Modularized tandem repeat recombinant III-type collagen with high platelet adhesion and application of modular tandem repeat recombinant III-type collagen

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to modular tandem repeat recombinant III-type collagen with high platelet adhesion and application thereof. An amino acid sequence of the recombinant III-type collagen comprises repeating units, and the number of the repeating units is larger than or equal to 3; and a functional module is inserted into the repeating unit. According to the invention, a plurality of discrete platelet binding sites in III-type collagen are integrated, a tandem repeat structure (repeat unit > = 3) of different module sequences is designed, and a space synergistic effect is optimized; a gene sequence is optimized by using codon preference, efficient secretory expression is performed through a pichia pastoris expression system, and a high-purity product is obtained through purification. The recombinant III type collagen prepared by the invention shows good biocompatibility, and can significantly promote the adhesion of red blood cells and platelets; the method can be widely applied to the fields of wound repair, hemostatic materials, tissue engineering and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a recombinant type III collagen with high platelet adhesion and modular tandem repeats and application thereof. BACKGROUND

[0002] Platelets play a key role in the process of wound hemostasis and repair. When blood vessels are damaged, platelets will quickly adhere to the injury site to form a platelet thrombus, achieving primary hemostasis. At the same time, platelets also release a variety of growth factors and cytokines to synergistically regulate local inflammatory response and tissue regeneration, promoting wound healing.

[0003] According to relevant research, collagen is the only ECM protein that promotes platelet generation and adhesion and mediates complete platelet activation. Three major platelet collagen receptors have been identified: alpha 2 beta 1, glycoprotein (GP) VI, and vWF. The interaction and cross-talk between the three platelet collagen receptors are crucial for effective thrombus formation.

[0004] Type III collagen is an important component of human extracellular matrix and plays a key role in maintaining the structure and function of tissues. It has good biocompatibility and biological activity, and can promote cell adhesion, proliferation and differentiation. However, natural type III collagen is mainly derived from animal tissues, which has the risk of immunogenicity, and the extraction process is complex and costly, making it difficult to meet the demand for large-scale, safe and controllable application. Therefore, the development of recombinant type III collagen with controllable expression, clear structure and high safety has become a research hotspot.

[0005] Currently, most of the existing recombinant type III collagens focus on mechanical properties or cell (such as fibroblasts, stem cells, etc.) adhesion optimization, but few are designed specifically for blood cells, especially platelet adhesion. In the existing designs targeting platelets, most recombinant type III collagen designs still use the natural full-length sequence (such as COL3A1), only retaining the original functional platelet binding sites (such as GPVI binding domain, alpha 2 beta 1 binding domain). However, these functional sites are sparsely distributed in the natural sequence, with limited receptor binding density, making it difficult to achieve efficient platelet recruitment and adhesion, which in turn leads to relatively low platelet adhesion efficiency of existing recombinant type III collagens. SUMMARY

[0006] To solve the problem of the original functional platelet binding sites in the natural sequence being sparsely distributed, the receptor binding density being limited, and the difficulty in achieving efficient platelet recruitment and adhesion, which in turn leads to relatively low platelet adhesion efficiency of existing recombinant type III collagens, the present application provides a recombinant type III collagen with high platelet adhesion and modular tandem repeats and application thereof.

[0007] The application provides a recombinant type III collagen with high platelet adhesion and a modular tandem repeat structure, wherein a plurality of receptor binding domains such as VWF / GPVI / α2β1 are integrated into a collagen framework by using a modular tandem structure (≥3 repeat units). The modular tandem structure design can increase the density of platelet binding sites, thereby further improving the adhesion performance of platelets.

[0008] Specifically, the amino acid sequence of the recombinant type III collagen comprises repeat units, and the number of the repeat units is ≥3.

[0009] The repeat unit comprises a type III collagen framework; the type III collagen framework comprises a repeat amino acid sequence represented by (Gly-X-Y)n, wherein X and Y represent any amino acid, and n represents the number of continuous repeats of the collagen characteristic sequence (Gly-X-Y), which is an integer ≥15. (Gly-X-Y)n is a typical structural feature of collagen, which endows collagen with unique physicochemical properties and biological functions.

