CN113429475A

Movatterモバイル変換

Info

Publication number: CN113429475A
Application number: CN202010207615.7A
Authority: CN
Inventors: 杜明春; 潘登科
Original assignee: Chengdu Zhongke Aoge Biotechnology Co ltd
Current assignee: Chengdu Zhongke Aoge Biotechnology Co ltd
Priority date: 2020-03-23
Filing date: 2020-03-23
Publication date: 2021-09-24

Abstract

The invention relates to the field of biological materials, in particular to a collagen material taken from a gene editing animal, a preparation method and application thereof. The immunogenicity of the collagen material processed and prepared by the method is reduced from the source of raw materials by a gene editing technology, and the immunogenicity is further reduced by using an optimized acellular processing technology and a crosslinking technology. The collagen material of the invention has low immunogenicity and good bioactivity, and is used for repairing and regenerating different tissues/organs clinically.

Description

Glue raw material and preparation method and application thereof

Technical Field

The invention relates to the field of biological materials, in particular to a glue raw material and a preparation method and application thereof.

Background

Collagen, collagen for short, is the main component of extracellular matrix, accounts for 20-30% of the total amount of protein in animal body, is widely present in connective tissues (bone, cartilage, skin, tendon, ligament, etc.) of animals, and plays roles of supporting, protecting, combining and forming septal and the like on organisms and organs. The preparation method is widely applied to the medical and health fields of general surgery, skin, orthopaedics, oral surgery, neurosurgery, cardiovascular surgery, cosmetology, drug transportation and the like.

Although collagen itself has low immunogenicity, it may cause immune rejection due to incomplete removal of foreign proteins and polysaccharides, etc. during the actual extraction process. Aiming at the potential problem, the invention completely knocks out certain specific antigens (such as a-Gal antigens) in the animal-derived collagen material by a gene editing technology, reduces the immunogenicity of the raw material from the source, and further reduces the immunogenicity by using an optimized acellular treatment technology and a crosslinking technology.

Disclosure of Invention

The invention provides a glue raw material and a preparation method and application thereof. The immunogenicity of the collagen material processed and prepared by the method is reduced from the source of raw materials by a gene editing technology, and the immunogenicity is further reduced by using an optimized acellular processing technology and a crosslinking technology. The collagen material of the invention has low immunogenicity and good bioactivity, and is used for repairing and regenerating different tissues/organs clinically.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of a glue raw material, which comprises the following steps:

step 1, breeding a gene editing animal;

step 2, collecting the collagen raw material of the animal in the step 1 as a raw material;

step 3, taking the raw material prepared in the step 2 to remove cells; the number of said decellularizations is at least 2;

step 4, decomposing the material prepared in the step 3; the number of said decompositions is at least 2;

step 5, taking the material obtained by decomposition in the step 4, and purifying the collagen material by ultrafiltration;

and 6, freeze-drying and purifying the material obtained in the step 5.

In some embodiments of the invention, the method of decellularizing in step 3 comprises physical, chemical, and/or biological methods; the physical method comprises repeated freezing and thawing and ultrasonic treatment; the chemical method comprises SDS treatment; the biological method comprises neutral protease enzymolysis;

the treatment times of the physical method, the chemical method or the biological method are respectively 0-10 times, and at least two treatment times of the decellularization method are not 0 at the same time.

In some embodiments of the invention, the freezing temperature of the repeated freeze-thaw is-20 to-196 ℃ (liquid nitrogen), the freezing time is 5min to 24h, the thawing temperature is 4 to 40 ℃, and the thawing time is 0.5 to 6 h.

In some specific embodiments of the invention, the low-frequency of the ultrasonic treatment is 10-40 KHz, and the low-frequency treatment time is 5 min-24 h; the high-frequency is 60-120 KHz, and the high-frequency processing time is 5 min-24 h; the ultrasonic power is 100W-10 KW, and the ultrasonic temperature is 0-40 ℃.

In some specific embodiments of the invention, the concentration range of the neutral protease is 0.001-5 wt.%, the enzyme activity is 500-130000U/g, the working concentration range is 0.6-2.4U/ml, the treatment temperature is 0-40 ℃, the oscillation frequency is 50-3000 rpm, and the treatment time is 0.5-24 h.

