Recombinant type iii collagen with pro-endothelization and low platelet adhesion and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a recombinant collagen type III with endothelialization promotion and low platelet adhesion, a preparation method and application thereof. The recombinant collagen type III retains a functional site in human natural collagen type III for promoting endothelial cell combination, and a key site for platelet combination is directionally replaced to reduce platelet adhesion, and the amino acid sequence is designed as a repeatable unit to enhance functional activity. The recombinant collagen can be expressed in a host cell through genetic engineering and purified to obtain. The obtained recombinant collagen type III has the characteristics of promoting endothelialization and reducing the risk of thrombosis, and is suitable for cardiovascular stent coating, artificial blood vessels and other blood contact instruments, and can be used as an active ingredient in the preparation of vascular repair or antithrombotic drugs.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a recombinant type III collagen that promotes endothelialization and has low platelet adhesion, as well as its preparation method and application. Background Technology

[0002] Blood contact devices pose a significant risk of thrombosis in clinical applications, primarily due to complications such as occlusion and embolism caused by platelet activation and aggregation. To reduce the incidence of thrombosis, existing devices typically employ anticoagulants; however, long-term use of anticoagulants can lead to bleeding complications. In contrast, endothelialization can provide long-term, dynamic regulation for blood contact devices without adverse effects. Endothelial cells can respond to various signals to regulate coagulation, releasing prothrombotic or antithrombotic factors, thereby maintaining vascular homeostasis.

[0003] Several extracellular matrix (ECM) components have been used in coatings of blood-contact materials to promote endothelialization, such as collagen, laminin, fibronectin, and gelatin. Among these, type III collagen, a key matrix component of the vascular wall, naturally possesses platelet-binding capabilities and plays a crucial role in vascular homeostasis and regenerative repair. However, the limited availability of natural type III collagen, its complex molecular structure, batch-to-batch variability, immunogenicity, and the risk of pathogen residue limit its widespread application. More critically, the inherent thrombotic tendency of type III collagen (platelet activation and aggregation properties) is a major factor restricting its use in vascular devices.

[0004] Therefore, there is an urgent need for a recombinant collagen that can both promote endothelialization and significantly reduce platelet adhesion in order to address the thrombosis problem inherent in existing collagen in blood-contact devices. Summary of the Invention

[0005] This invention provides a recombinant type III collagen that promotes endothelialization and reduces platelet adhesion, along with its preparation method and applications. This recombinant collagen, through precise modification of the functional domains of human type III collagen, possesses both the functions of promoting endothelialization and significantly reducing platelet adhesion, effectively reducing the inherent thrombotic tendency of existing collagens in blood-contact devices.

[0006] In a first aspect, the present invention provides a recombinant type III collagen that promotes endothelialization and has low platelet adhesion, the recombinant collagen comprising 1 to 4 sequentially linked repeating units, the amino acid sequence of each repeating unit being shown in SEQ ID NO. 1.

[0007] This invention designs a recombinant type III collagen fragment from amino acid 570-731 of natural human type III collagen, which is rich in key binding sites that interact with endothelial cells and platelets. Using genetic engineering techniques, endothelial cell binding sequences (such as GAPGER and GMPGER) are retained, while key platelet binding sequences (such as GVMGFP and GPPGPP) are directionally replaced with low-cell-binding sequences (GSPGGK) to reduce platelet adhesion. The modified amino acid sequences are used as repeating units, with the number of repeating units set to 1-4, thereby enhancing the functional activity of collagen. This constructs a recombinant type III collagen with high endothelial cell adhesion and proliferation activity and low platelet adhesion. Furthermore, the stability of the recombinant type III collagen decreases as the number of repeating units continues to increase.

[0008] In one specific embodiment, the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO.2.

[0009] Secondly, the present invention provides a nucleic acid molecule that encodes the recombinant type III collagen.

[0010] In one specific embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.3.

[0011] Thirdly, the present invention provides an expression vector comprising the nucleic acid molecule.

[0012] Fourthly, the present invention provides a recombinant cell containing the expression vector.

[0013] In one specific embodiment, the host cell of the recombinant cell is selected from one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0014] Fifthly, the present invention provides a method for preparing the recombinant type III collagen, comprising the following steps:

[0015] Culture the recombinant cells;

[0016] Recombinant type III collagen was induced to express and then purified to obtain recombinant type III collagen.

[0017] In a sixth aspect, the present invention provides the use of the recombinant type III collagen, the nucleic acid molecule, the expression vector, or the recombinant cell in the preparation of products for blood contact devices.