[0010] At least one functional module is inserted into the type III collagen framework; the functional module comprises at least one of the amino acid sequences represented by SEQ ID NO. 1-SEQ ID NO. 5.

[0011] SEQ ID NO. 1-SEQ ID NO. 5 are used for directly or indirectly combining with platelets.

[0012] The amino acid sequence represented by SEQ ID NO. 1 is used for indirectly combining with platelets after recognizing the von Willebrand factor VWF. Indirectly combining with platelets means that the collagen does not directly combine with platelets, but specifically binds to the A3 domain of VWF through the sequence SEQ ID NO. 1 of the collagen, to form a collagen-VWF complex, and then the complex is recognized by the GPIb-IX-V receptor on the platelet membrane surface, so as to realize the indirect adhesion of platelets to the collagen.

[0013] The amino acid sequence represented by SEQ ID NO. 2 is used for directly combining with platelets by recognizing the receptor of type III collagen (TIIICBP) on the platelets.

[0014] The amino acid sequence represented by SEQ ID NO. 3 is used for directly combining with platelets by recognizing the glycoprotein (GP) VI on the platelets.

[0015] The amino acid sequences represented by SEQ ID NO. 4 and SEQ ID NO. 5 are used for directly combining with platelets by recognizing the integrin α2β1 on the platelets.

[0016] The amino acid sequence of SEQ ID NO. 1 is RGQPGVMGF; the amino acid sequence of SEQ ID NO. 2 is KPGEPGPK; the amino acid sequence of SEQ ID NO. 3 is GAPGLRGGAGPPGPEGGKGAAGPPGPP; the amino acid sequence of SEQ ID NO. 4 is GAPGER; and the amino acid sequence of SEQ ID NO. 5 is GMPGER.

[0017] The above functional modules can directly or indirectly interact with platelet receptors, and enhance the adhesion capacity of platelets. Compared with the traditional structure, the original functional platelet binding sites in the natural sequence are sparsely distributed, the receptor binding density is insufficient, and it is difficult to achieve efficient platelet recruitment and adhesion, thereby causing the defect that the platelet adhesion efficiency of the existing recombinant type III collagen is relatively low. The recombinant type III collagen provided by the application significantly improves the density of platelet binding sites by adopting a modular tandem design of ≥3 repeating units. Specifically, the VWF binding domain, the GPVI activation domain and the α2β1 binding domain are integrated into the collagen skeleton at an optimal interval, thereby enhancing the density of functional sites, realizing the synergistic effect of multiple receptors, forming a cascade effect of “capture-activation-aggregation”, significantly improving the binding and regulation efficiency of platelets, and thereby solving the problem that the platelet adhesion efficiency of the existing recombinant type III collagen is relatively low due to insufficient density of platelet binding sites.

[0018] Preferably, the number of repeating units is 3-10. The amino acid sequence of the repeating unit is shown in SEQ ID NO. 6.

[0019] Preferably, the number of repeating units is 10, and the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 7, which clearly defines the primary structure of the recombinant type III collagen, thereby providing a basis for its production and identification.

[0020] Preferably, the amino acid sequence shown in SEQ ID NO. 7 is encoded by the nucleotide sequence shown in SEQ ID NO. 8, and the nucleotide sequence can be used to construct an expression vector to realize the biosynthesis of the recombinant protein.

[0021] The application further provides an expression vector containing the nucleotide sequence.

[0022] Further, the expression vector comprises pPICZ alpha A. By inserting the nucleotide sequence such as SEQ ID NO. 8 into a suitable expression vector, the expression of the recombinant type III collagen is realized.

[0023] The application further provides a host cell containing the nucleotide sequence or the expression vector.

[0024] Specifically, the host cell includes but is not limited to microorganism, plant or animal cell, the vector of the application can be introduced into the host cell by methods known to those skilled in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like.

[0025] Preferably, the host cell includes at least one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis. The above-mentioned host cells have the advantages of fast growth, easy cultivation and genetic manipulation, and are suitable for the production of recombinant proteins.

[0026] The application also provides the use of the recombinant collagen type III, the nucleotide sequence, the expression vector or the host cell in the preparation of a product for tissue engineering.

[0027] Preferably, the product includes the recombinant collagen type III, the nucleotide sequence, the expression vector or the host cell.