In some embodiments of the invention, the concentration of SDS is in the range of 0.001 to 5 wt.%, the oscillation frequency is in the range of 100 to 3000rpm, the treatment temperature is in the range of 0 to 40 ℃, and the treatment time is in the range of 0.5 to 24 hours.

In some embodiments of the present invention, the decellularization method can be implemented by stacking physical methods, chemical methods, and/or biological methods;

the superposition use comprises superposing at least two of repeated freeze thawing, ultrasonic treatment, SDS treatment and neutral protease enzymolysis for simultaneous use;

the superposition using comprises superposing ultrasonic treatment and SDS treatment and simultaneously using;

the superposition using comprises superposing ultrasonic treatment and neutral protease enzymolysis and simultaneously using;

the superposition use comprises the simultaneous superposition of SDS treatment and neutral protease enzymolysis;

the superposition use comprises superposition of ultrasonic treatment, SDS treatment and neutral protease enzymolysis.

In some embodiments of the invention, the decomposing comprises at least one of pepsin digestion, acetolysis, or sonication;

the low-frequency range of ultrasonic treatment is 10-40 KHz, the low-frequency treatment time is 1-24 h, the high-frequency range is 60-120 KHz, the high-frequency treatment time is 1-24 h, the ultrasonic power is 1-10 KW, and the ultrasonic temperature is 0-40 ℃; in the enzymolysis of the pepsin, the enzyme activity of the pepsin is 800-100000U/g; the concentration range is 0.001-10 wt.%, the oscillation frequency is 100-3000 rpm, the treatment temperature is 0-40 ℃, and the treatment time is 0.5-48 h; the concentration of acetic acid in the acetic acid dissolution is 0.001-20 wt.%;

the treatment times of the pepsin enzymolysis, the acetic acid dissolution or the ultrasonic treatment are respectively 0-10 times, and the treatment times of at least two of the treatments in the decomposition are not 0 at the same time.

In some embodiments of the invention, step 3 is specifically:

immersing in 0.08 wt.% neutral protease solution, wherein the enzyme activity is 1000U/g, the temperature is 25 ℃, the oscillation frequency is 100rpm, and the processing time is 8 h;

ultrasonic cleaning, processing with 110KHz for 30min, with power of 1KW and temperature of 10 deg.C;

submersed in a 0.1 wt.% SDS solution for 12h with an oscillation frequency of 1000rpm at a temperature of 30 ℃;

ultrasonic cleaning, treating with 20KHz for 200min, with power of 5KW and temperature of 30 deg.C.

The step 4 specifically comprises the following steps: the collagen raw material is subjected to the following decomposition treatment:

immersing in 1 wt.% pepsin solution for 10h, wherein the enzyme activity is 2000U/g, the oscillation frequency is 500rpm, and the treatment temperature is 37 ℃;

ultrasonic treatment is carried out for 6 hours at 80KHz, the power is 8KW, and the temperature is 20 ℃;

immersing in 1 wt.% acetic acid solution for 12h, with oscillation frequency of 600rpm and temperature of 30 ℃;

ultrasonic treatment for 3h at 120KHz, power of 5KW, and temperature of 30 deg.C.

In some embodiments of the invention, step 3 is specifically:

submersed in a1 wt.% SDS solution for 6h with an oscillation frequency of 500rpm at a temperature of 20 ℃;

immersing the substrate in 0.2 wt.% neutral protease solution, wherein the enzyme activity is 800U/g, the temperature is 10 ℃, the oscillation frequency is 3000rpm, and the processing time is 12 h;

submersed in a 0.05 wt.% SDS solution for 3h with an oscillation frequency of 400rpm at a temperature of 20 ℃;

ultrasonic cleaning, treating for 3h at 100KHz, with power of 1KW and temperature of 25 deg.C.

submersed in a 0.2 wt.% acetic acid solution for 12h with an oscillation frequency of 300rpm and a treatment temperature of 25 ℃;

ultrasonic treatment is carried out for 10 hours at 50KHz, the power is 1KW, and the temperature is 30 ℃;

immersing in 0.02 wt.% pepsin solution for 8h, wherein the enzyme activity is 1000U/g, the oscillation frequency is 1000rpm, and the temperature is 20 ℃;

ultrasonic treatment is carried out for 4 hours at 80KHz, the power is 2KW, and the temperature is 30 ℃.