[0018] Furthermore, the recombinant type III collagen provided by this invention can be used as a material for blood contact devices, and applied to cardiovascular stents, coatings, artificial blood vessels, dialysis devices, etc.

[0019] In a seventh aspect, the present invention provides the use of the recombinant type III collagen, the nucleic acid molecule, the expression vector, or the recombinant cell in the preparation of vascular repair or antithrombotic drugs.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. Significantly reduces platelet adhesion:

[0022] This invention utilizes genetic engineering to modify recombinant type III collagen, significantly reducing platelet adhesion rate. Compared to natural collagen, it exhibits a markedly lower tendency to form thrombi, thereby greatly reducing the risk of thrombosis and enhancing the safety and long-term stability of blood contact devices.

[0023] 2. Promotes endothelial cell proliferation and repair:

[0024] This invention retains key sequences in natural human collagen that promote endothelial cell adhesion and proliferation, effectively promoting the repair and regeneration of vascular endothelium, thereby accelerating the endothelialization process. Compared with existing technologies, this invention can more quickly restore the biological function of blood vessels and reduce the problem of delayed endothelialization.

[0025] 3. Optimize structural design and improve functionality:

[0026] This invention achieves the dual effects of reducing platelet adhesion and promoting endothelialization by precisely regulating the functional domains of collagen. Furthermore, by incorporating repeating units, the functional activity of collagen is enhanced. Compared to traditional natural collagen, this invention effectively avoids excessive platelet activation while ensuring endothelial repair, significantly improving antithrombotic properties.

[0027] 4. Enhances biocompatibility and stability:

[0028] The recombinant collagen of the present invention, after optimization, has significant advantages in biocompatibility, stability and durability, can provide longer-lasting protection, reduce thrombus formation, and avoid the problems of immune response and pathogen residue that may occur with traditional collagen.

[0029] 5. Expanded the application of type III collagen in anticoagulation:

[0030] The application of natural type III collagen in vascular devices is somewhat limited due to its inherent tendency to form blood clots. Compared with existing technologies, this invention effectively overcomes this problem through optimized recombinant collagen, which has significant advantages in reducing thrombus formation and promoting endothelial repair. This expands the application of collagen in anticoagulation and provides a new solution for the design and application of blood contact devices. Attached Figure Description

[0031] Figure 1 This is a diagram showing the colony PCR verification results of some transformants in this invention.

[0032] Figure 2 This is an SDS-PAGE analysis of proteins at various time points during the 5 L high-density induced fermentation in this invention.

[0033] Figure 3 This is an SDS-PAGE electrophoresis image of the protein purified in this invention.

[0034] Figure 4 This is the quantitative analysis result of endothelial cell viability in this invention.

[0035] Figure 5 This is the quantitative analysis result of endothelial cell adhesion in this invention.

[0036] Figure 6 This is the quantitative analysis result of platelet adhesion in this invention. Detailed Implementation

[0037] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0038] The inventive concept of this invention is as follows: A fragment of human natural type III collagen, amino acid 570-731, is selected. This fragment is rich in key binding sites that interact with endothelial cells and platelets. By retaining the endothelial cell binding sequences (such as GAPGER and GMPGER) of this fragment, and replacing the key platelet binding sequences (such as GVMGFP and GPPGPP) with the low cell binding sequence GSPGGK, the final amino acid sequence is shown in SEQ ID NO.1.

[0039] SEQ ID NO. 1: GPRGQPGSPGGKGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGSPGGKGAAGTPGLQGMPGER.

[0040] In this invention, the amino acid sequence of the recombinant type III collagen (referred to as recombinant collagen-C4) is based on the repetitive unit constructed above, and the number of the repetitive unit is set to 4 times, and its amino acid sequence is shown in SEQ ID NO.2, so as to enhance the functional activity of collagen.

[0041] SEQ ID NO.2:

[0042] This invention provides a recombinant type III collagen-C4 that promotes endothelialization and reduces platelet adhesion. Codon optimization is performed to target host cell expression, and signal peptide cleavage sites and EcoRI and KpnI restriction enzyme sites are added to both ends during the design process to facilitate subsequent gene manipulation. After the above optimization, its nucleotide sequence is shown in SEQ ID NO.3.

