 9 hemostatic sponge liver hemostatic test

Test article: 1) Sample: 20G05 recombinant type III humanized collagen sponge; 2) Control: ai Wei A microfiber hemostatic collagen (sponge) and Bonus medical collagen sponge (available from Bonus technologies (Beijing) were purchased from Corp.).

The test method comprises the following steps:

1) Cutting hemostatic sponge and sterile gauze into square blocks with the side length of 1.5cm and the thickness of similar, weighing, and placing into an operation tray for standby.

2) Rats were weighed. The rats were fixed by injecting 10% chloral hydrate anesthetic intraperitoneally on body weight and performing general anesthesia. Picking off 4X 4cm of chest ² Area hair, 75% medical alcohol disinfection, spread disinfection hole piece of cloth after disinfecting, whole experimental process is aseptic operation.

3) The abdomen was opened layer by layer under the rib arch along the middle of the upper abdomen by about 3cm, and the muscle layer was cut to expose the liver. Sterile gauze was placed under the incision and the middle lobe of the liver was released from the abdominal cavity onto the gauze.

4) The surface of the liver and the fluid accumulation around the abdominal cavity were washed with gauze. And (3) performing partial liver excision on the surface of the hepatic lobe by using a surgical knife, and immediately covering the hemostatic material on the surface of the bleeding incision and slightly pressing after the wound surface is obviously bleeding and blood is quickly dipped by using gauze.

5) And (3) after covering the hemostatic material for 1min, slightly taking down the material, observing the bleeding condition of the wound surface, if active bleeding occurs, continuing to cover and press, if the material is completely soaked by blood, replacing the new same material, and repeatedly applying pressure until the wound surface has no blood exudation. The bleeding amount of the liver wound surface is recorded according to the standard that the active bleeding is not existed in 30s after the sponge is removed.

Evaluation of results

Bleeding amount (g) =post hemostatic (gauze and hemostatic material weight) -pre hemostatic (gauze and hemostatic material weight)

TABLE 7

	Gauze and hemostatic material weight (g) before hemostasis	Hemostatic gauze and hemostatic Material weight (g)	Bleeding amount (g)
				Ai Wei stop	19.2	20.7	1.5
20G05	19.5	19.7	0.2
				Bona plug	126.4	127	0.6

Ai Wei stop: the end of the trial failed to complete coagulation and the wound still had active bleeding.

20G05: the wound size is similar to Ai Wei, the blood coagulation is completed after the test is finished, the hemostatic process is not needed to be pressed, the wound can be attached by itself, and the wound can be easily taken down after the active bleeding is avoided, so that the adhesion phenomenon is avoided.

Bonus plug: the size of the wound is similar to Ai Wei, and after the test, the wound still has slight active bleeding, can not be attached to the wound by itself and needs to be pressed.

EXAMPLE 10 evaluation of hemostatic sponge safety

In order to verify that the hemostatic sponge prepared from 20G05 recombinant humanized collagen meets the requirements of biosafety standards, the following safety evaluation was performed, and the commercial "Ai Wei stop microfiber hemostatic collagen (sponge)" was used as a control.

< cytotoxicity evaluation >

The leaching method comprises the following steps: leaching ratio: 3cm ² /mL, leaching solution: MEM medium+10% fbs, leaching time (24±2) h, leaching temperature: (37+ -1) deg.C. The experimental method is briefly described:

TABLE 8

Cell name	L929
		Number of plates	5000 holes/well
Administration mode	Administration of leaching solutions
		Co-cultivation time	24h
Detection method	CCK8 method
		Determination method	Survival rate > 70%, no cytotoxicity was judged

Cytotoxicity evaluation tests of 20G05 sponge samples and a control "Ai Wei stop microfibril hemostatic collagen (sponge)" were respectively carried out, and the results are shown in the following table.

From the cytotoxicity results, the hemostatic sponge prepared from 20G05 collagen also had no significant cytotoxicity compared to the commercial control "Ai Wei stop microfibril hemostatic collagen (sponge)".