In some embodiments of the invention, step 3 is specifically:

immersing in 0.4 wt.% neutral protease solution, wherein the enzyme activity is 2000U/g, the temperature is 10 ℃, the oscillation frequency is 400rpm, and the processing time is 12 h;

ultrasonic cleaning, processing for 4h at 40KHz, with power of 10KW and temperature of 30 ℃;

submersed in 11 wt.% SDS solution for 8h with an oscillation frequency of 600rpm at a temperature of 20 ℃;

ultrasonic cleaning, treating with 60KHz for 30min, with power of 1KW and temperature of 30 deg.C.

immersing in 0.6 wt.% pepsin solution for 12h, wherein the enzyme activity is 1000U/g, the oscillation frequency is 1000rpm, and the treatment temperature is 20 ℃;

ultrasonic treatment for 3h at 100KHz, with power of 1KW and temperature of 20 deg.C;

submersed in 0.3 wt.% acetic acid solution for 8h with an oscillation frequency of 800rpm at a temperature of 30 ℃;

ultrasonic treatment is carried out for 5 hours at 60KHz, the power is 10KW, and the temperature is 30 ℃.

In some embodiments of the invention, the gene editing comprises at least one of meganucleases (Meganuclease), Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR/Cas) systems.

In some embodiments of the invention, the gene editing comprises gene knock-out and/or gene transfer;

the gene knocked out in the gene knock-out comprises at least one of GGTA1, CMAH, β 4GalNT2, or PERV;

the gene transferred by the gene transfer comprises at least one of hCD46, hCD55, hCD47, LEA29Y, hTBM, hTFPI or EPCR.

In some embodiments of the invention, the animal comprises at least one of a pig, a cow, a sheep, a horse, a monkey, a dog, a rabbit, a chicken, a mouse, a silkworm, a fish, a marine organism, a donkey; the animals are stably passaged for more than 3 generations after gene editing. In other embodiments, animals are selected for stable passage 4 after gene editing.

The fish comprises freshwater fish.

In some embodiments of the invention, the collagen raw material is taken from the group consisting of dermis, fascia, sclera, capsule, tendon, fibrocartilage, bone, dentin; hyaline cartilage and elastic cartilage; reticular fibers, smooth muscle, endoneurium, artery, liver, spleen, kidney, lung, uterus; basement membrane, lens capsule; at least one of a membrane, a muscle, and a tendon sheath.

On the basis of the research, the invention also provides the collagen material prepared by the method.

The invention also provides the application of the collagen material in preparing tissue engineering materials, regenerative medical materials and transformation medical materials.

On the basis of the above research, the present invention also provides a composition comprising the collagen material of the present invention and a medically or pharmaceutically acceptable active molecule or living cell, the active molecule comprising a growth factor; the living cells include stem cells.

The invention also provides the application of the composition in preparing tissue engineering materials, regenerative medical materials and transformation medical materials.

The invention provides a preparation method of a collagen material, the collagen material prepared by the method and application thereof. (1) The gene editing technology is used for obtaining animals with low immunogenicity through the gene editing technology, and collecting collagen raw materials, namely reducing the immunogenicity of the collagen materials from material sources through the gene editing technology; (2) the gradual cell removal technology is different from the conventional method (such as one-time cell removal of a high-concentration reagent), and the cell removal treatment is carried out by adopting the principle of a small amount of times, for example, the active ingredients such as cells, polysaccharides and the like in the raw materials are removed by repeatedly using the steps of neutral protease solution soaking, Sodium Dodecyl Sulfate (SDS) solution soaking, repeated freeze thawing, ultrasound and the like, so that the immunogenicity of the collagen raw materials is reduced, and the influence on the activity of the materials in the treatment process is reduced as much as possible; (3) the gradual decomposition technology gradually decomposes the collagen raw material by repeatedly using pepsin solution for soaking, dissolving with acetic acid, performing ultrasound and the like. The collagen material is used for repairing and regenerating different tissues/organs clinically, and active molecules and/or living cells can be combined in the using process.

