Recombinant type iii humanized collagen microsphere with innovative spatial structure, and design, preparation process and use thereof

Abstract

A recombinant type III humanized collagen microsphere with an innovative spatial structure, and a design, a preparation process and the use thereof. The gene sequence of type III humanized collagen is designed and constructed, cleaved with a cleavage enzyme and then cyclized with a cyclase, so that the recombinant type III humanized collagen microsphere with an innovative spatial structure is obtained without introducing any exogenous insertion gene or modification gene. The collagen microsphere has a long degradation time, and a good stability and durability, is capable of exerting various biological effects for a long time, and thus can better adapt to applications in the medicine and cosmetic industries. Moreover, the gene of recombinant type III humanized collagen, the gene of a TEV cleavage enzyme, and the gene of Butelase cyclase are introduced into an engineered strain for the first time for fermentation and expression, so that the recombinant type III humanized collagen microsphere is obtained by the "one-step method", which has a good practical value and economic value.

Description

Recombinant type III humanized collagen microspheres with innovative spatial structure and their design, preparation process and application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 22, 2023, with application number 202311780528.0 and application name “A Recombinant Type III Humanized Collagen Microsphere with an Innovative Spatial Structure and Its Design, Preparation Process and Application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of biotechnology, and specifically relates to a recombinant type III humanized collagen microsphere with an innovative spatial structure and its design, preparation process and application. Background Art

[0003] Type III collagen, a subtype of the collagen family, is a crucial component of human connective tissue and a structural protein responsible for providing elasticity and support to skin, bones, muscles, and other tissues. Type III collagen hydrolysate has been shown to possess significant antioxidant, antihypertensive, lipid-lowering, and skin-damage repair activities. It also possesses biocompatibility unmatched by other polymer materials, leading to its widespread use in the pharmaceutical and cosmetics industries.

[0004] The traditional way of obtaining collagen is mainly through extraction from animal tissues (such as the skin, bones or hooves of animals such as cows and pigs). However, there are some potential risks in animal-derived collagen. Animal tissues may carry some pathogens, including bacteria, viruses and other microorganisms. If they are not adequately sterilized and tested during the extraction and processing process, these pathogens may be present in the final collagen product. If these collagen products are used, there may be a risk of related diseases, such as mad cow disease. In addition, animal-derived collagen may also trigger immune or allergic reactions. Some people are allergic to animal proteins, and people who are allergic to animal-derived collagen products are not suitable for use.

[0005] Genetic engineering technology utilizes molecular biology and genetics to modify or reorganize the genes of an organism. This advancement has enabled the widespread development and application of recombinant humanized type III collagen, which not only avoids the potential risks of animal-derived collagen but also ensures that the production process meets safety and quality requirements. Existing recombinant humanized type III collagen is produced by cloning the gene encoding type III collagen from a human genome, then introducing it into engineered bacteria for fermentation expression, and then extracting it through purification. Research has shown that the molecular structure of type III collagen consists of three helical polypeptide chains arranged in a specific pattern to form a linear triple helix. In particular, the collagenous domain of fibrillar collagen is composed of long, uninterrupted triple helices. Primary structural analysis reveals that long segments of the type III collagen polypeptide chain consist of repeating amino acid sequences: GLy-xy. Here, x is typically proline, and y is typically hydroxyproline or hydroxylysine. This tripeptide repeat plays a significant role in the structure of collagen. Hydroxyproline and hydroxylysine are rarely found in other proteins. The hydroxyl group of hydroxyproline participates in interchain hydrogen bonding, promoting the formation of the triple helix structure and conferring excellent intracellular stability to collagen. However, the linear triple helix structure exposes the ends of type III collagen to the external environment, making it more accessible to degrading enzymes, leading to degradation. Furthermore, during fermentation expression in engineered bacteria and subsequent extraction and purification, recombinant humanized type III collagen can be easily degraded or denatured due to environmental differences inside and outside the cell. Compared to the intracellular environment, the extracellular environment is subject to higher oxygen concentrations, varying temperatures, ion concentrations, and pH values. Glycine (Gly), hydroxyproline (HyPro), and hydroxylysine (HyLys), the amino acids that make up type III collagen, are all susceptible to oxidation. When exposed to high oxygen concentrations, they are susceptible to oxidation reactions, which damage interchain hydrogen bonds, compromise the stability of the type III collagen helix structure, and lead to a decrease in its overall properties, including its biological activity. In addition, high temperatures, ion concentration, and pH in the environment can also affect the stability of hydrogen bonds and helical structures, leading to collagen denaturation and degradation. Consequently, existing recombinant humanized type III collagen has always suffered from poor stability and susceptibility to degradation.

[0006] Protein cyclization is a chemical or biological method that converts a protein's linear structure into a ring. Cyclization can improve protein stability, degradation resistance, and biological activity; it can also create novel protein conformations to meet specific research or application needs. A commonly used protein cyclization method involves introducing a linker enzyme to connect the N-terminus and C-terminus of a protein, forming a ring structure. For example, Chinese patent application CN116888272A discloses a method for tandem polypeptide ligation and cyclization, using enzymes with Asx-specific ligase and cyclase activities (i.e., butelase-1, VyPAL2, and OaAEP1b) to provide sufficient orthogonality for protein tandem ligation and cyclization. However, for type III collagen, the introduction of redundant components can compromise its original function and activity. In particular, in the pharmaceutical and cosmetic fields, the definition of recombinant humanized type III collagen is strictly defined, requiring the absence of any exogenously inserted or modified genes. Existing enzymatic cyclization methods are clearly unsuitable for this cyclization.

[0007] Therefore, how to achieve the cyclization of recombinant humanized type III collagen, improve its stability, anti-degradation and biological activity, and create new protein conformations to better adapt to applications in the pharmaceutical and cosmetics industries is still a question worth exploring. Summary of the Invention

[0008] In response to the above-mentioned problems that the existing recombinant type III humanized collagen is poorly stable and easily degraded, and that the existing protein enzyme concatenation method is not suitable for the cyclization of recombinant type III humanized collagen, the present application provides a recombinant type III humanized collagen microsphere with an innovative spatial structure and its design, preparation process and application. The type III humanized collagen gene sequence is designed and constructed, and cyclized after cleavage by a cleavage enzyme and a cyclase, so as to obtain recombinant type III humanized collagen microspheres with an innovative spatial structure without introducing any exogenous inserted genes or modified genes. The specific technical solution is as follows:

[0009] First of all, one of the purposes of this application is to provide a recombinant type III humanized collagen microsphere with an innovative spatial structure. The collagen microsphere is formed by cyclizing a peptide segment whose amino acid sequence 100% covers the amino acid sequence of natural human type III collagen, and its amino acid sequence is shown in SEQ NO.1.

[0010] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPR, SEQ NO. 1 (72 amino acids).

[0011] The aforementioned recombinant type III humanized collagen microspheres with an innovative spatial structure have a particle size of 7 to 8 nanometers and are specifically formed by cyclizing two peptide segments with amino acid sequences such as those shown in SEQ NO.2 end to end.

[0012] GIPGEKGPAGERGAPGPAGPRGERGAPGFRGPAGPN, SEQ NO. 2 (36 amino acids).

[0013] Secondly, the second purpose of this application is to provide a method for designing recombinant type III humanized collagen microspheres with an innovative spatial structure. The method designs the type III humanized collagen gene sequence and constructs an expression vector. The method uses TEV cleavage and Butelase ligase cyclization to obtain novel recombinant type III humanized collagen microspheres without introducing any exogenous inserted genes or modified genes. The method specifically includes the following steps:

[0014] S1: Design of humanized type III collagen gene sequence:

[0015] A humanized type III collagen gene sequence for cyclization is designed based on the amino acid sequence of the existing human type III collagen, as shown in SEQ NO. 3;

[0016] S2: Construction of expression vector:

[0017] The pET28a plasmid was selected as the expression basis, MBP-His6 was used as the plasmid tag, and the specific target protein sequence ENLYFQ was designed according to the designed type III humanized collagen sequence to construct the expression vector pET28a-MBP-His6-ENLYFQ.

[0018] S3: Recombinant Expression

[0019] After inserting the type III humanized collagen gene designed in step S1 into the specific target protein gene of the expression vector constructed in step S2, a recombinant plasmid containing the target protein gene is obtained; the recombinant plasmid is transferred into an engineered bacterium for cultivation and induction of expression to obtain the target protein, whose amino acid sequence is shown in SEQ NO.4;

[0020] Right now:

[0021] S4: Enzyme digestion:

[0022] The target protein obtained in step S3 is digested with TEV cleavage enzyme to obtain a peptide sequence that can be cyclized, the amino acid sequence of which is shown in SEQ NO.5;

[0023] Namely: GIPGEKGPAGERGAPGPAGPRGERGAPGFRGPAGP-NHV.

[0024] S5: Cyclization:

[0025] The two peptide segments cleaved in step S4 were subjected to enzymatic ligation using Butelase ligase to obtain a circular protein connected end to end by two amino acids GN, the sequence of which is shown in SEQ NO.6, which is the recombinant type III humanized collagen microspheres with the innovative spatial structure shown in SEQ NO.1.

[0026] Again, the third purpose of this application is to provide a preparation process for recombinant type III humanized collagen microspheres with an innovative spatial structure. The preparation processes provided in this application are actually two:

[0027] First, the "step-by-step method": design the gene sequence of human type III collagen and construct a suitable expression vector, insert the designed gene sequence of human type III collagen into the expression vector to form a recombinant plasmid, and then introduce it into the engineered bacteria for fermentation expression; extract the target protein expressed by the recombinant plasmid, treat it with a suitable shearing enzyme, and then use a ligase to cyclize it to obtain recombinant type III humanized collagen microspheres with an innovative spatial structure.