< experiment for hemolysis >

The leaching method comprises the following steps: leaching ratio: 3cm ² /mL, leaching solution: normal saline, leaching time (72+/-2) h, leaching temperature: (37+ -1) deg.C.

The specific method comprises the following steps:

1) Preparing a test sample: (1) sample leaching solution preparation: configuring according to the leaching method; (2) positive control: purified water, no configuration is required; (3) negative control: 0.9% sodium chloride injection, and no configuration is needed.

2) Preparation of erythrocyte suspension: taking sterilized defibrinated rabbit blood, adding 10 times of 0.9% sodium chloride injection, and mixing well. After centrifugation for 300g,15min, the pellet was removed and the supernatant discarded until the supernatant did not appear red. The obtained erythrocytes were diluted to a 2% suspension of erythrocytes with 0.9% sodium chloride injection for use.

3) Sample addition and incubation:

table 10

	Sample leaching liquor/mL	0.9% sodium chloride injection/mL	Purified water/mL	2% erythrocyte suspension/mL
					Sample group	1	0	0	1
Positive control	0	0	1	1
					Negative control	0	1	0	1

Sample addition was performed according to the above table, and after mixing, the mixture was immediately placed in a 37℃water bath for incubation for 3h.

4) Results observation and evaluation: (1) the results of the water bath of each tube in the water solution are observed visually for 15min, 30min, 45min, 1h, 2h and 3h respectively, the liquid in the tube is poured out, centrifuged for 800g and 5min, and 200 mu L of supernatant is taken in a 96-well plate. The 96-well plate was placed in a microplate reader and its absorbance was measured at 540 nm. (2) The hemolysis rate is less than 5 percent and meets the test requirement.

HR＝(A-B)/(C-B)×100％

Wherein: HR: test sample hemolysis rate; a: testing the absorbance of the sample group; b: absorbance of negative control group; c: absorbance of positive control group

The prepared 20G05 collagen sponge and the negative control normal saline and the positive control purified water are subjected to in vitro hemolysis experimental evaluation, and experimental results are shown in the following table:

TABLE 11

According to an in-vitro hemolysis experiment, the in-vitro hemolysis evaluation result of the 20G05 recombinant III type humanized collagen sponge meets the requirements of the national standard (the hemolysis rate is less than 5 percent), and the in-vitro hemolysis evaluation method is safe and reliable.

EXAMPLE 11 hemostatic sponge muscle implant experiments

The specific method comprises the following steps:

experimental animal rabbit (New Zealand rabbit, 2.0-3.0kg, male)

The operation steps are as follows: 1) The implants were implanted intramuscularly in parallel along the long axis of the muscle fiber using a trocar implantation procedure. 2) When the paraspinal muscles of rabbits are adopted, 4 test material samples are implanted into one side muscle of the spinal column of each rabbit, and are parallel to the spinal column and are 25-50 mm away from the midline, and the implants are spaced by about 25mm. The same procedure was followed to implant 4 control material samples on the other side of the spine. The implantation site is shown in fig. 14.

Implantation specification: part 6 of the biological evaluation of medical devices with reference to GB 16886.6-2015: the requirement on the block material in the post-implantation local reaction test is that the test sample with the diameter of 1-3 mm, the length of 10mm and round ends is prepared.

Clinical observation: the normal tissue structure of each implantation site should be examined, preferably including assessment of regional draining lymph nodes, preferably using a low magnification magnifying glass. The nature and extent of any tissue reaction observed, such as hematoma, edema, cyst lumen, and/or other gross findings, is recorded. And record the presence, morphology and location of the implant, including possible residues of resolvable material. The general photo should form a file.

In addition, in examining the implantation site, a general necropsy should be performed as appropriate once the animal shows signs of unhealthy or reaction to the implant.

And (5) collecting and evaluating an implantation sample: collecting tissue at the implantation site, performing fixation, dehydration, embedding and other operations to prepare a wax block, slicing the wax block, HE staining, and performing histological evaluation on the sample through observation by an optical microscope.