The invention relates to a preparation method of a collagen material, which relates to a gene editing technology, is different from a conventional method (treating the tissues/organs of an existing animal by physical, chemical and biological methods), and the gene editing technology is used for carrying out fixed-point 'editing' on a target gene, realizing the modification of a specific DNA fragment and reducing the immunogenicity of the collagen material from the source (collecting the animal) of the biological material. In the process, according to the characteristics of specific tissue materials, an editing system, a target gene, an animal species and the like are determined through screening, wherein the target gene range comprises knockout genes such as GGTA1, CMAH, beta 4GalNT2, PERV and the like, and transgenes such as hCD46, hCD55, hCD47, LEA29Y, hTBM, hTFPI, EPCR and the like. Taking alpha-Gal as an example, when xenoantigen remained in animal tissues or animal-derived collagen materials enters human bodies, hyperacute immune rejection is initiated, and the main target antigen of the immune rejection is considered to be caused by a-Gal antigen existing in animal tissues, wherein the a-Gal antigen exists in most mammals except human and higher primates; the immunogenicity of the alpha-Gal antigen knock-out (GTKO) animal-derived collagen material can be obviously reduced by developing the alpha-Gal antigen knock-out (GTKO) animal-derived collagen material through a gene editing technology.

On the other hand, the preparation method of the collagen material relates to a decellularization technology and a crosslinking technology, and is different from the conventional method (such as single decellularization of a high-concentration SDS solution or single crosslinking of high-concentration glutaraldehyde), the decellularization treatment and the crosslinking treatment are optimized according to the effect of reducing the immunogenicity of the collagen material by a gene editing technology, for example, the decellularization of a low-concentration SDS solution for multiple times or the crosslinking of low-concentration glutaraldehyde for multiple times, and the influence on the bioactivity and the like of the collagen material in the treatment process is reduced as much as possible by a mild treatment mode.

Therefore, the glue raw material, the preparation method and the application thereof provided by the invention have important practical significance.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the results of immunohistochemical assays of example 1 and comparative example 1;

FIG. 2 shows the results of the histocompatibility test of example 1 and comparative example 1;

FIG. 3 shows the results of immunohistochemical assays of example 2 and comparative example 2;

FIG. 4 shows the results of the histocompatibility test of example 2 and comparative example 2;

FIG. 5 shows the results of immunohistochemical assays of example 3 and comparative example 3;

fig. 6 shows the results of the histocompatibility test of example 3 and comparative example 3.

Detailed Description

The invention discloses a glue raw material and a preparation method and application thereof, and a person skilled in the art can realize the glue raw material by properly improving process parameters by referring to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

a. breeding the gene editing animals;

b. collecting collagen raw materials;

c. performing multiple circulating cell-removing treatments on the collagen raw material, wherein the treatment modes comprise repeated freeze thawing, ultrasonic treatment, soaking in a neutral protease solution and soaking in a Sodium Dodecyl Sulfate (SDS) solution;

d. performing multiple circulating decomposition treatment on the collagen raw material, wherein the treatment modes comprise pepsin solution soaking, acetic acid dissolution and ultrasonic treatment;

e. purifying the collagen material by ultrafiltration;

f. freeze-drying the purified collagen material.

In some embodiments, the gene editing techniques include at least one of meganucleases (Meganuclease), Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas) systems.

In some embodiments, the gene editing techniques include both gene knock-out and gene transfer, wherein the knocked-out gene comprises at least one of GGTA1, CMAH, β 4GalNT2, and PERV, and the transferred gene comprises at least one of hCD46, hCD55, hCD47, LEA29Y, hTBM, hTFPI, and EPCR.

In some embodiments, the animal comprises at least one of a pig, a cow, a sheep, a monkey, a dog, a rabbit, a mouse, a fish, a marine organism, a donkey; the animal used for preparing the collagen material is an animal which is subjected to stable passage for more than 3 generations after gene editing, and further, the animal which is subjected to stable passage for 4 generations after gene editing is selected to prepare the collagen material.