[0028] Preferably, the product includes a product for wound repair and hemostasis. Specifically, it includes a wound repair dressing and a hemostatic material.

[0029] Preferably, the method for preparing the recombinant collagen type III using the host cell includes the following steps: Culturing the host cell in a culture medium, purifying after inducing the expression of the recombinant collagen type III, and obtaining the recombinant collagen type III.

[0030] Further, the method for preparing the recombinant collagen type III using the host cell includes the following steps: The codon-optimized gene sequence is cloned into the pPICZαA vector, and the codon optimization can improve the expression efficiency of the gene in the host cell.

[0031] The Pichia pastoris X33 strain is electroporated and methanol-induced expression is performed. Pichia pastoris X33 is a commonly used eukaryotic expression system that can fold and modify the recombinant collagen protein correctly. Methanol-induced expression can initiate the synthesis of the recombinant protein.

[0032] The target protein is purified from the culture supernatant. By suitable purification methods such as ultrafiltration, ion exchange, dialysis and the like, high-purity recombinant collagen type III can be obtained.

[0033] Preferably, the product further includes a pharmaceutically acceptable adjuvant.

[0034] Preferably, the pharmaceutically acceptable adjuvant includes any one or more of diluents, excipients, fillers, wetting agents, disintegrants, flavoring agents and binders.

[0035] Compared with the prior art, the present application has the following advantages: 1. The present application provides a recombinant type III collagen with high platelet adhesion and a modular tandem repeat structure, which has an amino acid sequence comprising repeat units, the number of which is ≥3; the repeat units comprise a type III collagen framework; the type III collagen framework comprises an amino acid repeat sequence represented by (Gly-X-Y)n, wherein X and Y represent any amino acid, and n represents the number of consecutive repeats of the collagen characteristic sequence (Gly-X-Y), which is generally an integer ≥15; at least one functional module is inserted into the type III collagen framework; the functional module comprises at least one of the following discrete platelet-related binding sites in type III collagen: a site related to the binding of von Willebrand factor (VWF), a platelet-binding octapeptide binding site, a site related to the binding of glycoprotein VI (GPVI), and a site related to the binding of integrin α2β1. Compared with the original functional platelet binding sites in the traditional structure, which are sparsely distributed in the natural sequence and have insufficient receptor binding density, it is difficult to achieve efficient platelet recruitment and adhesion, thereby resulting in the problem of relatively low platelet adhesion efficiency of existing recombinant type III collagen. The present application provides a recombinant type III collagen with the advantage of significantly improved platelet binding site density. By using a modular tandem design of ≥3 repeat units, the VWF binding domain, GPVI activation domain, and α2β1 binding domain are integrated into the collagen framework at an optimal spacing, which significantly improves the density of functional platelet binding sites. At the same time, through the synergistic effect of multiple receptors, the cascade effect of "capture-activation-aggregation" is achieved, thereby solving the problem of the original functional platelet binding sites in the prior art, which are sparsely distributed in the natural sequence and have limited receptor binding density, making it difficult to achieve efficient platelet recruitment and adhesion, thereby resulting in the problem of relatively low platelet adhesion efficiency of existing recombinant type III collagen.

[0036] 2. Compared with existing natural type III collagen, the recombinant type III collagen provided by the present application has the following advantages: (1) High platelet adhesion: by integrating multiple discrete platelet binding sites in type III collagen and designing a (Gly-X-Y)n modular tandem repeat structure, the spatial synergistic effect is optimized, so that the recombinant type III collagen of the present application has significantly higher platelet adhesion capacity than natural full-length type III collagen.

[0037] (2) Good biocompatibility: the recombinant type III collagen of the present application is designed and modified based on the natural type III collagen framework, which retains its good biocompatibility and reduces the immune response when used in vivo.

[0038] (3) High expression and high purity: the codon bias optimization of the gene sequence is used, and the high-efficiency secretory expression is carried out through the Pichia pastoris expression system, and then the affinity chromatography purification technology is combined, so that the high-purity recombinant collagen type III product can be obtained. This not only ensures the stability of the product quality, but also is beneficial to the large-scale application in the medical and tissue engineering fields.