[0028] The second is the "one-step method": design the gene sequence of human type III collagen, construct a suitable expression vector, insert the designed gene sequence of human type III collagen into the expression vector to form a recombinant plasmid; introduce the recombinant plasmid together with the cleavage enzyme gene and the cyclase gene into the engineered bacteria for fermentation expression; and complete the process of expressing the target protein from the recombinant plasmid in the engineered bacteria and enzymatically cutting and cyclizing the target protein, and then extract and purify it to obtain recombinant type III humanized collagen microspheres with an innovative spatial structure.

[0029] That is, the first "step-by-step method" is to introduce the recombinant plasmid containing the gene sequence of human type III collagen into the engineered bacteria for fermentation, extract the protein, and then perform enzymatic digestion and cyclization treatment to obtain cyclized type III humanized collagen microspheres; the second "one-step method" is to introduce the recombinant plasmid containing the gene sequence of human type III collagen together with the shearing enzyme and ligase genes into the engineered bacteria, realizing the "one-step method" to directly express in the engineered bacteria to obtain cyclized type III humanized collagen microspheres.

[0030] It should be noted that the design of the gene sequence of human type III collagen and the design of the expression vector are not unique. They can be designed based on the amino acid sequence characteristics of the selected existing human type III collagen, the characteristics of the cleavage enzyme used, and the characteristics of the ligase, so as to achieve 100% coverage of the amino acid sequence of natural human type III collagen, retain its functional region (with biological activity), and achieve the purpose of cyclization without introducing any foreign genes or modifications.

[0031] Both processes in this application are preferred. The designed humanized type III collagen gene sequence is shown in SEQ NO. 3; the constructed expression vector is pET28a-MBP-His6-ENLYFQ; the selected cleavage enzymes are all TEV cleavage enzymes, and the selected cyclases are all Butelase ligases. The amino acid sequence of the recombinant humanized type III collagen microspheres with a novel spatial structure prepared under these conditions is shown in SEQ NO. 1.

[0032] In the aforementioned process for preparing recombinant type III humanized collagen microspheres with innovative spatial structure, the engineering bacteria include but are not limited to one of Escherichia coli, Bacillus subtilis, and yeast, preferably Escherichia coli.

[0033] In the preparation process of the aforementioned innovative spatial structure recombinant type III humanized collagen microspheres, the TEV shearing enzyme can be purchased from the market, or the His-TEVp recombinant protein gene can be inserted into the pET-21a vector to obtain a recombinant plasmid, which is then introduced into an engineered bacterium for expression.

[0034] In the aforementioned process for preparing recombinant type III humanized collagen microspheres with an innovative spatial structure, the Butelase ligase can be purchased commercially or expressed by integrating the Butelase ligase gene into an expression site in an engineered bacterium. For example, the Butelase ligase gene can be designed to have the sequence shown in SEQ NO. 7 and then integrated into the lacZ site of BL21 using CRISPR / Cas9 technology for expression.

[0035] The preparation process of the aforementioned innovative spatially structured recombinant type III humanized collagen microspheres, the first "step-by-step" process includes the following steps:

[0036] SA1: Culture the bacteria: Inoculate the engineered bacteria introduced with the recombinant plasmid into the culture medium and culture at 32-37°C with shaking for 20-24 hours to obtain the expression bacteria seed solution;

[0037] SA2: Transfer fermentation: Transfer the expression bacteria seed solution to a new culture medium at an inoculum concentration of 5% to 15% by volume and continue fermentation at 32-37°C.

[0038] SA3: Induced expression: When the density of the engineered bacteria in the fermentation broth reaches the predetermined concentration, IPTG inducer is added and the target protein expression is induced at 37°C;

[0039] SA4: Extraction of target protein: After the fermentation expression is completed, the fermentation product is collected, the precipitate is centrifuged, and ultrasonically disrupted on ice. After denaturation and renaturation, the protein is purified to obtain the target protein;

[0040] SA5: Enzymatic digestion: Resuspend the target protein in TEV cleavage enzyme solution and incubate the enzyme digestion reaction at pH 6.0-9.0 and temperature 29-34°C to obtain peptides that can be cyclized;

[0041] SA6: Enzymatic cyclization: Butelase ligase is added to the protein solution after enzyme digestion. The enzyme ligation reaction conditions are pH 5.0-7.0 and reaction temperature 37-45°C to obtain cyclized recombinant humanized type III collagen.

[0042] SA7: Collection and purification: Collect the cyclized recombinant type III humanized collagen for purification to obtain recombinant type III humanized collagen microspheres with innovative spatial structure.

[0043] The specific process of the second "one-step method" includes the following steps:

[0044] SB1: Culture of bacterial strains: Introduce the recombinant plasmid containing the designed humanized type III collagen gene sequence and the recombinant plasmid containing the TEV shearing enzyme gene into an engineered bacterium integrated with the Butelase ligase gene, and inoculate the engineered bacterium into a culture medium. Culture the culture at 32-37°C with shaking for 20-24 hours to obtain a seed solution of the expression bacteria.

[0045] SB2: Transfer fermentation: Transfer the expression bacteria seed solution to a new culture medium at an inoculum concentration of 5% to 15% by volume and continue fermentation at 32-37°C.

[0046] SB3: Induced expression: When the density of the engineered bacteria in the fermentation broth reaches the predetermined concentration, IPTG inducer is added and the target protein is induced to express and undergo enzymatic cleavage and cyclization at 37°C.

[0047] SB4: Collection and purification: After the fermentation expression is completed, the fermentation product is collected, the precipitate is centrifuged and ultrasonically broken on ice, and then the protein is purified after denaturation and renaturation to obtain recombinant type III humanized collagen microspheres with an innovative spatial structure.

[0048] The culture medium used in the preparation process of the two aforementioned innovative spatial structures of recombinant type III humanized collagen microspheres is M9 culture medium, the specific composition of which is 20 g / L glucose, 10 g / L sodium chloride, and 20 g / L ammonium sulfate.

[0049] In the preparation processes of the two aforementioned innovative spatially structured recombinant type III humanized collagen microspheres, the added inducers are both IPTG inducers with a concentration of 0.5-10%; and the concentration of the inducers in the culture system is both 0.1-1 mM. In the "one-step method", the concentration of the IPTG inducer in the culture system is preferably 0.5 mM.

[0050] In the preparation processes of the two aforementioned innovative spatial structures of recombinant type III humanized collagen microspheres, when the IPTG inducer was added, the density of the engineered bacteria reached OD600 of 0.6 to 0.7.

[0051] In addition, the fourth purpose of this application is to provide an application of recombinant type III humanized collagen microspheres with an innovative spatial structure, that is, the application of the aforementioned recombinant type III humanized collagen microspheres with an innovative spatial structure in the preparation of hemostatic materials, bone repair materials, skin repair materials or medical devices; wherein the medical devices include Class II medical devices such as dressings, and Class III medical devices such as hyaluronic acid injections or filling implant materials.

[0052] The innovative features and beneficial effects of this application are as follows:

[0053] 1) The recombinant type III humanized collagen microspheres of this application are a new protein conformation. Compared with the linear structure of natural human type III collagen, the recombinant type III humanized collagen microspheres of this application change the original linear spatial structure of collagen through cyclization, effectively delaying the degradation of collagen by proteases, improving its stability and durability, and being able to exert its various biological effects for a long time, and can better adapt to applications in the pharmaceutical and cosmetics industries.

[0054] 2) The protein sequence of the recombinant humanized type III collagen microspheres in this application 100% covers the natural human type III collagen sequence, retains the core functional region of collagen and does not introduce any exogenous inserted genes or modified genes. It can promote the proliferation and repair of tissue cells and has better biological activity than natural human type III collagen.

[0055] 3) The particle size of the recombinant humanized type III collagen microspheres of the present application is only 7 to 8 nanometers, which can easily pass through the skin barrier and maximize its biological efficacy.

[0056] 4) The recombinant type III humanized collagen microspheres of the present application have a protein microsphere structure after cyclization and recombination, have a more stable molecular structure and a longer half-life, and are more suitable for applications in products such as drug delivery and transdermal preparations.

[0057] 5) The recombinant humanized type III collagen microspheres of the present application also have good bioactivity and biocompatibility, and can be widely used in the medical field, such as treating tissue damage, repairing trauma, etc., and have important medical and economic value.

[0058] 6) The method for preparing recombinant type III humanized collagen microspheres in this application, with the purpose of not introducing any exogenous inserted genes or modified genes, constructed a pET28a-MBP-His6-ENLYFQ expression vector, which enables the target gene sequence to be inserted into the recombinant plasmid and then cloned into the engineered bacteria for expression; and realizes that the collagen obtained by the expression of the engineered bacteria can be cleaved by TEV cleavage enzyme. After removing exogenous genes such as tags, the remaining protein peptides retain the core functional region of the collagen and have a coverage rate of 100% with the amino acid sequence of natural human type III collagen, which fully meets the definition requirements of my country for recombinant type III humanized collagen type A (i.e., without any exogenous inserted genes or modified genes).

[0059] 7) The method for preparing recombinant type III humanized collagen microspheres of the present application designs the natural human type III collagen gene for the purpose of cyclization, and adds three amino acids NHV after its protein sequence, so that the collagen expressed by the engineered bacteria is cleaved by TEV shearing enzyme to form a peptide sequence that promotes water solubility and can be linked head to tail, and then the two amino acids HV are removed under the action of Butelase ligase and GN is linked to achieve head to tail connection. The final cyclic collagen sequence consisting of two peptides connected head to tail fully meets the definition requirements of recombinant type III humanized collagen type A; the collagen is cyclized to form a ring structure, and the 3D spatial structure presents a spherical structure with a hollow middle.

[0060] 8) The method for preparing recombinant type III humanized collagen microspheres of the present application is the first to introduce the designed recombinant type III humanized collagen gene, the cleavage enzyme TEV cleavage enzyme gene, and the cyclase Butelase ligase gene into the same engineered bacteria for fermentation expression, thereby completing the expression, enzymatic cleavage and cyclization of the target gene during the fermentation expression process, and achieving the "one-step method" to obtain the recombinant type III humanized collagen microspheres in production.