Evaluation index: 1) The degree of inflammation; 2) Material parameters such as rupture, and/or presence of fragments, shape and location of degradable material residues.

Local response test after intramuscular implantation of rabbits was performed on 20G05 collagen sponge and positive control "Ai Wei stop microfibril hemostatic collagen (sponge)" with the following experimental results:

clinical observation and evaluation: after 1 day of implantation, one rabbit died (caused by postoperative infection), and the skin at the implantation points of the rest rabbits implanted for 1, 3 and 5 days had no bleeding, redness and swelling and sample removal. During the implantation period, the general state of the animal is observed, and no abnormal condition is found.

Anatomic observation assessment: after 1, 2, 4 and 8 weeks of implantation, the muscle tissue at the implantation site has no inflammatory reaction and other abnormal conditions.

Microscopic evaluation: the microscopic examination results are shown in FIG. 15.

The 20G05 sponge sample and the positive control substance Ai Wei stop show normal degradation states in the muscle of the rabbit, and the degradation period of the 20G05 sample in the muscle of the rabbit is primarily judged to be 4-8 weeks.

The results of local reaction experiments after intramuscular implantation of rabbits show that the 20G05 collagen sponge meets the requirements of national standards.

EXAMPLE 12 hemostatic sponge stimulation or intradermal response test

The fixing method comprises the following steps: the breathable adhesive tape is used for fixation (the adhesive tape and gauze fall off after fixation, and observation is needed).

Dosing animals: rabbit

The specific method comprises the following steps:

1) Preparing a test sample: (1) test article: sample leaching solution. (2) Positive control: 20% SDS physiological saline solution (as prepared). (3) Negative control group: physiological saline.

2) The administration method comprises the following steps: (1) the absorbent gauze is cut into a size of 2.5cm×2.5cm, and 5-6 layers are suitably stacked. (2) The corresponding extract is applied to gauze pieces, preferably in an amount to saturate the gauze pieces, typically 0.5mL per gauze drop, and applied to both sides of the animal's back as shown in fig. 16. (3) The patch was secured with a bandage (semi-occlusive or occlusive) for at least 4 hours. (4) And after the contact period is over, the application patch is taken down, and the contact position is marked by using the durable ink. (5) The residual test material is removed by a suitable method, such as washing with warm water or other suitable non-irritating solvents and drying. (6) Route of administration: skin contact. (7) Frequency and rate of administration: the patch was fixed for at least 4h in a single contact test.

3) Observation and scoring: (1) frequency and time of observation: in a single contact test, the contact sites were recorded (1.+ -. 0.1) h, (24.+ -. 2) h, (48.+ -. 2) h and (72.+ -. 2) h, respectively, after removal of the patch. (2) The content is observed: the animal skin was observed for erythema and edema caused by the test material over a defined period of time and scored. (3) Animal reaction observation and judgment: the scores for each test site are recorded according to the following table.

TABLE 12 skin reaction scoring System

4) Evaluation of results:

(1) calculation of primary stimulation index (PII)

After 72h scoring, the total erythema and edema primary stimulation scores of each animal caused by the test material at (24±2) h, (48±2) h and (72±2) h were added, and the sum of all scores was divided by 6 (two test/observation sites, 3 time points) to obtain PII.

Note that: calculations were performed using only observation data for (24.+ -. 2) h, (48.+ -. 2) h and (72.+ -. 2) h.

(2) Reporting the corresponding reaction type

The following table reports the type of response for each set of test samples based on the primary stimulation index, and can be used to evaluate the potential of a hemostatic sponge sample to produce a skin stimulation response under test conditions.