[0039] (4) Wide application range: due to the high platelet adhesion and good biocompatibility, the recombinant collagen type III of the application can be widely used in wound healing, hemostasis and other fields of tissue engineering. In the aspect of wound healing, the hemostasis and repair process of the wound can be accelerated; in the hemostatic material, the hemostasis efficiency can be significantly improved; in other fields of tissue engineering, it can be used as a high-quality material for constructing tissue engineering scaffolds, etc., to provide a good support and microenvironment for tissue repair and regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 PCR verification results of part of the transformed colonies in the application.

[0041] Figure 2 SDS-PAGE electrophoresis diagram of the protein in the supernatant in the application.

[0042] Figure 3 SDS-PAGE electrophoresis detection diagram of protein purification in the application.

[0043] Figure 4 Quantitative analysis results of biocompatibility evaluation in the application, 1 in the figure is Control (positive control 1% (v / v) Triton X-100), 2 is 6010 (RCIII-6010) and 3 is PBS (negative control), wherein Control is positive control 1% (v / v) Triton X-100; 6010 is RCIII-6010; PBS is negative control.

[0044] Figure 5 Quantitative analysis results of red blood cell adhesion in the application.

[0045] Figure 6 Quantitative analysis results of platelet adhesion in the application. DETAILED DESCRIPTION

[0046] The application will be described in detail below in combination with the drawings and specific embodiments, but should not be understood as limiting the application. If not specially stated, the technical means used in the following examples are the conventional means familiar to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specially stated, can be obtained from commercial channels.

[0047] The inventive concept of the present application is as follows: selecting the sequences related to the binding of von Willebrand factor VWF in collagen type III-RGQPGVMGF, the platelet binding octapeptide-KPGEPGPK, the sequence related to the binding of glycoprotein VI-GAPGLRGGAGPPGPEGGKGAAGPPGPP, and the sequence related to the binding of integrin α2β1-GAPGER, GMPGER, multiple discrete platelet binding site related sequences, integrating them into a repeating unit under the premise of ensuring the skeleton (Gly-X-Y)n, such as the sequence of 60 amino acids shown in SEQ ID NO. 6, and then setting the number of repeating units to 3 times or more to form a modular tandem repeat structure.

[0048] wherein the sequence shown in SEQ ID NO. 6 is: GPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGER.

[0049] The present application provides a recombinant collagen type III-6010 with high platelet adhesion, and the amino acid sequence thereof is the repeating unit shown in SEQ ID NO. 7, with the number of repeating units set to 10.

[0050] wherein the sequence shown in SEQ ID NO. 7 is: GPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGERGPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGERGPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGERGPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGERGPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGERGPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGERGPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGERGPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGERGPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGERGPRGQPGVMGFPGKPGEPGPKGAPGERGAPGLRGGAGPPGPEGGKGAAGPPGPPGMPGER.

[0051] The application provides a type III recombinant collagen-6010 with high platelet adhesion, which is optimized in codon for host cell expression, and signal peptide cleavage sites and EcoR I and Kpn I enzyme cleavage sites are added at both ends in the design process to facilitate later gene operation. After the above optimization, the nucleotide sequence is shown as SEQ ID NO. 8.

[0052] The sequence shown as SEQ ID NO. 8.

[0053] In one specific embodiment, the host cell is selected from any of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0054] Example 1 I. Expression of recombinant collagen type III According to the preference of Pichia pastoris codon, the DNA sequence is codon-optimized by using codon optimization software without changing the amino acid sequence such as SEQ ID NO. 6, and a EcoR I enzyme cutting site and a signal peptide cleavage site are introduced at the 5' end of the optimized target gene sequence, and a Kpn I enzyme cutting site is introduced at the 3' end to obtain a nucleotide sequence as shown in SEQ ID NO. 7 for full gene synthesis, and it is connected to the expression vector pPICZ alpha A vector and transformed into Escherichia coli to obtain Top10-pPICZ alpha A-RCIII recombinant strain.

[0055] The Top10-pPICZ alpha A-RCIII recombinant strain is inoculated into LB liquid medium containing blasticidin resistance and cultured overnight. The supernatant medium is removed by centrifugation, and the recombinant plasmid pPICZ alpha A-RCIII is extracted by using a plasmid small extraction kit. After linearization by restriction enzyme Sac I, the linearized plasmid is recovered by alcohol precipitation. Among them, overnight refers to 14h. The manufacturer of the plasmid small extraction kit is Tiangen Biochemical Company.