[0061] 9) This application designs the Butelase ligase gene and achieves the purpose of introducing it into engineered bacteria for expression, laying the foundation for achieving a "one-step" method to obtain recombinant type III humanized collagen microspheres.

[0062] 10) The present application discloses a method for preparing recombinant type III humanized collagen microspheres, wherein the "one-step method" involves cloning the target gene into an engineered bacterium for expression, enzymatic cleavage, and cyclization. The expressed protein is purified to obtain the product. The production operation is simple, the process flow is simplified, and it is more suitable for large-scale industrial production, with good practical value and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG1 is a schematic diagram of the 3D spatial structure of the recombinant type III humanized collagen microspheres with innovative spatial structure of this application;

[0064] FIG2 is a schematic diagram of the target protein cleavage by TEV cleavage enzyme of the present application;

[0065] FIG3 is a schematic diagram of the cyclization process of the recombinant humanized type III collagen with an innovative spatial structure of the present application;

[0066] Figure 4 is the protein electrophoresis detection results in Example 4 of the present application (in the figure, lane 1 is a marker, lane 2 is the product 1-1 before IPTG induction of control group 1, lane 3 is the product 1-2 after IPTG induction of control group 1, lane 4 is the purified product 1-3 of control group 1 (5 μL sample load), lane 5 is the target protein product collected and purified in Example 3 (5 μL sample load), lane 6 is the product of control group 2, lane 7 is the product after extraction and purification in Example 2, and lane 8 is the product after final extraction and purification in Example 3);

[0067] FIG5 is a Western Blot verification result of Example 4 of the present application (in the figure, lane 1 is a marker, lane 2 is the product obtained in Example 2, and lane 4 is the product obtained in Example 3);

[0068] FIG6 is a negative staining photograph of annular collagen in Example 5 of the present application;

[0069] FIG7 is one of the photos of negative staining data collected in Example 5 of the present application;

[0070] FIG8 shows the results of two-dimensional classification in Example 5 of the present application;

[0071] FIG9 is a diagram showing the result of three-dimensional reconstruction in Example 5 of the present application;

[0072] FIG10 is a diagram showing the results of three-dimensional classification in Example 5 of the present application;

[0073] FIG11 is an electron density map showing annular collagen from two viewing angles in Example 5 of the present application;

[0074] FIG12 is a diagram showing the diameter measurement in Example 5 of the present application;

[0075] FIG13 shows the effect of collagen microspheres on HSF cell migration in Example 7 of the present application;

[0076] Figure 14 is a flow chart of the injection and sacrifice of collagen microsphere animals in Example 8 of the present application. DETAILED DESCRIPTION

[0077] In order to make the purpose, technical solutions and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with embodiments.

[0078] Example 1

[0079] This embodiment addresses the problems of existing linear recombinant humanized type III collagen, which is unstable and easily degraded, and the incompatibility of existing protein enzyme cyclization methods with recombinant type III humanized collagen cyclization. It proposes a novel spatially structured recombinant type III humanized collagen microsphere and its design, preparation process, and application. By designing the amino acid sequence of existing human type III collagen and constructing a suitable expression vector, the recombinant plasmid can be expressed in engineered bacteria, and the expressed target protein can be cyclized after cleavage by cleavage enzymes and cyclases, thereby achieving the goal of obtaining novel recombinant type III humanized collagen microspheres without introducing any exogenous inserted genes or modified genes.

[0080] The amino acid sequence of the recombinant type III humanized collagen microspheres with an innovative spatial structure obtained in this example is as follows:

[0081] As shown in Figure 1, the recombinant type III humanized collagen microspheres with an innovative spatial structure have a total of 72 amino acids, and the particle diameter is about 7 to 8 nanometers. It is a new protein conformation, presenting a spherical structure with a hollow center in the 3D spatial structure. Compared with the linear structure of natural human type III collagen, the recombinant type III humanized collagen microspheres of this application are spherical structures formed by cyclization. The ends of the protein chains are cyclized and closed to avoid contact with degradative enzymes, effectively delaying the degradation of collagen by proteases, improving its stability and durability, and being able to exert its various biological effects for a long time, and can better adapt to applications in the pharmaceutical and cosmetic industries. For example, due to the cyclization of the protein microsphere structure, it has a more stable molecular structure and a longer half-life, making it more suitable for applications such as drug delivery and transdermal preparations.

[0082] In addition, the amino acid sequence of the recombinant type III humanized collagen microspheres with an innovative spatial structure described in this embodiment covers 100% of the amino acid sequence of human natural type III collagen, retaining the core functional region of collagen without introducing any exogenous inserted genes or modified genes. It has all the functions of natural type III collagen and has better biological activity than human natural type III collagen, and can promote the proliferation and repair of tissue cells. Moreover, since the particle size of the collagen microspheres is only 7 to 8 nanometers, it is very easy to pass through the skin barrier and maximize its biological efficacy. In addition, the collagen microspheres described in this embodiment, like natural type III collagen, also have good biological activity and biocompatibility, and can be widely used in the medical field, such as treating tissue damage, repairing trauma, etc., and have important medical value and economic value.

[0083] The recombinant type III humanized collagen microspheres with an innovative spatial structure described in this embodiment are specifically formed by cyclizing two peptide segments with 36 amino acids connected end to end. The amino acid sequence is as follows:

[0084] To achieve this cyclization result, this embodiment also provides a method for designing recombinant type III humanized collagen microspheres with an innovative spatial structure. In this embodiment, the expression vector designed and constructed for the type III humanized collagen gene sequence is based on the ability to be cleaved by TEV cleavage enzyme and cyclized by Butelase ligase, thereby achieving recombinant type III humanized collagen microspheres with an innovative spatial structure without introducing any exogenous inserted genes or modified genes. The specific steps include the following:

[0085] S1: Design of humanized type III collagen gene sequence:

[0086] Based on the amino acid sequence of the existing human type III collagen, the gene sequence of humanized type III collagen for cyclization is designed as follows:

[0087] S2: Construction of expression vector:

[0088] In order to avoid introducing any exogenous inserted genes or modified genes, this example selected the pET28a plasmid as the expression basis and used MBP-His6 as the plasmid tag, as shown in Figure 2; and based on the designed type III humanized collagen sequence, that is, the cleavage enzyme and cyclase properties used, a specific target protein sequence ENLYFQ was designed to construct the expression vector pET28a-MBP-His6-ENLYFQ.

[0089] S3: Recombinant Expression

[0090] After the humanized type III collagen gene designed in step S1 is inserted into the specific target protein gene of the expression vector constructed in step S2, a recombinant plasmid containing the target protein gene is obtained; the recombinant plasmid is transferred into engineered bacteria for culture and induction of expression to obtain the target protein, whose amino acid sequence is shown in SEQ NO. 4, specifically as follows:

[0091] S4: Enzyme digestion:

[0092] The target protein obtained in step S3 is digested with TEV cleavage enzyme to remove exogenous genes such as tags, thereby obtaining a peptide sequence that can be cyclized. The amino acid sequence is shown in SEQ NO. 5, which is as follows:

[0093] S5: Cyclization:

[0094] The two peptides digested in step S4 were ligated using Butelase ligase to remove the two amino acids HV, thereby obtaining a circular protein connected end-to-end by the two amino acids GN, as shown in FIG3 . The amino acid sequence of the obtained circular protein is as follows:

[0095] That is, the recombinant type III humanized collagen microspheres with an innovative spatial structure shown in SEQ NO.1.

[0096] Of course, the design method of this embodiment is only one of them, and other design methods are not excluded to achieve the purpose of 100% coverage of the amino acid sequence of natural human type III collagen, retaining its functional region (with biological activity), and being able to cyclize without introducing any foreign genes or modifications.

[0097] In addition, in order to obtain the recombinant type III humanized collagen microspheres with the innovative spatial structure, this embodiment also provides two preparation processes. The first is to introduce the recombinant plasmid containing the gene sequence of human type III collagen into the engineering bacteria for fermentation, extract the protein, and then perform enzyme digestion and cyclization treatment to obtain the cyclized type III humanized collagen microspheres in a "step-by-step method". The second is to introduce the recombinant plasmid containing the gene sequence of human type III collagen into the engineering bacteria together with the shearing enzyme and ligase genes, and realize the "one-step method" to directly express in the engineering bacteria to obtain the cyclized type III humanized collagen microspheres. That is:

[0098] "Step-by-step method": design the gene sequence of human type III collagen and construct a suitable expression vector. Insert the designed gene sequence of human type III collagen into the expression vector to form a recombinant plasmid, and then introduce it into the engineered bacteria for fermentation expression; extract the target protein expressed by the recombinant plasmid, treat it with a suitable shearing enzyme, and then use a ligase to cyclize it to obtain recombinant type III humanized collagen microspheres with an innovative spatial structure.

[0099] "One-step method": Design the gene sequence of human type III collagen and construct a suitable expression vector, insert the designed gene sequence of human type III collagen into the expression vector to form a recombinant plasmid; introduce the recombinant plasmid together with the shearing enzyme gene and the cyclase gene into the engineered bacteria for fermentation expression; and complete the process of expressing the target protein from the recombinant plasmid and performing enzymatic cutting and cyclization in the engineered bacteria, and then extract and purify to obtain recombinant type III humanized collagen microspheres with an innovative spatial structure.