TABLE 13 Rabbit primary or cumulative stimulation index types

Average score	Reaction type
		0～0.4	Very slight
0.5～1.9	Mild and mild
		2～4.9	Poisoning of
5～8	Heavy weight

Sample 20G05 collagen sponge and negative control physiological saline, positive control 20% SDS are used for rabbit stimulation or intradermal reaction test, and the experimental results are shown in the following table:

TABLE 14 Experimental results of stimulation or intradermal reaction in rabbits 1

TABLE 15 Experimental results of stimulation or intradermal reaction in rabbits 2

The results of the rabbit stimulation or intradermal reaction test show that: the stimulation index of the 20G05-DHT sample leaching solution to the skin of the rabbit is consistent with that of a negative control physiological saline, so that the stimulation or intradermal reaction test evaluation result of the 20G05 collagen sponge rabbit meets the requirements of the national standard.

EXAMPLE 13 acute toxicity test of hemostatic sponge

The leaching method comprises the following steps: leaching ratio: 3cm ² /mL, leaching solution: normal saline/vegetable oil, leaching time (72+ -2) h, leaching temperature: (37+ -1) deg.C.

Experimental animals: (1) species/strain: ICR mice; (2) gender: a male; animal grade: (3) SPF stage; (4) experimental animal specification: 17-23g

The specific method comprises the following steps: 1) Frequency and rate of administration: the administration was carried out according to the body weight of the animals, and the injection rate was constant and did not exceed 0.1mL/s for single administration.

TABLE 16 grouping and administration information Table

2) Clinical observation: (1) detecting animals: 1-4 groups of animals; (2) frequency and time of observation: after the leaching solution is injected, the animals are observed to react in time, and the general state, toxicity performance and death number of each group of animals are observed and recorded at 4 hours, 24 hours, 48 hours and 72 hours. (3) The content is observed: including but not limited to, death or moribund animals, mental status, behavioral activity, eating status, fecal characteristics, respiratory status, eyelid status, and the like.

3) Weight of: (1) detecting animals: 1-4 groups of animals; (2) detection time: D1-D4, the body weight was measured at a fixed time daily. The dying animals are not weighed; (3) detection period: and D4, completing the plan, and making a plan which is properly adjusted according to the actual situation under the unknown condition, and making a scheme change record.

4) Evaluation of results: (1) and in the observation period of 72 hours, the reaction of the test group animals is not greater than that of the solvent control group animals, and the test sample is judged to have no acute systemic toxicity reaction. (2) And if 2 or more animals in the test group have moderate toxic symptoms or death and 3 or more animals have weight reduction of more than 10%, judging that the test sample has acute systemic toxic reaction. (3) The test animals showed slight toxic symptoms, or no more than 1 animal showed moderate toxic symptoms or death, or animals in the group showed general weight loss although no toxic symptoms, and 10 mice were subjected to retest.

Sample 20G05 collagen sponge was subjected to ICR mouse acute toxicity experiments, the experimental results are shown in the following table and fig. 17:

TABLE 17 acute toxicity test results in mice

TABLE 18

The acute toxicity test result of the ICR mice shows that: the body weight of the mice in the group of 20G05 normal saline/plant oil immersed extract is not different from that of the mice in the group of solvent control, and the mice in each group are in a normal state as observed by general behaviours. The acute toxicity experimental evaluation result of the sample 20G05 collagen sponge ICR mice meets the requirements of national standards.

EXAMPLE 14 preparation of hemostatic sponges with different collagen concentrations and evaluation of Water absorption

< sample preparation >

3%, 5% and 10% (wt/vol) of recombinant type III humanized collagen 20G05 were added to 1% (vol/vol) acetic acid solution, respectively, and magnetically stirred at room temperature until the protein was completely dissolved, and filtered using a filter membrane to prepare three collagen solutions.

And (3) placing the three freeze-dried recombinant type III humanized collagen sponge samples in a vacuum drying oven, and performing vacuum thermal crosslinking at 150 ℃ for 10 hours to obtain the recombinant type III humanized collagen sponge samples which are respectively marked as 20G05-1, 20G05-2 and 20G05-3.

< evaluation of Water absorption >

Specific experimental methods were performed with reference to example 6.