[0056] Pichia pastoris X33 competent cells are prepared, and the obtained linearized plasmid is transformed into Pichia pastoris X33 competent cells by electroporation to construct Pichia pastoris X33-pPICZ alpha A-RCIII expression strain. Among them, the Pichia pastoris X33 strain is purchased from Xi'an Juzi Biological Gene Technology Co., Ltd.

[0057] After the positive transformants grown on the plate after electroporation are screened by using YPD plates containing different concentrations of blasticidin, high-copy transformants are picked for colony PCR identification, and the PCR products are analyzed by agarose gel electrophoresis. The agarose gel electrophoresis map of the PCR verification result of the Pichia pastoris X33-pPICZ alpha A-RCIII expression strain is as shown in Figure 1 , which is sent for sequencing verification.

[0058] As can be seen from Figure 1 , the colony PCR identification shows that there is a target band at 2300bp which is consistent with the theoretical size, proving that the target gene has been successfully introduced into the Pichia pastoris genome, and the recombinant strain is successfully constructed.

[0059] The sequencing verified correct Pichia pastoris X33-pPICZ alpha A-RCIII expression strain was cultured in 5 mL YPD medium at 30°C and 220 rpm overnight, then 1 mL culture was transferred into 25 mL BMGY liquid medium and grown at 30°C and 250 rpm until OD 600 6, at this time the Pichia pastoris X33-pPICZ alpha A-RCIII expression strain will be in the logarithmic phase of growth. The bacteria were collected by centrifugation and transferred to BMMY liquid medium (methanol concentration of 1% (v / v)) with OD 600 value of 1, and the induction culture was shaken. Methanol was added to the medium every 24 h to a final concentration of 1% (v / v). The supernatant was collected by centrifugation at 8000 rpm for 5 min at 0 h, 24 h, 48 h and 72 h after induction, and the expression of the target protein-6010 was analyzed. The results of SDS-PAGE electrophoresis detection of supernatant protein collected at 24 h from different transformants are shown in Figure 2 . Among them, the composition of YPD liquid medium is 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose; the composition of BMGY liquid medium is 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 10% (v / v) phosphate buffer with pH of 6.0 and concentration of 1M, 1% (v / v) glycerol; the composition of BMMY liquid medium is 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 10% (v / v) phosphate buffer with pH of 6.0 and concentration of 1M, 1% (v / v) methanol.

[0060] As can be seen from Figure 2 , the molecular weight of the protein is 70 kDa, which is close to the theoretical value of recombinant type III collagen-6010, proving that the recombinant collagen protein is successfully expressed in Pichia pastoris.

[0061] II. Purification of recombinant type III collagen 1. The fermentation broth obtained from the strain was centrifuged at 8000 rpm for 15 min to collect the supernatant, which was filtered through a 0.22 μm filter membrane, then ultrafiltration was used for preliminary purification and concentration: the fermentation supernatant was filtered through a 30 kDa ultrafiltration membrane to remove part of the host protein, nucleic acid and other impurities, and a concentrated collagen filtrate was obtained. This step can significantly reduce the load of the subsequent chromatography column and improve the initial purity of the target protein.

[0062] 2. Pretreatment of MMC cation exchange chromatography column: Equilibrate the MMC column with 20mM potassium phosphate buffer (pH 6.0) to ensure that the electrostatic and hydrophobic interactions between the stationary phase and the target protein are optimal.

[0063] Sample loading conditions: Adjust the pH of the protein filtrate to 6.0 before loading.

[0064] Gradient elution: A gradient elution was performed using 0-100% (v / v) solution B. Solution B was the eluent with a pH of 6.0, consisting of 20 mM potassium phosphate buffer and 1 M NaCl. A 30% (v / v) solution B elution was used to remove weakly bound proteins; a linear gradient elution of 30%-70% (v / v) solution B was used to separate medium-bound impurities; and an isocratic elution of 80% (v / v) solution B was used to specifically recover recombinant collagen. The elution peak was monitored in real-time using UV absorption to ensure concentrated elution of the target protein. The eluent was collected and sampled for SDS-PAGE electrophoresis analysis. The purified SDS-PAGE electrophoresis results are shown below. Figure 3 As shown.