In some embodiments, the collagen raw material is taken from dermis, fascia, sclera, capsule, tendon, fibrocartilage, bone, dentin; hyaline cartilage and elastic cartilage; reticular fibers, smooth muscle, endoneurium, artery, liver, spleen, kidney, lung, uterus; basement membrane, lens capsule; at least one of a membrane, a muscle, and a tendon sheath.

In some embodiments, in the multiple-cycle decellularization treatment, the freezing temperature range in the repeated freeze-thaw treatment is-20 to-196 ℃ (liquid nitrogen), the freezing time is 5min to 24h, the unfreezing temperature range is 4 to 40 ℃, and the unfreezing time is 0.5 to 6 h; the low-frequency range of the ultrasonic treatment is 10-40 KHz, the low-frequency treatment time is 5 min-24 h, the high-frequency range is 60-120 KHz, the high-frequency treatment time is 5 min-24 h, the ultrasonic power is 100W-10 KW, and the ultrasonic temperature is 0-40 ℃; the concentration range of the neutral protease in the neutral protease treatment is 0.001-5 wt.%, and the enzyme activity of the neutral protease is 500 to E

130000U/g, the working concentration range of 0.6-2.4U/ml, the treatment temperature of 0-40 ℃, the oscillation frequency of 50-3000 rpm, and the treatment time of 0.5-24 h; in the SDS treatment, the concentration range of SDS is 0.001-5 wt.%, the oscillation frequency is 100-3000 rpm, the treatment temperature is 0-40 ℃, and the treatment time is 0.5-24 h; the multiple-cycle treatment is to combine the treatment methods for use, and the use frequency of each treatment method is 0-10 times.

In some embodiments, in the multiple cyclic decomposition treatment, the low-frequency range in the ultrasonic treatment is 10-40 KHz, the low-frequency treatment time is 1-24 h, the high-frequency range is 60-120 KHz, the high-frequency treatment time is 1-24 h, the ultrasonic power is 1-10 KW, and the ultrasonic temperature is 0-40 ℃; in the treatment of the pepsin solution, the concentration of the pepsin ranges from 0.001 wt% to 10 wt%, the enzyme activity ranges from 800U/g to 100000U/g, the oscillation frequency ranges from 100rpm to 3000rpm, the treatment temperature ranges from 0 ℃ to 40 ℃, and the treatment time ranges from 0.5 h to 48 h; the concentration of acetic acid in the acetic acid treatment is 0.001-20 wt.%. The multiple-cycle treatment is to combine the treatment methods for use, and the use frequency of each treatment method is 0-10 times.

The invention also provides the collagen material prepared by the method.

The collagen material provided by the invention can be combined with active molecules and/or living cells in the using process, and the active molecules comprise growth factors. Living cells include stem cells.

The collagen material provided by the invention, the preparation method and the reagent used in the application can be purchased from the market.

The invention is further illustrated by the following examples:

example 1

A glue material and its preparation method and tissue engineering application are provided.

The preparation method comprises the following steps:

a. preparing a knockout GGTA1 through TALEN system gene editing, transferring the knockout GGTA1 into hCD55, and editing fish by using genes with stable passage of 5 generations;

b. collecting gene-edited fish skin as a collagen extraction raw material;

c. the collagen raw material was subjected to decellularization as follows:

c1. immersing in 0.08 wt.% neutral protease solution, wherein the enzyme activity is 1000U/g, the temperature is 25 ℃, the oscillation frequency is 100rpm, and the processing time is 8 h;

c2. immersing in an ultrasonic cleaner, processing for 30min at 110KHz with power of 1KW and temperature of 10 deg.C;

c3. submersed in a 0.1 wt.% SDS solution for 12h with an oscillation frequency of 1000rpm at a temperature of 30 ℃;

c4. immersing in ultrasonic cleaner, treating with 20KHz for 200min at 5KW power and 30 deg.C.

d. The collagen raw material is subjected to the following decomposition treatment:

d1. immersing in 1 wt.% pepsin solution for 10h, wherein the enzyme activity is 2000U/g, the oscillation frequency is 500rpm, and the treatment temperature is 37 ℃;

d2. immersing in an ultrasonic device, and processing for 6h at 80KHz with power of 8KW and temperature of 20 ℃;

d3. immersing in 1 wt.% acetic acid solution for 12h, with oscillation frequency of 600rpm and temperature of 30 ℃;

d4. immersing in an ultrasonic device, processing for 3h at 120KHz with 5KW power and 30 ℃;

e. purifying the collagen material by ultrafiltration;

f. and (4) freeze-drying the collagen material.