The water absorption test of the three samples was performed separately and compared with the control, and the results are shown in the following table.

TABLE 19

The experimental results show that the water absorption of the three samples is higher than the water absorption requirement of 2000% specified by the industry standard, wherein the water absorption of 20G05-1 is the highest, and the other two samples also have higher water absorption and are higher than the reference substances, so that the hemostatic effect is achieved.

EXAMPLE 15 preparation of hemostatic sponges under different crosslinking conditions and Water absorption evaluation

1% (wt/vol) of recombinant type III humanized collagen 20G05 was added to 3% (vol/vol) acetic acid solution, and the mixture was magnetically stirred at room temperature until the protein was completely dissolved, and filtered using a filter membrane to prepare a collagen solution.

And (3) respectively injecting the collagen solution into 100mm moulds, and freeze-drying in a freeze dryer according to a freeze-drying curve (-20 ℃ for 5 hours and gradually heating to room temperature within 30 hours) to obtain the collagen sponge sample.

And (3) placing the collagen sponge sample subjected to freeze drying in a vacuum drying oven, and performing vacuum thermal crosslinking at 140 ℃ and 150 ℃ and 160 ℃ for 10 hours respectively to obtain three recombinant type III humanized collagen sponge samples which are respectively marked as 20G05-140, 20G05-150 and 20G05-160.

taking a certain amount of sponge, weighing dry weight of the sponge to be m1, immersing the sponge into purified water, slightly pressing the sponge with tweezers until the sponge is completely soaked, standing the sponge for 2 hours, ensuring that all air is completely removed, ensuring the integrity of a sample, slightly clamping a corner of the sponge with small tweezers after the sample absorbs enough water, taking the sponge out of the water without squeezing the sample, slightly holding the tweezers, draining the sponge on the water surface for 1min, weighing the wet weight of the sample again to be m2, and calculating the water absorption rate according to the pressing method. The test was repeated 5 times and the average was taken. The calculation formula is as follows: water absorption (%) = (m 2-m 1)/m 1. Times.100% the water absorption test of each of the three samples was performed and compared with the control, and the results are shown in the following table.

Table 20

The experimental results show that the water absorption of the three samples is higher than the water absorption requirement of 2000% specified by the industry standard, wherein the water absorption of 20G05-150 is the highest, and the other two samples also have higher water absorption and are higher than the reference substances, so that the hemostatic effect is achieved.

EXAMPLE 16 preparation of bone repair Material

The hemostatic sponge related to the above embodiment is mainly obtained by directly lyophilizing a collagen solution, wherein no other raw materials are added, so that the applicant tries to add other raw materials (such as hydroxyapatite) to prepare a composite material on the basis of the above, and surprisingly, compared with natural collagen, the hydroxyapatite powder in the collagen solution of the present invention has better dispersion, is more favorable for simulating the structure of the natural bone, and the bone repair composite material prepared by the process has better actual repair effect. The collagen has more advantages in the preparation of composite materials such as bone repair and the like.

Specific sample preparation and evaluation were as follows:

To each of the above three collagen solutions, 1% (wt/vol) of 200nm hydroxyapatite powder (Macklin, cat# H875580, batch # C10963596) was added, and each was stirred in an ice water bath for 30 minutes to prepare three slurries containing hydroxyapatite.

And respectively injecting the three slurries into a 100mm mold, freeze-drying in a freeze dryer according to a freeze-drying curve (-20 ℃ for 5 hours, gradually heating to room temperature within 30 hours), and performing vacuum thermal crosslinking for 10 hours at 150 ℃ in a vacuum drying box of the freeze-dried sample to obtain the collagen-hydroxyapatite composite bone repair material sample. The three bone repair materials prepared were designated 20G05-HA, 19G291-HA and COL-HA, respectively.

EXAMPLE 17 microscopic morphology observations of bone repair materials

Autologous bone repair is a gold standard for bone repair technology, but autologous bone repair is subject to many limitations due to limited sources, so developing an ideal bone repair material is key to achieving better bone repair technology. The ideal bone biological material is the bone repairing material which simulates the components and the structure of the natural bone as far as possible, and the bone repairing material prepared by the process is consistent with the natural bone in terms of components, so that the microstructure of the sample needs to be further evaluated.