[0065] Depend on Figure 3 It can be seen that when the elution liquid fraction increases to 80%, the target protein is specifically eluted, impurities are effectively removed, and the target protein is separated and purified.

[0066] It is important to note the advantages of this gradient elution: high resolution – separating proteins with different binding strengths through salt concentration gradients, reducing co-elution; high recovery rate – avoiding protein residue or loss caused by one-step elution; and process robustness – adapting to differences in samples from different batches.

[0067] 3. Desalting and Lyophilization: Excess salt is removed by dialysis to avoid affecting protein stability. Lyophilization yields solid-state recombinant collagen, preserving its native conformation and activity. This solid-state recombinant collagen is recombinant type III collagen (recombinant collagen 6010).

[0068] Example 2: Bioactivity of recombinant type III collagen 1. Blood compatibility of recombinant type III collagen was determined by in vitro hemolysis test: (1) Preparation of red blood cell suspension: Take the anticoagulant rabbit whole blood, centrifuge at 3000 rpm for 10 min, take the lower red blood cell precipitate (RBCs), wash with phosphate buffer solution (PBS) for 3 times, then take 1 mL RBCs and add 19 mL PBS to dilute to obtain a red blood cell suspension with a volume ratio of 5%. The concentration of the phosphate buffer solution (PBS) is 0.01M, the pH is 7.4, the components are Na2HPO4·12H2O: 2.9g / L, KH2PO4: 0.2g / L, and NaCl is added to 0.9% w / v (9g / L). (2) Dissolve the recombinant collagen type III in PBS, filter to obtain a dissolved solution, then add 0.5 mL of the dissolved solution to 0.5 mL of the red blood cell suspension. At the same time, the negative control uses 0.5 mL of PBS, and the positive control uses 0.5 mL of 1% (v / v) Triton X-100. Then incubate all samples in a 37°C constant temperature incubator for 1 h, take out and centrifuge at 3000 rpm / min at room temperature, so that the intact red blood cells RCBs settle at the bottom, and the color of the upper solution is observed.

[0069] (3) Measure the absorbance of the obtained upper solution at 540nm using a UV-visible spectrophotometer. The calculation formula of hemolysis rate is as follows:

[0070] Hemolysis ratio= (OD sample -OD negative control ) / (OD positive control - OD negative control ) x 100%; In the formula, Hemolysis ratio represents the hemolysis rate, with a unit of %; OD sample , OD negative contol , OD positive control represent the absorbance of the experimental sample, the absorbance of the negative control group and the absorbance of the positive control group, respectively.

[0071] The hemolytic activity (hemolysis rate) determination shows that, as shown in Figure 4 , the positive control 1% (v / v) Triton X-100 is red after centrifugation, while the transparency of RCIII-6010 is similar to that of the negative control (PBS), indicating that no red blood cell rupture is visible. The quantitative results show that RCIII-6010 only causes 0.32% hemolysis, with a hemolysis rate of <5%, which meets the biological material standard (ISO 10993-4 (15)).

[0072] From the above results, it can be seen that the qualitative observation and quantitative data are consistent, indicating that the recombinant collagen protein RCIII-6010 has no significant hemolytic activity, indicating that the collagen protein of the application has good blood compatibility.

[0073] 2. For RC III RBC (red blood cell) adhesion assay: BSA, full-length collagen and recombinant collagen type III (recombinant collagen 6010) were uniformly coated in the well plate and incubated at 4°C overnight, then the well plate was blocked with 1% (v / v) heat-denatured BSA at room temperature for 1h to complete the coating of the well plate, then 100 μL of the prepared 5% red blood cell suspension was dropped into the well plate, and incubated at 37°C for 1h. The full-length collagen refers to the purified full-length recombinant collagen type I and type III, which is composed of full-length α1 chain sequences from human collagen type I and type III, respectively, and is from Xi'an Juzi Biological Gene Technology Co., Ltd.