The collagen material prepared by the steps is used as a hemostatic material.

Comparative example 1

Collecting common fish skin to prepare collagen material. Adding 0.5M acetic acid into fish skin to swell for 10h, oscillating at 500rpm and 37 deg.C to obtain acid crude extract of fish skin collagen. Extracting by ultrafiltration at 10 deg.C and lyophilizing to obtain collagen material.

Effect example 1 immunogenicity test

1.1 immunohistochemical detection: the samples were implanted subcutaneously in rats and after a specified period of time, the experimental rats were sacrificed and the implant material and surrounding skin tissue were removed, fixed with 4 wt.% paraformaldehyde fixative and immunohistochemically stained with CD3 monoclonal antibody.

The results are shown in FIG. 1:

CD3 mab was subjected to immunohistochemical staining, primarily to identify T cells (dark brown) present in the test sample due to immune rejection. The results show that the sample of example 1 found substantially a small number of T cells after subcutaneous implantation in rats for 1 month, whereas the sample of comparative example 1 found a large number of T cells, indicating that the sample of example 1 was less immunogenic than the sample of comparative example 1.

Effect example 2 biological Activity test

2.1 histocompatibility assay: the samples were implanted into rat muscles and after a specific time the experimental rats were sacrificed and the implant material and surrounding muscle tissue were removed and fixed with 4 wt.% paraformaldehyde fixing solution and HE stained.

The results are shown in FIG. 2:

HE staining showed that after 1 month of rat muscle implantation, the sample of example 1 was completely fused with the surrounding muscle tissue and cells were grown into the sample, whereas the sample of comparative example 1 and the surrounding muscle tissue had distinct boundaries, indicating that the sample of example 1 was more bioactive.

Example 2

The preparation method comprises the following steps:

a. preparing a knockout GGTA1 through CRISPR/Cas system gene editing, transferring the knockout GGTA1 into a pig with hCD46, and editing the pig by using a gene with stable passage of 3 generations;

b. collecting gene-edited pig dermis as a collagen extraction raw material;

c. the collagen raw material was subjected to decellularization as follows:

c1. submersed in a1 wt.% SDS solution for 6h with an oscillation frequency of 500rpm at a temperature of 20 ℃;

c2. immersing the substrate in 0.2 wt.% neutral protease solution, wherein the enzyme activity is 800U/g, the temperature is 10 ℃, the oscillation frequency is 3000rpm, and the processing time is 12 h;

c3. submersed in a 0.05 wt.% SDS solution for 3h with an oscillation frequency of 400rpm at a temperature of 20 ℃;

c4. immersing in ultrasonic cleaner, treating at 100KHz for 3 hr, power 1KW and temp 25 deg.C.

d1. submersed in a 0.2 wt.% acetic acid solution for 12h with an oscillation frequency of 300rpm and a treatment temperature of 25 ℃;

d2. immersing in an ultrasonic device, treating for 10h at 50KHz with power of 1KW and temperature of 30 ℃;

d3. immersing in 0.02 wt.% pepsin solution for 8h, wherein the enzyme activity is 1000U/g, the oscillation frequency is 1000rpm, and the temperature is 20 ℃;

d4. immersing in an ultrasonic device, processing for 4h at 80KHz with power of 2KW and temperature of 30 ℃;

e. purifying the collagen material by ultrafiltration;

f. and (4) freeze-drying the collagen material.

The collagen material prepared by the steps is used for the oral filling material.

Comparative example 2

Collecting common pigskin to prepare collagen material. Adding 1M acetic acid into pigskin to swell for 12h, wherein the oscillation frequency is 1000rpm, and the treatment temperature is 20 ℃, thus obtaining the acid crude extraction liquid of pigskin collagen. Extracting by ultrafiltration at 20 deg.C and lyophilizing to obtain collagen material.