Natural bone is a natural composite material composed of extracellular matrix and cells embedded therein. The extracellular matrix is composed of mineralized collagen fibers, the closely arranged nano structure and the precise three-dimensional space structure of the hydroxyapatite crystals in the collagen fibers provide a strong support for organisms, and the unique loading property of bones is determined by skillfully combining the high tensile strength and the compressive nano crystals of the collagen fibers. The hydroxyapatite crystals form a framework structure of bone salt due to the overlapping of the collagen layers. The orientation structure has strong binding force, is not easy to generate dislocation and stacking fault, and is beneficial to improving the stability and performance of the machine body. From the structural analysis of bone, there are two different structural layers of composites, namely hydroxyapatite reinforced collagen fibers constituting a 3-7 μm coaxial layer ring structure and ossicular reinforcing interstitial bone on the millimeter to micrometer scale. The subtle structure and arrangement mode of the bone matrix can meet the requirement of not only the stability of the structure, but also the normal biological absorption and exchange.

The microscopic morphology of the three bone repair materials is observed by using a scanning electron microscope, the advantages and disadvantages of the microscopic structure in the aspect of simulating natural bones are compared and analyzed, and the experimental results are shown in fig. 18-20:

Natural collagen has poor solubility and high solution viscosity, so that hydroxyapatite powder is poorly dispersed, the inside of a sample is uneven, and the structure of natural bone is difficult to simulate, as shown in fig. 20; both recombinant collagens 20G05 and 19G291 have better solubility and can be combined more uniformly, as shown in FIGS. 18 and 19. Compared with the microcosmic appearance of the two bone repair materials of 20G05-HA and 19G291-HA, the microcosmic structure of 20G05-HA is more uniform, the nano-scale combination of hydroxyapatite crystal grains and collagen fibers is realized, and meanwhile, a multi-scale pore structure exists in the material, so that the purpose of better simulating the preparation of the natural bone composite material is achieved. The multi-stage structure bionic structure is realized, the natural bone structure is further simulated, the hydroxyapatite crystal grains are embedded into the collagen fiber and are tightly combined, and the hydroxyapatite crystal is tightly arranged in the collagen fiber and has a nano structure and a precise three-dimensional space structure. This is mainly derived from the molecular design of 20G05 collagen, which allows it to better complex with hydroxyapatite to mimic the composition and structure of natural bone.

EXAMPLE 18 evaluation of the biological Functivity of bone repair Material

In order to verify the effectiveness of the material in repairing, MC3T3 cells are subjected to a cell inoculation test, and the attached growth condition of the cells is observed by adopting a scanning electron microscope. Three bone repair material samples were cut into 6mm x 3mm pieces for cell seeding experiments, and the comparative analysis of biofunctionality was performed as follows:

Inoculating cells on the material: (1) cell state observation: taking a bottle of cells, observing the cell state under a microscope, and visually observing that the cell growth state is good and the cells grow and fuse to more than 85% so as to be digestible; (2) digestion: sucking the culture medium, cleaning the culture medium once according to the size of a culture flask with a proper amount of PBS, sucking the PBS, adding pancreatin for digestion (the pancreatin addition amount is determined by shaking and the bottom surface of the culture flask can be paved, so that excessive culture is not suitable), incubating at 37 ℃, observing under a microscope, immediately adding a culture medium with twice the pancreatin amount for stopping digestion when cells begin to fall off, repeatedly blowing to enable the cells in the flask to fall off, transferring the liquid in the culture flask into a centrifuge tube, centrifuging 250g and 5min (taking care of balancing), discarding the supernatant, sucking the culture medium into the centrifuge tube, and fully and uniformly mixing to form a cell suspension; (3) cell count: adding 10 mu l of trypan blue into 10 mu l of cell suspension, uniformly mixing, and counting under a microscope; (4) preparing a cell suspension: taking the cell suspension in step (2) and adding the cell suspension to a culture medium. (5) Inoculating cells: the samples were wetted with medium prior to inoculation until the samples were spread out in the medium. After wetting was completed, sterile forceps were used to add to the well plate and the medium was blotted dry. In the cell suspension fully mixed, the cell suspension is sucked and dripped on the surface of a sample membrane, and the cell suspension needs to be uniformly paved on the surface of the membrane, so that the situation that the cell suspension is too concentrated is avoided, and an inoculation point is difficult to find during shooting. (6) After the cells and the materials are fully adhered, adding a culture medium, and culturing for 2, 5 and 7 days.