[0100] It should be noted that the design of the gene sequence of human type III collagen and the design of the expression vector are not unique. They can be designed based on the amino acid sequence characteristics of the selected existing human type III collagen, the characteristics of the cleavage enzyme used, and the characteristics of the ligase, so as to achieve 100% coverage of the amino acid sequence of human natural type III collagen, retain its functional region (with biological activity), and be able to cyclize without introducing any foreign genes or modifications. As a preferred embodiment, in this embodiment, the designed type III humanized collagen gene sequence is shown in SEQ NO.3; the constructed expression vector is pET28a-MBP-His6-ENLYFQ; the cleavage enzyme is TEV cleavage enzyme; the cyclase is Butelase ligase; the amino acid sequence of the recombinant type III humanized collagen microsphere with an innovative spatial structure is shown in SEQ NO.1. In other embodiments, those skilled in the art can make similar designs based on the inspiration of this application, such as replacing the cleavage enzyme, constructing the corresponding expression vector, etc.

[0101] The specific preparation process of the "step-by-step method" in this embodiment includes the following steps:

[0102] SA1: Culture the bacteria: Inoculate the engineered bacteria introduced with the recombinant plasmid into the culture medium and culture at 32-37°C with shaking for 20-24 hours to obtain the expression bacteria seed solution;

[0103] SA2: Transfer fermentation: Transfer the expression bacteria seed solution to a new culture medium at an inoculum concentration of 5% to 15% by volume and continue fermentation at 32-37°C.

[0104] SA3: Induced expression: When the density of the engineered bacteria in the fermentation broth reaches the predetermined concentration, IPTG inducer is added and the target protein expression is induced at 37°C;

[0105] SA4: Extraction of target protein: After the fermentation expression is completed, the fermentation product is collected, the precipitate is centrifuged, and ultrasonically disrupted on ice. After denaturation and renaturation, the protein is purified to obtain the target protein;

[0106] SA5: Enzymatic digestion: Resuspend the target protein in TEV cleavage enzyme solution and incubate the enzyme digestion reaction at pH 6.0-9.0 and temperature 29-34°C to obtain peptides that can be cyclized;

[0107] SA6: Enzymatic cyclization: Butelase ligase is added to the protein solution after enzyme digestion. The enzyme ligation reaction conditions are pH 5.0-7.0 and reaction temperature 37-45°C to obtain cyclized recombinant humanized type III collagen.

[0108] SA7: Collection and purification: Collect the cyclized recombinant type III humanized collagen for purification to obtain recombinant type III humanized collagen microspheres with innovative spatial structure.

[0109] The specific process of the "one-step method" in this embodiment includes the following steps:

[0110] SB1: Culture of bacterial strains: Introduce the recombinant plasmid containing the designed humanized type III collagen gene sequence and the recombinant plasmid containing the TEV shearing enzyme gene into an engineered bacterium integrated with the ligase gene. Inoculate the engineered bacterium into the culture medium and culture with shaking at 32-37°C for 20-24 hours to obtain the seed solution of the expression bacteria.

[0111] SB2: Transfer fermentation: Transfer the expression bacteria seed solution to a new culture medium at an inoculum concentration of 5% to 15% by volume and continue fermentation at 32-37°C.

[0112] SB3: Induced expression: When the density of the engineered bacteria in the fermentation broth reaches the predetermined concentration, IPTG inducer is added and the target protein is induced to express and undergo enzymatic cleavage and cyclization at 37°C.

[0113] SB4: Collection and purification: After the fermentation expression is completed, the fermentation product is collected, the precipitate is centrifuged and ultrasonically broken on ice, and then the protein is purified after denaturation and renaturation to obtain recombinant type III humanized collagen microspheres with an innovative spatial structure.

[0114] The preparation process for the two innovative spatially structured recombinant type III humanized collagen microspheres utilizes different culture media; appropriate culture media can be selected based on culture needs. The addition of inducers is also not exclusive; these can be added based on specific experiments, as long as they can induce expression of the designed target protein.

[0115] In this example, M9 culture medium was selected, specifically composed of 20 g / L glucose, 10 g / L sodium chloride, and 20 g / L ammonium sulfate. The added inducer was IPTG, with a concentration of 0.5-10%. When the IPTG inducer was added, the density of the engineered bacteria was preferably controlled to an OD600 of 0.6-0.7. The concentration of the inducer in the culture system was 0.1-1 mM. In the "one-step method," the concentration of the IPTG inducer in the culture system was preferably 0.5 mM, to ensure that both the recombinant plasmid containing the designed humanized type III collagen gene sequence and the recombinant plasmid containing the TEV shearing enzyme gene were well expressed, achieving in vivo shearing and circularization.

[0116] The preparation process for the novel spatially structured recombinant type III humanized collagen microspheres described in this embodiment utilizes engineered bacteria including, but not limited to, Escherichia coli, Bacillus subtilis, and yeast. For example, in one embodiment, the recombinant plasmid can be transformed into competent BL21 (DE3) E. coli cells via heat shock. In other embodiments, other engineered bacteria can be used, primarily to achieve fermentative expression of the recombinant plasmid.

[0117] It should be noted that in the preparation process of the innovative spatially structured recombinant type III humanized collagen microspheres described in this example, the TEV cleavage enzyme used can be purchased from the market, or the His-TEVp recombinant protein gene can be inserted into the pET-21a vector to obtain a recombinant plasmid, which can then be introduced into an engineered bacterium for expression; the Butelase ligase used can also be purchased from the market, or the Butelase ligase gene can be integrated into the expression site of the engineered bacterium for expression. However, it should be clarified here that Butelase ligase is a new class of Asx-specific peptide ligases from butterfly peas, which can efficiently cyclize non-native polypeptides in various organisms. However, this enzyme has only been shown to efficiently connect the ends of amino acid cyclic chains to form cyclic peptides in plants, and there are no reports of its cloning, fermentation expression in engineered bacteria, and protein cyclization. In order to integrate Butelase ligase into the expression site of the engineered bacteria, this example also designed its gene sequence. The designed nucleotide sequence is shown in SEQ NO.7, and then it is integrated into the lacZ site of BL21 using CRISPR / Cas9 technology.

[0118] In addition to inserting the His-TEVp recombinant protein gene into the pET-21a vector, the expression vector containing the shearing enzyme gene described in this embodiment can also be obtained by using other vectors that can express active TEV shearing enzyme in engineered bacteria in other embodiments.

[0119] In this embodiment, the protein collection and purification operation is as follows: after the fermentation expression is completed, the fermentation product is collected, the precipitate is centrifuged and ultrasonically disrupted on ice, and then the protein is purified after denaturation and renaturation. Specifically, the centrifugation conditions are: 4°C, 6000-10000r / min, centrifugation for 15-30min; the bacteria are collected by centrifugation. The bacterial sludge is resuspended in lysis buffer and a mixture of protease inhibitors is added. Ultrasonic disruption is performed on ice. The supernatant is centrifuged and the insoluble inclusion bodies are resuspended in Triton buffer. After incubation at room temperature for 30 minutes, the supernatant is centrifuged and the precipitate is resuspended in lysis buffer containing 6M urea and incubated overnight to dissolve the protein. Protein purification is performed using a His-tag nickel column to remove residual tags and other exogenous genes. In other embodiments, the culture medium and purification method can be used, the main purpose of which is to cultivate the above-mentioned functional engineering bacteria to achieve the purpose of fermentation expression and extraction and purification.

[0120] Finally, this embodiment proposes an application of recombinant type III humanized collagen microspheres with an innovative spatial structure, that is, the application of the aforementioned recombinant type III humanized collagen microspheres with an innovative spatial structure in medicine or cosmetics, including the application in the preparation of Class II medical devices such as dressings, hyaluronic acid injections or filling implants, Class III medical devices, hemostatic materials, bone repair and drug delivery carrier materials, or sensitive skin repair products.

[0121] Example 2

[0122] This example uses a one-step method to prepare recombinant type III humanized collagen microspheres with the innovative spatial structure described in Example 1. The engineering bacteria used in this example is Escherichia coli BL21 (DE3), and the specific preparation process is as follows:

[0123] The first step is genetic design.

[0124] Based on the amino acid sequence of existing human type III collagen, a humanized type III collagen gene sequence for cyclization was designed, as shown in SEQ NO. 3. At the same time, a Butelase ligase gene sequence was designed, as shown in SEQ NO. 7.

[0125] The second step is to construct the expression vector and engineering bacteria.

[0126] Using pET28a plasmid as the expression base and MBP-His6 as the plasmid tag, a specific target protein sequence ENLYFQ was designed to construct the expression vector pET28a-MBP-His6-ENLYFQ; after inserting the designed type III humanized collagen gene into the constructed pET28a-MBP-His6-ENLYFQ expression vector specific target protein gene, a recombinant plasmid 1 containing the target protein gene was obtained for future use.

[0127] The His-TEVp recombinant protein gene was inserted into the pET-21a vector to obtain a recombinant plasmid 2 containing the TEV shearing enzyme gene for future use.

[0128] The Butelase ligase gene sequence shown in SEQ NO.7 was integrated into the lacZ site of BL21 using CRISPR / Cas9 technology, and BL21 cells expressing cyclase were obtained for later use.

[0129] The third step is to assemble the engineered bacteria for production.

[0130] First, the recombinant plasmid 1 containing the target protein gene is transformed into the competent cell BL21 (DE3) integrated with the Butelase ligase gene through heat shock, and the first generation of recombinant genetically engineered bacteria is screened; then the recombinant plasmid 2 containing the TEV shearing enzyme gene is transformed into the first generation of recombinant genetically engineered bacteria, and the second generation of recombinant genetic engineering that can express recombinant type III humanized collagen microspheres with innovative spatial structure is screened again, which is the engineered bacteria for production.

[0131] The fourth step is fermentation expression.

[0132] 1) Culture: Inoculate the engineered bacteria obtained from assembly and screening into a 500 mL Erlenmeyer flask containing 50 mL of M9 medium. Shake and culture at 35°C and 200 rpm for 24 h to obtain the expression strain seed solution.

[0133] 2) Transfer fermentation: The expression strain seed solution was transferred to a 10 L shake flask containing 3 L of M9 medium at a volume concentration of 10% and fermentation was continued at 35°C and 200 rpm.