Effect example 3 immunogenicity test

3.1 immunohistochemical detection: the samples were implanted subcutaneously in rats and after a specified period of time, the experimental rats were sacrificed and the implant material and surrounding skin tissue were removed, fixed with 4 wt.% paraformaldehyde fixative and immunohistochemically stained with CD68 monoclonal antibody.

The results are shown in FIG. 3:

the CD68 mab was subjected to immunohistochemical staining, primarily to identify macrophages present in the test sample (dark brown) due to immune rejection. The results show that the example 2 sample found substantially a small number of macrophages over a2 month period of subcutaneous implantation in rats, whereas the comparative example 2 found a large number of macrophages, indicating that the example 2 sample was less immunogenic than the comparative example 2.

Effect example 4 biological Activity test

4.1 histocompatibility assay: the samples were implanted into rat muscles and after a specific time the experimental rats were sacrificed and the implant material and surrounding muscle tissue were removed and fixed with 4 wt.% paraformaldehyde fixing solution and HE stained.

The results are shown in FIG. 4:

HE staining showed that after 2 months of rat muscle implantation, the sample of example 2 was completely fused with the surrounding muscle tissue and cells were grown into the sample, whereas the sample of comparative example 2 and the surrounding muscle tissue had distinct boundaries, indicating that the sample of example 2 was more bioactive.

Example 3

The preparation method comprises the following steps:

a. preparing cattle with GGTA1 knocked out by gene editing of ZFNs system, and editing the cattle by using genes with stable passage of 3 generations;

b. collecting gene-edited bovine achilles tendon as a collagen extraction raw material;

c. the collagen raw material was subjected to decellularization as follows:

c1. immersing in 0.4 wt.% neutral protease solution, wherein the enzyme activity is 2000U/g, the temperature is 10 ℃, the oscillation frequency is 400rpm, and the processing time is 12 h;

c2. immersing in an ultrasonic cleaner, processing for 4h at 40KHz, with power of 10KW and temperature of 30 ℃;

c3. submersed in 11 wt.% SDS solution for 8h with an oscillation frequency of 600rpm at a temperature of 20 ℃;

c4. immersing in ultrasonic cleaner, treating with 60KHz for 30min at 1KW power and 30 deg.C.

d1. immersing in 0.6 wt.% pepsin solution for 12h, wherein the enzyme activity is 1000U/g, the oscillation frequency is 1000rpm, and the treatment temperature is 20 ℃;

d2. immersing in an ultrasonic device, processing for 3h at 100KHz with power of 1KW and temperature of 20 ℃;

d3. submersed in 0.3 wt.% acetic acid solution for 8h with an oscillation frequency of 800rpm at a temperature of 30 ℃;

d4. immersing in an ultrasonic device, and treating for 5h at 60KHz with power of 10KW and temperature of 30 ℃;

e. purifying the collagen material by ultrafiltration;

f. and (4) freeze-drying the collagen material.

The collagen material prepared by the steps is used for bone repair materials.

Comparative example 3

Collecting common bovine achilles tendon to prepare collagen material. Adding 1M acetic acid into the bovine achilles tendon, swelling for 24h, oscillating at 1000rpm and 20 deg.C to obtain acid crude extract of bovine achilles tendon collagen. Extracting by ultrafiltration at 20 deg.C and lyophilizing to obtain collagen material.

Effect example 5 immunogenicity test

5.1 immunohistochemical detection: the samples were implanted subcutaneously in rats and after a specified period of time, the experimental rats were sacrificed and the implant material and surrounding skin tissue were removed, fixed with 4 wt.% paraformaldehyde fixative and immunohistochemically stained with CD3 monoclonal antibody.

The results are shown in FIG. 5:

CD3 mab was subjected to immunohistochemical staining, primarily to identify T cells (dark brown) present in the test sample due to immune rejection. The results show that the sample of example 3 found substantially a small number of T cells over 1 month of subcutaneous implantation in rats, whereas the sample of comparative example 3 found a large number of T cells, indicating that the sample of example 3 was less immunogenic than comparative example 3.