Fixing: and taking out the cultured material. (1) 2.5% glutaraldehyde fixation: original medium was aspirated off, PBS was added, material was purged, PBS was aspirated off 3 times, and 2.5% glutaraldehyde was added for fixation overnight (room temperature). (2) Alcohol gradient elution: glutaraldehyde, which immobilizes the cells, was pipetted off and the samples were dehydrated using gradient alcohol. Soaking with 30%, 50%, 70%, 80%, 90% and 95% alcohol for 10-15 min respectively, and soaking with 100% alcohol for 3 times and 10 min/time. (3) And (3) drying a sample: vacuum drying at 60deg.C, and storing the dried sample in a dryer.

Scanning electron microscope observation: the surface scanning electron microscope was used to observe the cell attachment growth and proliferation on the sample.

The experimental results are shown in fig. 21 to 23.

The 19G291-HA sample is cultured for 2d, the attached growth of cells is hardly observed on the surface, the sample is seriously disintegrated, a better structure is difficult to maintain, the 5d and 7d experiments cannot be continuously carried out, and the biological functionality is poor.

Comparing 20G05-HA with COL-HA, 20G05-HA was significantly better than COL-HA samples in terms of cell number and proliferation rate. The 20G05-HA sample can be better attached to MC3T3 cells, the cell morphology and the growth condition are better, the sample can be embodied in a system to have the function of better promoting the growth of osteoblasts, and the 20G05 sample realizes better bionic preparation due to the molecular design of the sample and also HAs obvious advantages in the aspect of biological functionality.

EXAMPLE 19 evaluation of biosafety of bone repair Material

In order to verify whether the 20G05-HA recombinant III humanized collagen-hydroxyapatite composite bone repair material meets the requirement of biosafety standard, the following safety evaluation is carried out, and COL-HA is used as a reference substance.

< cytotoxicity evaluation >

Specific experimental methods were performed as described in example 10.

Cytotoxicity evaluation tests were conducted on 20G05-HA and COL-HA samples, respectively, and the results are shown in the following table.

Cytotoxicity results indicated that: cytotoxicity of the 20G05-HA and the COL-HA meets the requirements of national standards, and obvious safety problems do not exist in the aspect of cytotoxicity.

< experiment for hemolysis >

Specific experimental methods were performed as described in example 10.

In vitro hemolysis experimental evaluation is carried out on two samples of 20G05-HA and COL-HA and purified water of a negative control physiological saline and a positive control, and the experimental results are shown in the following table:

table 22

According to in-vitro hemolysis experiments, the in-vitro hemolysis evaluation results of the 20G05-HA and COL-HA samples meet the requirements of national standards (the hemolysis rate is less than 5 percent), and the in-vitro hemolysis method is safe and reliable.

EXAMPLE 20 bone repair Material stimulation or intradermal reaction experiment

The fixed position was performed as in fig. 24, and other experimental methods were performed with reference to example 12.