[0074] The unadhered red blood cells were washed away with PBS, and the hemoglobin was released by lysis with deionized water. The absorbance was measured at 540 nm by an enzyme-labeled instrument. The calculation formula of red blood cell adhesion is as follows: RBC adhesion = OD hemostat / OD reference value ×100%; In the formula, RBC adhesion represents the adhesion of red blood cells, and the unit is %; OD hemostat , OD reference value represent the absorbance of the hemostatic agent and the absorbance of the reference value, respectively.

[0075] The red blood cell adhesion assay showed as Figure 5 .

[0076] As can be seen from Figure 5 , compared with BSA, natural full-length collagen type I and type III, the number of red blood cells adsorbed by the recombinant collagen type III-6010 of the present application is significantly increased, showing good red blood cell aggregation effect.

[0077] 3. The platelet adhesion experiment was performed as follows: BSA, full-length collagen and recombinant collagen type III (recombinant collagen 6010) were uniformly coated in the well plate and incubated at 4°C overnight, then the well plate was blocked with 1% (v / v) heat-denatured BSA at room temperature for 1h to complete the coating of the well plate, then 100 μL of the prepared 5% red blood cell suspension was dropped into the well plate, and incubated at 37°C for 1h. The full-length collagen refers to the purified full-length recombinant collagen type I and type III, which is composed of full-length α1 chain sequences from human collagen type I and type III, respectively, and is from Xi'an Juzi Biological Gene Technology Co., Ltd.

[0078] Adhered platelet = OD hemosta t / OD reference value × 100 %. In the above formula, adhered platelet represents absorbance, unit: %; OD hemostat , OD reference value respectively represent absorbance of hemostatic agent and absorbance of reference value.

[0079] The platelet adhesion assay shows that the recombinant collagen-6010 has Figure 6 .

[0080] As can be seen from Figure 6 , compared with BSA, natural full-length type I and type III collagen, the platelet adsorption capacity of the recombinant type III collagen-6010 of the present application is significantly improved, and the platelet adhesion performance is excellent.

[0081] The above results show that the recombinant type III collagen of the present application has good blood compatibility, can significantly promote platelet adhesion and red blood cell aggregation, and has higher adhesion effect on related blood cells than natural collagen.

[0082] It should be noted that when numerical ranges are involved in the present application, both endpoints of each numerical range and any number between the two endpoints can be selected, and in order to prevent redundancy, the present application describes preferred embodiments.

[0083] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept, and these changes and modifications all fall within the scope of all changes and modifications of the present application.

Claims

1. A recombinant type III collagen with high platelet adhesion of modular tandem repeats, characterized in that, The amino acid sequence thereof comprises a repeating unit, and the number of the repeating unit is ≥3; The repeating unit comprises a type III collagen skeleton; the type III collagen skeleton comprises a repeating amino acid sequence as shown in (Gly-X-Y)n, wherein X and Y represent any amino acid, and n is an integer ≥15; At least one functional module is inserted into the type III collagen skeleton; the functional module comprises at least one of the amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 5; The amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 5 are used for directly or indirectly combining with platelets.

2. The recombinant collagen type III according to claim 1, characterized in that, The number of the repeating unit is 3-10; The amino acid sequence of the repeating unit is shown in SEQ ID NO.

6.

3. The recombinant collagen type III according to claim 2, characterized in that, The number of the repeating unit is 10, and the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO.

7.

4. The recombinant collagen type III of claim 3, characterized in that, The amino acid sequence shown in SEQ ID NO. 7 is encoded by the nucleotide sequence shown in SEQ ID NO.

8.

5. An expression vector, characterized by, The nucleotide sequence of claim 4 is contained.

6. A host cell containing the nucleotide sequence of claim 4 or the expression vector of claim 5.

7. The host cell of claim 6, wherein, The host cell comprises at least one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

8. Use of the recombinant type III collagen of any one of claims 1-4, the nucleotide sequence of claim 4, the expression vector of claim 5 or the host cell of claim 6 in the preparation of a product for tissue engineering.

9. Use according to claim 8, characterized in that, The product comprises a product for wound repair and hemostasis.

10. Use according to claim 8, characterized in that, A method for preparing the recombinant type III collagen by using the host cell, comprising the following steps: The host cell is cultured in a culture medium, and after the expression of the recombinant type III collagen is induced, the recombinant type III collagen is purified to obtain the recombinant type III collagen.

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