[0134] 3) Induced expression: When the engineered bacteria density in the fermentation broth reached 0.6 (OD600), 2% IPTG was added to the culture at a concentration of 0.5 mM. Protein expression and cyclization were induced at 37°C to obtain a fermentation broth containing recombinant humanized type III collagen microspheres with an innovative spatial structure.

[0135] Step 5: extraction and purification.

[0136] After fermentation and expression, the fermentation product was collected and centrifuged at 8000 rpm for 20 minutes at 4°C. The precipitated bacterial sludge was then resuspended in lysis buffer (50 mM phosphate, 300 mM NaCl, 5 mM imidazole, pH 8.0) and a protease inhibitor cocktail (primarily composed of leupetin, pepstatin A, aprotinin, E-64, etc., EDTA-free) was added. The cells were then sonicated on ice. The insoluble inclusion bodies were resuspended in Triton buffer and incubated at room temperature for 30 minutes. The supernatant was removed by centrifugation under the same centrifugation conditions as above. The precipitate was resuspended in lysis buffer containing 6 M urea and incubated overnight to solubilize the protein. After denaturation and renaturation, the protein was purified using a His-tag nickel column. After two chromatography steps using an ion chromatography column and a hydrophobic chromatography column, recombinant type III humanized collagen microspheres with an innovative spatial structure exceeding 95% purity were obtained. The amino acid sequence is shown in SEQ NO.1.

[0137] Example 3

[0138] This example uses a step-by-step method to prepare recombinant type III humanized collagen microspheres with the innovative spatial structure described in Example 1. The engineering bacteria used in this example is Escherichia coli BL21 (DE3). The specific preparation process is as follows:

[0139] The first step is genetic design.

[0140] Based on the amino acid sequence of existing human type III collagen, a humanized type III collagen gene sequence for cyclization was designed, as shown in SEQ NO. 3. At the same time, a Butelase ligase gene sequence was designed, as shown in SEQ NO. 7.

[0141] The second step is to construct the expression vector and engineering bacteria.

[0142] Using the pET28a plasmid as the expression vector and MBP-His6 as the plasmid tag, a specific target protein sequence, ENLYFQ, was designed to construct the expression vector pET28a-MBP-His6-ENLYFQ. The designed humanized type III collagen gene was inserted into the constructed pET28a-MBP-His6-ENLYFQ expression vector, generating recombinant plasmid 1 containing the target protein gene for future use. Simultaneously, the His-TEVp recombinant protein gene was inserted into the pET-21a vector, generating recombinant plasmid 2 containing the TEV shearing enzyme gene for future use.

[0143] The Butelase ligase gene sequence shown in SEQ NO.7 was integrated into the lacZ site of BL21 using CRISPR / Cas9 technology, and BL21 cells expressing cyclase were obtained for later use.

[0144] The third step is to assemble the engineered bacteria for production.

[0145] The recombinant plasmid 1 containing the target protein gene was transformed into the competent cell BL21 (DE3) by heat shock, and the target protein production engineered bacteria 1 were screened;

[0146] The recombinant plasmid 2 containing the TEV shearing enzyme gene was transformed into another competent cell BL21 (DE3) to screen and obtain the shearing enzyme-producing engineered bacteria 2;

[0147] BL21 with Butelase ligase gene integrated was used as ligase production engineered bacteria 3.

[0148] The fourth step is fermentation expression.

[0149] 1) Cultivation: Inoculate each of the three engineered bacteria into 500 mL of M9 medium in a 500 mL Erlenmeyer flask. Shake and culture at 35°C and 200 rpm for 24 h to obtain expression strain seed solution 1, seed solution 2, and seed solution 3.

[0150] 2) Transfer Fermentation: The three expression strain seed solutions were transferred to 10 L shake flasks containing 3LM9 medium at a 10% volume concentration and fermented at 35°C and 200 rpm.

[0151] 3) Induction of expression: When the density of engineered bacteria in the fermentation broth reached 0.6 (OD600), 1.5% IPTG inducer was added and protein expression was induced at 37°C to obtain crude products containing target protein, TEV cleavage enzyme, and Butelase ligase.

[0152] Step 5: Extract protein.

[0153] Collect each fermentation product and purify it separately. The specific method can refer to the purification method of Example 2, but is not limited to this method. Other methods in the prior art can also be referred to. After purification, the target protein to be cleaved, TEV cleavage enzyme, and Butelase ligase are obtained and set aside.

[0154] Step 6: Enzyme digestion.

[0155] The TEV cleavage enzyme was prepared into an enzyme solution with a mass concentration of 10 U / ul, and the target protein to be cleaved was added at a material-liquid ratio of 3%. The enzymatic cleavage reaction conditions were controlled as follows: pH 7.4, temperature 30°C, and incubation for 60 minutes to obtain a peptide segment that could be cyclized.

[0156] Step 7: enzyme concatenation.

[0157] The pH value of the protein solution after enzyme digestion was adjusted to 6.0, and Butelase ligase was added at an amount of 0.03% of the mass of the protein solution. The cyclization reaction conditions were controlled as follows: pH 6.0, temperature 42° C., and incubation for 30 min to obtain cyclized recombinant humanized type III collagen.

[0158] Step 8: Collection and purification.

[0159] The cyclized recombinant humanized type III collagen was collected and purified. The purification method can refer to the extraction and purification method of Example 2, but is also not limited to this method. The specific operation is: centrifugation to remove the supernatant, centrifugation conditions at 4°C, 8000r / min, centrifugation for 20min, resuspending the precipitate in lysis buffer containing 6M urea, and incubating overnight to dissolve the protein. After denaturation and renaturation, the protein was purified using a His-tag nickel column. After two chromatography steps using an ion chromatography column and a hydrophobic chromatography column, recombinant type III humanized collagen microspheres with an innovative spatial structure were obtained, whose amino acid sequence is shown in SEQ NO.1.

[0160] The "step-by-step method" of this embodiment is to introduce recombinant plasmid 1 and recombinant plasmid 2 into engineering bacteria and culture them separately, and culture the engineering bacteria integrated with the Butelase ligase gene separately to obtain the target protein, TEV shearing enzyme and Butelase ligase for use. It is not excluded that in other embodiments, the engineering bacteria producing TEV shearing enzyme and Butelase ligase are mixed and cultured with the engineering bacteria producing the target protein (including different mixed forms), or the recombinant plasmid 1, recombinant plasmid 2 and the engineering bacteria integrated with the Butelase ligase gene are combined in different forms (including different combinations) than in Example 2 to control the experimental conditions to achieve the process of target protein shearing and enzyme concatenation.

[0161] Example 4

[0162] This example is to verify whether the recombinant type III humanized collagen microspheres with innovative spatial structure were successfully prepared in Example 2 and Example 3.

[0163] This example includes two verification experiments. The first is to detect the protein products expressed in each stage of fermentation by electrophoresis to verify whether the recombinant type III humanized collagen is cyclized; the second is to verify by Western Blot whether the product obtained in Example 2 is the human collagen of the recombinant type III humanized collagen microspheres.

[0164] The amino acid sequences of the recombinant humanized type III collagen microspheres with innovative spatial structures obtained in Example 2 and Example 3 are both shown in SEQ NO.1.

[0165] After analysis, the molecular weight of the peptide ring represented by this sequence is 6.68kDa. Since type III collagen has a triple helical structure, the formed protein microsphere product should contain three peptide ring structures. Therefore, the molecular weight of the protein microsphere is predicted to be approximately 18kDa. The specific verification test is as follows:

[0166] 1. SDS-PAGE protein electrophoresis experiment

[0167] In this experiment, the protein products expressed in each stage of fermentation were detected by electrophoresis to verify whether the recombinant type III humanized collagen was expressed and cyclized. Two groups of controls were set up in this experiment. The fermentation product of the first-generation recombinant genetically engineered bacteria in Example 2 was used as control 1 to verify whether the recombinant plasmid 1 of the constructed target protein gene can be expressed in the engineered bacteria; the fermentation products taken from the first-generation recombinant genetically engineered bacteria were divided into IPTG-induced pre-product 1-1 and IPTG-induced post-product 1-2 for verification, and the induced products were recovered and purified. At the same time, the purified target protein was collected and compared with Example 3. In Example 2, the culture product 2 of the engineered bacteria into which the Butelase ligase gene was not integrated was introduced was used as control group 2 to verify whether the target protein expressed by the recombinant plasmid 1 can be cleaved by the TEV shearing enzyme expressed by the plasmid 2.

[0168] The two control groups differed in terms of the plasmids contained in the engineered bacteria and whether the Butelase ligase gene was integrated. The plasmid introduction process, engineered bacteria culture method, and protein purification method were the same as in Example 2. The specific experimental procedures are as follows:

[0169] 1. Sample processing: Collect all proteins and add loading buffer to mix evenly, boil in boiling water bath for 10 minutes, cool naturally and set aside.

[0170] 2. Electrophoresis: Use GenScript SurePAGE™ precast gel (15%), add the treated samples to the sample wells, and run electrophoresis at 140V for 50 minutes until the bromophenol blue band runs to the bottom of the gel.

[0171] 3. Coomassie Brilliant Blue R-250 staining using a microwave oven:

[0172] 1) Prepare staining solution: Dissolve Coomassie Brilliant Blue R250 to a final concentration of 0.1% (W / V) in 40% ethanol and 10% acetic acid solution.

[0173] 2) Prepare decolorizing solution: Dissolve ethanol and acetic acid at a final concentration of 10% (V / V).

[0174] 3) After electrophoresis is complete, pry open the gel sheet, remove the gel, and place it in a staining container containing 100 mL of staining solution.

[0175] 4) Cover the container and microwave on high for 8 minutes. To avoid danger, be careful not to let the solution boil.

[0176] 5) Remove the dye container from the microwave oven and place it on a decolorization shaker at room temperature for 5 minutes.

[0177] 6) Pour off the staining solution and carefully wash the gel with deionized water.