Effect example 6 biological Activity test

6.1 histocompatibility assay: the samples were implanted into rat muscles and after a specific time the experimental rats were sacrificed and the implant material and surrounding muscle tissue were removed and fixed with 4 wt.% paraformaldehyde fixing solution and HE stained.

The results are shown in FIG. 6:

HE staining showed that after 1 month of rat muscle implantation, the example 3 sample was completely fused with the surrounding muscle tissue and cells were grown into the sample, whereas the comparative example 3 sample and the surrounding muscle tissue had distinct boundaries, indicating that the example 3 sample was more bioactive.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The preparation method of the glue raw material is characterized by comprising the following steps:

step 1, breeding a gene editing animal;

step 6, freeze-drying and purifying the material obtained in the step 5;

wherein, the cell removing method in the step 3 comprises a physical method, a chemical method and/or a biological method; the physical method comprises repeated freezing and thawing and ultrasonic treatment; the chemical method includes Sodium Dodecyl Sulfate (SDS) treatment; the biological method comprises neutral protease enzymolysis;

the treatment times of the physical method, the chemical method or the biological method are respectively 0-10 times, and at least two treatment times of the decellularization method are not 0 at the same time;

the freezing temperature of the repeated freeze thawing is-20 to-196 ℃ (liquid nitrogen), the freezing time is 5min to 24 hours, the thawing temperature is 4 to 40 ℃, and the thawing time is 0.5 to 6 hours;

the low-frequency of the ultrasonic treatment is 10-40 KHz, and the low-frequency treatment time is 5 min-24 h; the high-frequency is 60-120 KHz, and the high-frequency processing time is 5 min-24 h; the ultrasonic power is 100W-10 KW, and the ultrasonic temperature is 0-40 ℃;

the concentration range of the neutral protease is 0.001-5 wt.%, the enzyme activity is 500-130000U/g, the working concentration range is 0.6-2.4U/ml, the treatment temperature is 0-40 ℃, the oscillation frequency is 50-3000 rpm, and the treatment time is 0.5-24 h;

the concentration range of the SDS is 0.001-5 wt.%, the oscillation frequency is 100-3000 rpm, the treatment temperature is 0-40 ℃, and the treatment time is 0.5-24 h;

wherein the decomposing in step 4 comprises at least one of pepsin enzymolysis, acetic acid dissolution or ultrasonic treatment;

2. The method according to claim 1, wherein step 3 is specifically:

ultrasonic cleaning, treating with 20KHz for 200min, with power of 5KW and temperature of 30 deg.C;

or

ultrasonic cleaning, processing for 3h at 100KHz, with the power of 1KW and the temperature of 25 ℃;

or

immersing in an ultrasonic cleaner, processing for 4h at 40KHz, with power of 10KW and temperature of 30 ℃;

3. The method according to claim 1, wherein step 4 is specifically:

ultrasonic treatment for 3h at 120KHz, with power of 5KW and temperature of 30 deg.C;

or

ultrasonic treatment for 4 hours at 80KHz, with power of 2KW and temperature of 30 ℃;

or

4. The method of any one of claims 1 to 3, wherein the gene editing comprises at least one of meganucleases (Meganucleae), Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR/Cas) systems.

5. The method according to any one of claims 1 to 4, wherein the gene editing comprises gene knock-out and/or gene transfer;

6. The method of any one of claims 1 to 5, wherein the animal comprises at least one of a pig, a cow, a sheep, a horse, a monkey, a dog, a rabbit, a chicken, a mouse, a silkworm, a fish, a marine organism, a donkey; the animals are stably passaged for more than 3 generations after gene editing; preferably, animals are selected for stable passage 4 after gene editing.

7. The method of any one of claims 1 to 6, wherein the collagen raw material is taken from the group consisting of dermis, fascia, sclera, capsule, tendon, fibrocartilage, bone, dentin; hyaline cartilage and elastic cartilage; reticular fibers, smooth muscle, endoneurium, artery, liver, spleen, kidney, lung, uterus; basement membrane, lens capsule; at least one of a membrane, a muscle, and a tendon sheath.

8. A collagen material produced by the method of any one of claims 1 to 7.

9. Use of the collagen material according to claim 8 for the preparation of tissue engineering materials, regenerative medical materials, transformation medical materials.