Sample 20G05-HA bone repair material and negative control physiological saline, positive control 20% SDS are used for rabbit stimulation or intradermal reaction test, and the experimental results are shown in the following table:

TABLE 23 Experimental results of stimulation or intradermal reaction in rabbits 1

Table 24 Experimental results of stimulation or intradermal reaction in rabbits 2

The results of the rabbit stimulation or intradermal reaction test show that: the stimulation index of the 20G05-HA sample leaching solution to the skin of the rabbit is consistent with that of a negative control physiological saline, so that the stimulation or intradermal reaction test evaluation result of the 20G05-HA bone repair material rabbit meets the requirements of the national standard.

EXAMPLE 21 acute toxicity test of bone repair Material

Grouping and administration information is performed with reference to the following table and other experimental methods are performed with reference to example 13.

TABLE 25 grouping and administration information Table

Sample 20G05-HA bone repair material ICR mice acute toxicity test, the experimental results are shown in the following Table and FIG. 25:

table 26 acute toxicity test results in mice

/>

Table 27

The acute toxicity test result of the ICR mice shows that: the weight of the mice in the physiological saline/plant oil extract group of the bone repair material is not different from that of the mice in the solvent control group, and the mice in each group are in a normal state as observed by general behavioural. The evaluation result of the acute toxicity experiment of the sample bone repair material ICR mice meets the requirements of national standards.

EXAMPLE 22 preparation of samples of bone repair Material with different collagen concentrations and evaluation of their biological functionality

< sample preparation >

1 percent, 2 percent and 3 percent (wt/vol) of recombinant type III humanized collagen 20G05 are respectively added into 1 percent (vol/vol) acetic acid solution, and are respectively magnetically stirred at normal temperature until the protein is completely dissolved, and are filtered by using a filter membrane to prepare three collagen solutions.

5% (wt/vol) of 200nm hydroxyapatite powder (Macklin, cat# H875580, batch # C10963596) was added to each of the above three collagen solutions, and stirred in an ice water bath for 30 minutes, respectively, to prepare three slurries containing hydroxyapatite.

And respectively injecting the three slurries into a 100mm mold, freeze-drying in a freeze dryer according to a freeze-drying curve (-20 ℃ for 5 hours, gradually heating to room temperature within 30 hours), and performing vacuum thermal crosslinking for 10 hours at 150 ℃ in a vacuum drying box of the freeze-dried sample to obtain the collagen-hydroxyapatite composite bone repair material sample. The three bone repair materials prepared were designated 20G05-HA-1, 20G05-HA-2 and 20G05-HA-3, respectively.

< evaluation of biological Functions >

In order to verify the effectiveness of the material in repairing, MC3T3 cells are subjected to a cell inoculation test, and the attached growth condition of the cells is observed by adopting a scanning electron microscope. Three bone repair material samples were cut into 6mm x 3mm pieces for cell seeding assays, for comparative analysis of biofunctionality, see example 18 for specific procedures.

The experimental results are shown in FIGS. 26 to 31.

The surface and side cell attachment growth and proliferation conditions can show that the 20G05-HA-1/2/3 sample can attach MC3T3 cells well, the cell morphology and growth and proliferation conditions are good, the system can show that the sample HAs better osteoblast growth promoting function, and the 20G05-HA-2 is optimal and can be used as a preferable process.

Claims

1. A recombinant humanized III-type collagen is characterized in that the amino acid sequence is shown as SEQ ID NO. 4.

2. The recombinant humanized type III collagen according to claim 1, wherein the encoding gene is as shown in SEQ ID No. 5.

3. A method for preparing recombinant humanized type III collagen, comprising the steps of:

(1) Cloning the gene sequence as claimed in claim 2 into an expression vector, transforming Pichia pastoris engineering bacteria, and screening to obtain engineering bacteria capable of efficiently expressing recombinant humanized collagen;

(3) Purifying the fermentation broth to obtain the recombinant humanized collagen.

4. The method of claim 3, wherein the expression vector is pgapzαa.

5. The method of claim 3, wherein the pichia pastoris engineered strain is pichia pastoris GS115.

6. Use of recombinant humanized type III collagen according to any one of claims 1 to 2 for the preparation of hemostatic sponges, bone repair materials, medical or medical filler materials, cosmetics.