[0178] 7) Pour off the deionized water and add 100 mL of decolorizing solution.

[0179] 8) Cover the container with a lid and heat in a microwave on high for 8 minutes.

[0180] 9) Pour away the decolorizing solution, add new decolorizing solution, and repeat step 8.

[0181] 10) Remove from the microwave oven and place on a decolorization shaker at room temperature and shake gently until the background is clear.

[0182] The results are shown in Figure 4, where lane 1 is the marker, lane 2 is the product 1-1 before IPTG induction of control group 1, lane 3 is the product 1-2 after IPTG induction of control group 1, lane 4 is the purified product 1-3 of control group 1 (5 μL loading), lane 5 is the target protein product collected and purified in Example 3 (5 μL loading), lane 6 is the product of control group 2, lane 7 is the product extracted and purified in Example 2, and lane 8 is the product finally extracted and purified in Example 3.

[0183] The electrophoresis results show that compared to the control group 1 pre-IPTG induction product 1-1, the control group 1 post-IPTG induction product 1-2 showed a significantly enhanced band around 60 kD, and the purified product 1-3 of the expression product after induction in the control group 1 showed a single band of high purity, the same as the target protein induced and expressed in Example 3, verifying that the target protein contained in the recombinant plasmid 1 was induced and specifically expressed. Product 4 of the control group 2 showed a protein fragment with a molecular weight of approximately 9 kD, which verified that the target protein expressed by the recombinant plasmid 1 could be cleaved by TEV cleavage enzyme, and the tag protein could be accurately cleaved and removed to obtain a peptide segment with an amino acid sequence as shown in SEQ NO. 5. Finally, the product obtained in Example 2 and the product finally extracted and purified in Example 3 had significantly increased molecular weight, both of which were about 18kD. This shows that the peptide fragments after cleavage by TEV shearing enzyme can be cyclized by Butelase ligase, and it is precisely two peptide fragments that are cyclized, verifying that both Example 2 and Example 3 successfully obtained recombinant type III humanized collagen with an innovative spatial structure, namely, the circular protein microspheres with an amino acid sequence as shown in SEQ NO.1.

[0184] 2. Western Blot Verification

[0185] This experiment was conducted to verify the human collagen antibody Western Blot of the final product obtained in Example 2 and Example 3, the recombinant type III humanized collagen microspheres with innovative spatial structure. The specific experimental process is as follows:

[0186] 1. Use the eBlot rapid wet transfer instrument to transfer proteins to the membrane:

[0187] 1) Use scissors to cut a piece of filter paper and a piece of PVDF membrane that are similar in size to the electrophoresis gel. Mark one corner of the PVDF membrane with a pencil.

[0188] 2) After activating the PVDF membrane with methanol, soak the filter paper and PVDF membrane with transfer buffer.

[0189] 3) Add transfer buffer (5.8 g Tris, 2.9 g glycine, 0.37 g SDS, 200 ml methanol, and double-distilled water to 1 L) to the tray and place the sponge, PVDF membrane, filter paper, and transfer cassette.

[0190] 4) Place a sponge on the black plate of the transfer cassette, then place the filter paper and gel. Once aligned, use a glass rod to remove any bubbles.

[0191] 5) Use a micropipette to take a small amount of transfer buffer and place it on the run electrophoresis gel (SDS-PAGE protein electrophoresis gel of the product obtained in Example 2 and the product after final extraction and purification in Example 3), then cover it with a PVDF membrane, and then cover it with filter paper and sponge; after alignment, use a glass rod to remove all bubbles.

[0192] 6) After clamping the transfer clamp, place it into the transfer fixture with the black side facing the black side.

[0193] 7) Place the transfer fixture and ice box in the transfer tank. Fill the transfer tank with transfer buffer.

[0194] 8) Turn on the power supply, constant voltage 110V, 1h.

[0195] 2. Blocking and Antibody Incubation:

[0196] 1) Turn off the power, open the transfer clip, take out the PVDF membrane, and rinse with double distilled water.

[0197] 2) Place the PVDF membrane in blocking solution and block on a shaker at 37°C for 1 hour.

[0198] 3) Discard the blocking solution, wash with PBST buffer, incubate with primary antibody working solution, and incubate at 37°C on a shaker for 1 hour.

[0199] 4) Discard the primary antibody working solution, wash with PBST buffer, incubate with secondary antibody working solution, and incubate at 37°C on a shaker for 1 hour.

[0200] 5) Discard the secondary antibody working solution and wash with PBST buffer. Wash the membrane four times on a shaker for 5 minutes each time.

[0201] 3. Development and exposure:

[0202] 1) Use flat paper to absorb the residual liquid on the membrane and lay the PVDF membrane flat.

[0203] 2) Use a micropipette to take equal volumes of Solution A and Solution B in the ECL reagent and place them in an EP tube and return them to room temperature.

[0204] 3) After mixing, add the mixture evenly to the membrane and allow to react for 60 seconds in the dark.

[0205] 4) Discard the ECL mixture and place it in a dark box for exposure and development. The exposure time should be controlled at about 30 seconds.

[0206] The experimental results are shown in Figure 5, which shows that a clear band appears near 18kD (lane 1 is the marker, lane 2 is the product obtained in Example 2, and lane 4 is the product obtained in Example 3), indicating that the protein microsphere products finally obtained in Examples 2 and 3 are recombinant type III humanized collagen.

[0207] Example 5

[0208] This example further examines the spatial structure of the recombinant type III humanized collagen microspheres with an innovative spatial structure obtained in Example 2. The specific experimental scheme and test results are as follows:

[0209] 1. Negative Staining Sample Preparation and Observation

[0210] 1.1 Experimental methods

[0211] 1) Sample information: The collagen product finally obtained in Example 2 was dissolved in PBS buffer solution, and the sample concentration was 10 mM.

[0212] 2) Negative staining sample preparation and observation:

[0213] The protein sample was diluted to 0.1 mg / mL with PBS buffer. 8 μL was dropped onto a copper grid that had been glow-discharged (15 mA for 90 seconds, PELCO easiGlow™) and incubated for 40 seconds. The excess sample was removed by aspiration, and the grid was quickly rinsed with 0.75% uranium formate. 10 μL of 0.75% uranium formate was then added, incubated for 40 seconds, and the excess solution was removed by aspiration and allowed to dry.

[0214] The prepared copper grid containing the sample was placed on a Talos 120 electron microscope (Thermo Fisher Scientific, USA) at 120 kV for sample observation. Negative staining photos were collected at 92,000x magnification using a Ceta-D camera (Thermo Fisher Scientific, USA). The camera has a resolution of

[0215] 1.2 Experimental Results

[0216] The negative staining results are shown in FIG6 . As can be seen from the figure, the negative staining photo of the annular collagen shows that the sample particles are uniform and not aggregated.

[0217] 2 Three-dimensional reconstruction of negative staining data

[0218] 2.1 Data Collection and Processing

[0219] Negative staining photos were collected, as shown in FIG7 , a total of 60 photos. The negative staining data were processed using CryoSPARC v4.0.3 software for 2D classification, 3D reconstruction, and 3D classification.

[0220] 2.2 Results of two-dimensional classification

[0221] After 2D classification of negatively stained data, 45 good classifications were finally selected, totaling 8,047 particles, as shown in Figure 8. These selected particles were used for subsequent 3D reconstruction.

[0222] 2.3 Three-dimensional reconstruction results of annular collagen

[0223] The selected particles were first subjected to ab-initio reconstruction, generating five types of ab-initio models, as shown in Figure 9. The final selected annular collagen three-dimensional density map was the fourth type (3244 particles, accounting for approximately 40%).

[0224] Then, the three-dimensional classification (i.e., heterogeneous refinement) was performed using the five types of three-dimensional reconstruction models generated above. The results are shown in Figure 10. In the figure, there are 3007 particles corresponding to the fourth type of annular collagen three-dimensional density map, accounting for 37.4%, with a resolution of approximately The electron density map of the annular collagen is shown from two perspectives, i.e., the three-dimensional spatial conformation, as shown in FIG11 ; the size measurement result, i.e., the diameter measurement result, is shown in FIG12 . ).

[0225] Through verification, it can be seen that the three-dimensional spatial conformation of the recombinant type III humanized collagen microspheres with an innovative spatial structure obtained in Example 2 is a ring-shaped spherical structure with a diameter of about 7 nm and a pore size of about 3 nm. The specific spatial conformation is shown in Figure 1, that is, the recombinant type III humanized collagen microspheres with an innovative spatial structure present a spherical structure with a hollow center in the 3D spatial structure.

[0226] Example 6

[0227] This example uses a time-of-flight high-resolution tandem mass spectrometry system to analyze and detect the recombinant type III humanized collagen microspheres with innovative spatial structure obtained in Example 2. The specific amino acid primary structure (peptide map) pre-treatment scheme is as follows:

[0228] (1) Rinse: Add 100 μL of 50 mM ammonium bicarbonate to a 10k ultrafiltration tube for rinse.

[0229] (2) Solution replacement (replace the original storage buffer of the sample): Centrifuge at 16,000 r / min for 10 min. Add 110 μg of sample to 5 μL (22 μg / μL) of sample, add 95 μl of 7 M guanidine hydrochloride, vortex, and mediate. Centrifuge at 16,000 g to dryness.

[0230] (3) Denaturation: Add 100 μL 7 M guanidine hydrochloride.

[0231] (4) Reduction: Add 4 μL of 1 M DTT and incubate at 42°C for 1 h.

[0232] (5) Add 10 μL of 1 M IAA and incubate at room temperature in the dark for 30 min.

[0233] (6) Centrifugation, add 100 μL of 50 mM ammonium bicarbonate, and centrifuge at 16,000 g for 15 min until dry. Repeat three times.

[0234] (7) Enzymatic hydrolysis: sample and trypsin 30:1 (mass ratio), oven at 37°C overnight.

[0235] (8) Invert and centrifuge for 1 min, then add FA to a final concentration of 1% in the solution.

[0236] (9) The pre-treated samples are analyzed by time-of-flight mass spectrometry or high-resolution mass spectrometry for amino acid sequence confirmation.

[0237] The amino acid sequence of the recombinant type III humanized collagen microspheres with innovative spatial structure obtained in Example 2 was verified to be:

[0238] The amino acid coverage of the designed recombinant type III humanized collagen was 100%, which once again verified that the present application successfully synthesized recombinant type III humanized collagen microspheres with an innovative spatial structure as shown in the amino acid sequence of SEQ NO.1.

[0239] Example 7

[0240] This example conducts a cell biology experiment on the recombinant type III humanized collagen microspheres with an innovative spatial structure obtained in Example 2. The specific experimental process and results are as follows:

[0241] 1. Purpose of the experiment

[0242] The cell biological activity of the recombinant type III humanized collagen microspheres with innovative spatial structure obtained in Example 2 was investigated by MTT assay and cell scratch test.

[0243] 2. Experimental Materials

[0244] Human skin fibroblasts (HSF cells), high-glucose DMEM medium, fetal bovine serum (FBS), trypsin, PBS, penicillin-streptomycin solution, MTT, DMSO, Hoechst 33342 staining solution, bovine serum albumin, pipette tips (5 ml, 1 ml, 200 μl, 10 μl), 96-well plates, centrifuge tubes (50 ml, 15 ml, 2 ml), etc.

[0245] Test sample: recombinant type III humanized collagen microspheres with innovative spatial structure obtained in Example 2;

[0246] Reference substance: Commercial recombinant humanized type III collagen freeze-dried fibers

[0247] 3. Experimental Methods

[0248] 3.1 Solution preparation

[0249] Culture medium preparation: 90% high-glucose DMEM medium + 10% FBS, plus 1% penicillin-streptomycin solution.

[0250] Preparation of MTT solution: Concentration: 5 mg / ml. Specific preparation method: Weigh 0.5 g of MTT and dissolve it in 100 ml of PBS (dissolution can be enhanced by placing it in a 60°C water bath). Sterilize the solution with a 0.22 μm filter and store at 4°C in the dark. Wrap the solution in aluminum foil during preparation and storage.

[0251] PBS formula: NaCl 8g, KCl 0.2g, Na 2HPO 4 1.44g, KH 2PO 4 0.24g, adjust pH to 7.4, and adjust volume to 1L.

[0252] 3.2 Detection methods (MTT assay, cell scratch assay)

[0253] The MTT assay works by reducing exogenous MTT to water-insoluble, blue-purple crystalline formazan by succinate dehydrogenase in the mitochondria of living cells, which then deposits within the cells. Dead cells, however, do not exhibit this effect. Dimethyl sulfoxide (DMSO) dissolves the formazan in cells, and its absorbance, measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA), indirectly reflects the number of viable cells. Within a certain cell population range, the amount of MTT crystals formed is proportional to the cell number.

[0254] The principle of the scratch test is to use a micropipette tip to scratch the cell growth area on a monolayer of adherent cells cultured in an in vitro culture dish or plate, aspirate the cells in the scratch area, and then continue to culture for a set time (for example, 24 hours) to observe whether the surrounding cells migrate to the central scratch area to determine the cell growth and migration ability.

[0255] 4. Experimental Procedure

[0256] 4.1 MTT assay to detect cell growth activity

[0257] Collect logarithmic-phase cells, adjust the cell suspension concentration, and add 100 μl to each well. Plate the cells to a density of 10,000 cells / well. Fill the edge wells with sterile PBS. Incubate at 37°C with 5% CO2 until the cell monolayer covers the bottom of the well. Add the drug in a gradient of concentrations. In principle, drug addition can be performed once the cells have attached to the wall. Repeat the procedure in five replicates. Incubate at 37°C with 5% CO2 for 24 or 48 hours and observe under an inverted microscope. Add 20 μl of MTT solution (5 mg / ml, i.e., 0.5% MTT) to each well and continue incubation for 4 hours. If the drug reacts with MTT, centrifuge and discard the culture medium. Carefully rinse the wells with PBS 2-3 times before adding the MTT-containing culture medium. Terminate the incubation and carefully aspirate the culture medium. Add 150 μl of dimethyl sulfoxide to each well and shake on a shaker at low speed for 10 minutes to fully dissolve any crystals. Measure the absorbance of each well at OD 450 nm using an enzyme-linked immunosorbent assay (ELISA). At the same time, zero adjustment wells (culture medium, MTT, dimethyl sulfoxide) and control wells (cells, drug dissolution medium of the same concentration, culture medium, MTT, dimethyl sulfoxide) were set.

[0258] 4.2 Cell scratch assay to investigate cell migration

[0259] First, use a marker pen and a ruler to evenly draw horizontal lines across the back of a 6-well plate, approximately every 0.5-1 cm across the wells, making three lines per well. Add approximately 5 × 10⁵ cells to each well and culture in an incubator. Observe cell growth the next day. Once cells have filled the bottom of the well, use a pipette tip and a ruler to draw a line as close as possible to the horizontal line on the back. Wash the cells three times with PBS, remove the scratched cells, and add culture medium containing 1% FBS. Incubate the plate at 37°C with 5% CO₂. After 24 hours, take a photograph to record the confluence of each scratched well.

[0260] 5. Cell Proliferation and Migration Assay

[0261] 5.1 Effect of collagen on HSF cell growth. Specific data are shown in Table 1.

[0262] Table 1. Effects of recombinant humanized type III collagen on HSF cell growth

[0263] The experimental results show that under the experimental conditions of this example, the recombinant type III humanized collagen microspheres with an innovative spatial structure obtained in Example 2 exhibited a growth-promoting effect on HSF cells at concentrations of 0.01% and 0.02% at 24 hours, which is comparable to the performance of the commercial collagen product in promoting cell proliferation as a control.

[0264] 5.2 Effects of recombinant humanized type III collagen microspheres on HSF cell migration are as follows:

[0265] This experiment investigated the effect of recombinant humanized type III collagen microspheres with the innovative spatial structure obtained in Example 2 on HSF cell migration. The primary objective was to observe the degree of confluence in the scratched area 24 hours after the addition of collagen. The results were obtained by sampling three fields of view at the time of scratching and three fields of view in each group 24 hours later. The results are shown in Figure 13.

[0266] The results of the cell scratch test showed that the recombinant type III humanized collagen microspheres with an innovative spatial structure obtained in Example 2 had significant cell migration promoting activity when added at concentrations of 0.01% and 0.02% and cultured for 24 hours. The migration activity of HSF cells (human skin fibroblasts) indicated that collagen has a promoting effect on skin repair and wound healing.

[0267] Example 8

[0268] This example investigates the in vivo degradability of recombinant humanized type III collagen microspheres with innovative spatial structures obtained in Example 2. The specific experimental process is as follows:

[0269] 1. Selection of Experimental Animals

[0270] Mouse model: C57BL / 6J female mice, weighing 20 g each.

[0271] 2. Experimental Animal Grouping

[0272] The experimental animals were divided into experimental group, control group and blank group, with 12 animals in each group, totaling 36 animals. Each group had 3 experiments, with 3 animals in each experiment, as shown in Table 2.

[0273] The experimental group received injections of recombinant humanized type III collagen microspheres with the innovative spatial structure obtained in Example 2; the control group received injections of commercially available freeze-dried recombinant humanized type III collagen fibers. Dissections were performed on days 1, 14, 28, and 56, with three animals dissected at each time point. A control group and a blank control group were also established, totaling 36 animals.

[0274] Table 2. Degradability of recombinant humanized type III collagen in animals

[0275] 3. Experimental Procedure

[0276] 3.1 Surgical operation:

[0277] 1) Weighing: Weigh the animal before injection and determine the total injection dose based on the animal's weight. The injection dose for each injection site is then determined based on the total injection volume and the number of injection sites.

[0278] 2) The product is implanted intradermally on the back of the animal, with the spine as the midline. There is one injection area on each side of the back. One animal has two injection points. The product is injected intradermally on the back. The injection dose at each injection point is 1 / 2 of the total injection dose.

[0279] Specific treatment: Inject mice 28 days and 56 days in advance 28 days; inject mice 14 days in advance 14 days; and inject mice 1 day in advance 24 hours. At time 0, mice at 1 day, 14 days, and 28 days were sacrificed for testing. At 56 days, mice were observed for another 28 days. The specific process is shown in Figure 14.

[0280] 4. Detection indicators and methods

[0281] 4.1 Safety evaluation indicators

[0282] 4.1.1. General observations

[0283] Observe the animals' activity, feeding, and mental state daily for 28 days after injection, and weigh them weekly. Observe the skin's appearance and morphology at the injection site for 7 days, including the presence of papular erythema, eschar, nodule, redness, swelling, or exudate. Observe the injection site for tactile sensation, including the presence of nodules, masses, and changes in skin smoothness. Closely monitor the animals' condition and behavior, noting any histological changes or behavioral abnormalities caused by vascular embolism.

[0284] The results of the skin injection local site test are shown in Table 3:

[0285] Table 3. Skin reaction scoring system

[0286] Table 4. Stimulus response scores

[0287] As shown in Table 4, the results of the skin injection local site test show that under the experimental conditions of this example, the recombinant type III humanized collagen microspheres with an innovative spatial structure obtained in Example 2 were observed for changes in the appearance and morphology of the skin at the injection site within 7 days after injection. The results were the same as those of the control group. Except for some erythema in the first two days after injection, there were no papular erythema or eschar nodules, redness, swelling, exudate, etc. on the third day, indicating that there was no obvious irritation to the skin.

[0288] 4.2. In vivo degradation observation and evaluation

[0289] Histopathological evaluation

[0290] The observation time nodes were 1d, 14d, 28d, and 56d after injection. After the experimental animals were sacrificed, the tissues at the injection site were fixed with 10% formalin and then embedded in paraffin for sectioning. HE staining and Sirius red staining were performed, and the content of human type III collagen was then detected.

[0291] Table 5. Residual rate of human type III collagen injected into mouse skin

[0292] The test results in Table 5 show that the residual rate of human type III collagen in the injection site tissue of the experimental group of mice was significantly higher than that of the control group, verifying that the recombinant type III humanized collagen microspheres with an innovative spatial structure obtained in Example 2 have the characteristic of significantly extending the degradation cycle in vivo, or promote the regeneration of their own collagen.

[0293] Based on the above examples, we can conclude that this application, for the first time, has obtained cyclase and recombinant humanized type III collagen through the fermentation technology of genetically engineered bacteria. Under the action of cyclase, the peptide segments cleaved by TEV cleavage enzyme are connected head-to-tail to obtain a new type of cyclic collagen with a globular 3D spatial structure. This protein sequence is not only completely consistent with the partial sequence of natural human type III collagen, but also has all the biological effects of type III collagen, excellent water solubility and biocompatibility. Moreover, through the change of spatial structure, the degradation cycle of the protein is greatly extended, achieving the goal of small protein with great efficacy.

[0294] Finally, it should be noted that: for those skilled in the art, it is obvious that the present application is not limited to the details of the above-mentioned exemplary embodiments, and that the present application can be implemented in other specific forms without departing from the spirit or basic features of the present application. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. In addition, it should be understood that although this specification is described in terms of implementation methods, it does not contain only one technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A recombinant type III humanized collagen microsphere with an innovative spatial structure, wherein: The collagen microspheres are formed by cyclizing a peptide segment whose amino acid sequence 100% covers the amino acid sequence of natural human type III collagen, and the amino acid sequence is shown in SEQ NO.

1.

2. The recombinant type III humanized collagen microspheres with an innovative spatial structure according to claim 1, wherein: The collagen microsphere has a particle size of 7 to 8 nanometers and is formed by cyclizing two peptide segments of an amino acid sequence such as that shown in SEQ NO.2 end to end.

3. A design method for recombinant type III humanized collagen microspheres with innovative spatial structure, wherein: The steps include: S1: Design of humanized type III collagen gene sequence: According to the amino acid sequence of the existing human type III collagen, a type III humanized collagen gene sequence for cyclization is designed, as shown in SEQ NO.3; S2: Construction of expression vector: The pET28a plasmid was selected as the expression basis, MBP-His6 was used as the plasmid tag, and the specific target protein sequence ENLYFQ was designed according to the designed type III humanized collagen sequence to construct the expression vector pET28a-MBP-His6-ENLYFQ; S3: Recombinant Expression After inserting the type III humanized collagen gene designed in step S1 into the specific target protein gene of the expression vector constructed in step S2, a recombinant plasmid containing the target protein gene is obtained; the recombinant plasmid is transferred into an engineering bacterium for culture and induction of expression to obtain a target protein, whose amino acid sequence is shown in SEQ NO.4; S4: Enzyme digestion: The target protein obtained in step S3 is digested with TEV cleavage enzyme to obtain a peptide sequence that can be cyclized, and its amino acid sequence is shown in SEQ NO.5; S5: Cyclization: The two peptides cleaved in step S4 were subjected to enzymatic ligation reaction using Butelase ligase to obtain a circular protein with two amino acids GN connected head to tail, whose sequence is shown in SEQ NO.6, i.e., the recombinant type III humanized collagen microspheres with the innovative spatial structure shown in SEQ NO.

1.

4. A process for preparing recombinant type III humanized collagen microspheres with an innovative spatial structure, wherein: The gene sequence of human type III collagen was designed as shown in SEQ NO.3, and an expression vector pET28a-MBP-His6-ENLYFQ was constructed. The gene sequence of human type III collagen was inserted into the expression vector to form a recombinant plasmid, which was then introduced into engineered bacteria for fermentation expression. The target protein expressed by the recombinant plasmid was extracted, treated with TEV shearing enzyme, and then cyclized with Butelase ligase to obtain recombinant type III humanized collagen microspheres with an innovative spatial structure.

5. The preparation process of the recombinant type III humanized collagen microspheres with innovative spatial structure according to claim 4, wherein: The process includes the following steps: SA1: Cultivate bacteria: Inoculate the engineered bacteria introduced with the recombinant plasmid into the culture medium, and culture at 32-37°C with shaking for 20-24 hours to obtain the seed solution of the expression bacteria; SA2: Transfer fermentation: transfer the expression bacteria seed solution to a new culture medium at an inoculum concentration of 5% to 15% by volume, and continue fermenting at 32 to 37°C; SA3: Induced expression: When the density of engineered bacteria in the fermentation broth reaches a predetermined concentration, an inducer is added and the target protein is induced to express at 37°C; SA4: Extraction of target protein: After the fermentation expression is completed, the fermentation product is collected, the precipitate is centrifuged, ultrasonically broken on ice, and then the protein is purified after denaturation and renaturation to obtain the target protein; SA5: Enzyme cleavage treatment: resuspend the obtained target protein in TEV cleavage enzyme solution, incubate the enzyme cleavage reaction at pH 6.0-9.0 and temperature 29-34°C to obtain peptide segments that can be cyclized; SA6: Enzyme ligation: Add Butelase ligase to the protein solution after enzyme cleavage, and control the enzyme ligation reaction conditions to: pH 5.0-7.0, reaction temperature 37-45°C, to obtain cyclized recombinant type III humanized collagen; SA7: Collection and purification: Collect the cyclized recombinant type III humanized collagen for purification to obtain recombinant type III humanized collagen microspheres with innovative spatial structure.

6. The preparation process of the recombinant type III humanized collagen microspheres with innovative spatial structure according to claim 4 or 5, wherein: The engineered bacteria include but are not limited to any one of Escherichia coli, Bacillus subtilis, and yeast.

7. The preparation process of the recombinant type III humanized collagen microspheres with innovative spatial structure according to claim 6, wherein: The TEV shearing enzyme is obtained by inserting the His-TEVp recombinant protein gene into the pET-21a vector to obtain a recombinant plasmid, and then introducing the recombinant plasmid into an engineering bacterium for expression.

8. The preparation process of the recombinant type III humanized collagen microspheres with innovative spatial structure according to claim 7, wherein: The Butelase ligase has a gene sequence as shown in SEQ NO.7, and is integrated into the gene expression site of the engineering bacteria through CRISPR / Cas9 technology for expression.

9. A process for preparing recombinant type III humanized collagen microspheres with an innovative spatial structure, wherein: The gene sequence of human type III collagen is designed as shown in SEQ NO.3, and an expression vector pET28a-MBP-His6-ENLYFQ is constructed, and the gene sequence of human type III collagen is inserted into the expression vector to form a recombinant plasmid; the recombinant plasmid is introduced into the engineering bacteria together with the TEV shearing enzyme gene and the Butelase cyclase gene for fermentation expression; and the target protein expressed by the recombinant plasmid is subjected to enzymatic cleavage and cyclization in the engineering bacteria, and then extracted and purified to obtain recombinant type III humanized collagen microspheres with an innovative spatial structure.

10. The preparation process of the recombinant type III humanized collagen microspheres with innovative spatial structure according to claim 9, wherein: The process includes the following steps: SB1: Culture strains: The recombinant plasmid containing the designed type III humanized collagen gene sequence and the TEV shearing enzyme gene are introduced into the engineered bacteria integrated with the Butelase ligase gene, and the engineered bacteria are inoculated into the culture medium, and cultured at 32-37°C with shaking for 20-24 hours to obtain the expression bacteria seed solution; SB2: Transfer fermentation: transfer the expression bacteria seed solution to a new culture medium at an inoculum concentration of 5% to 15% by volume, and continue fermenting at 32 to 37°C; SB3: Induced expression: When the density of engineered bacteria in the fermentation broth reaches the predetermined concentration, the inducer is added, and the target protein is induced to express and then digested and cyclized at 37°C; SB4: Collection and purification: After the fermentation expression is completed, the fermentation product is collected, the precipitate is centrifuged and ultrasonically broken on ice, and then the protein is purified after denaturation and renaturation to obtain recombinant type III humanized collagen microspheres with innovative spatial structure.

11. The preparation process of the recombinant type III humanized collagen microspheres with innovative spatial structure according to claim 9 or 10, wherein: The engineered bacteria include but are not limited to any one of Escherichia coli, Bacillus subtilis, and yeast.

12. The preparation process of the recombinant type III humanized collagen microspheres with innovative spatial structure according to claim 11, wherein: The TEV shearing enzyme gene is a His-TEVp recombinant protein gene, which is inserted into a pET-21a vector to form a recombinant plasmid, and then the recombinant plasmid and a recombinant plasmid containing a target protein are introduced into an engineering bacterium for expression.

13. The preparation process of the recombinant type III humanized collagen microspheres with innovative spatial structure according to claim 12, wherein: The nucleotide sequence of the Butelase ligase gene is shown in SEQ NO.7, and it is integrated into the gene expression site of the engineered bacteria through CRISPR / Cas9 technology for expression.

14. The preparation process of the recombinant type III humanized collagen microspheres with innovative spatial structure according to claim 4 or 9, wherein: The culture medium used for fermentation expression was M9 culture medium, the inducer used was IPTG inducer, and the concentration of the inducer added in the culture system was 0.1-1 mM.

15. Application of recombinant type III humanized collagen microspheres with innovative spatial structure, wherein: Use of the recombinant type III humanized collagen microspheres with an innovative spatial structure as described in claim 1 or 2, or the recombinant type III humanized collagen microspheres with an innovative spatial structure designed as claimed in claim 3, or the recombinant type III humanized collagen microspheres with an innovative spatial structure prepared as claimed in claim 4 or 9 in the preparation of hemostatic materials, bone repair materials, skin repair materials or medical devices.

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