[0043]

[0044] In one specific embodiment, the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0045] Example 1

[0046] I. Expression of recombinant type III collagen

[0047] 1. Based on the codon preference of Pichia pastoris, without altering the amino acid sequence (e.g., SEQ ID NO.2), codon optimization software was used to optimize the gene sequence, and a new codon was introduced at the 5' end of the optimized target gene sequence. EcoR I restriction site and signal peptide cleavage site were introduced at the 3' end. Kpn The nucleotide sequence obtained by the I restriction site is as shown in SEQ ID NO.3. The whole gene is synthesized and ligated into the expression vector pPICZα A. The gene is then transformed into Escherichia coli to obtain the Top10-pPICZα A-rCOLIII strain.

[0048] 2. The Top10-pPICZα A-rCOLIII strain was inoculated into bleomycin-resistant LB broth and cultured for 12 hours. The bacterial culture was centrifuged to remove the supernatant, and the recombinant plasmid pPICZα A-rCOLIII was extracted using a plasmid mini-extraction kit. Restriction endonuclease... Sac After linearization, the linearized plasmid was recovered using alcohol precipitation. The plasmid micro-extraction kit was manufactured by Tiangen Biotech Co., Ltd.

[0049] 3. Pichia pastoris X33 competent cells were prepared, and the obtained linearized plasmid was transformed into Pichia pastoris X33 competent cells by electroporation to construct the Pichia pastoris X33-pPICZα A-rCOLIII expression strain. The Pichia pastoris X33 strain was purchased from Xi'an Giant Biogene Technology Co., Ltd.

[0050] 4. Positive transformants after electroporation were screened using YPD plates containing different concentrations of bleomycin. High-copy transformants were selected for colony PCR identification, and the PCR products were analyzed by agarose gel electrophoresis. The colony PCR verification results of some transformants are shown below. Figure 1 As shown.

[0051] Depend on Figure 1 As can be seen from the colony PCR identification, there is a target band at 2400bp that matches the theoretical size, proving that the target gene has been successfully introduced into the Pichia pastoris genome and the recombinant strain has been successfully constructed.

[0052] 5. The high-copy Pichia pastoris X33--pPICZα A-rCOLIII expression strain, whose sequencing verification was correct, was scaled up in a 5 L fermenter for production. First, the target strain was picked and inoculated into 50 mL of BMGY liquid medium, and cultured at 30 ℃ and 220 rpm for 24 h with shaking. Then, the 24-h culture was transferred at a 2% inoculation ratio to three 500 mL Erlenmeyer flasks, each containing 100 mL of fresh BMGY liquid medium to ensure continued cell growth under suitable conditions. The flasks were cultured for another 48 h to ensure optimal seed viability. Under aseptic conditions, the culture was inoculated into a fermenter containing BSM medium, and the stirring speed was gradually increased. Glycerol was added at a rate of 80 mL / h. When the dissolved oxygen level gradually increased to 80%, the cells were starved for 1 h, and methanol was then added at a rate of 12 mL / h. The methanol addition phase lasted approximately 40 h. Samples were taken during the methanol induction phase, and the supernatant was analyzed by SDS-PAGE electrophoresis. The electrophoresis results are shown in Figure 2. The sampling times were 0h, 8h, 16h, 24h, 32h, 36h, and 40h after induction. The BMGY liquid medium consisted of 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 10% (v / v) phosphate buffer (pH 6.0, 1M), and 1% (v / v) glycerol. The BSM medium consisted of 0.46 g / L CaSO4·2H2O, 7.5 g / L MgSO4·7H2O, 2.1 g / L KOH, 9.1 g / L K2SO4, 13.4 mL / L H3PO4, and 40.0 g / L glycerol. After autoclaving at 121 ℃ for 20 min, 4.35 mL / L filtered trace element PTM1 was added.

[0053] Depend on Figure 2 It is known that the molecular weight of this protein is about 100 kDa, proving that the recombinant collagen was successfully expressed in Pichia pastoris.

[0054] II. Purification of Recombinant Type III Collagen

[0055] 1. Centrifuge the obtained fermentation broth at 8000 rpm for 20 min. Pass the supernatant obtained through a hollow fiber column to remove impurities and obtain the target protein enrichment solution.

[0056] 2. Pretreatment of MMC cation exchange chromatography column: Equilibrate the MMC column with 20mM potassium phosphate buffer (Solution A, pH 6.0) to ensure that the electrostatic and hydrophobic interactions between the stationary phase and the target protein are optimal.

[0057] Sample loading: Adjust the pH of the protein filtrate to 6.0 before loading the sample.

[0058] Gradient elution: A gradient elution was performed using a mixture of solutions A and B, where solution B consisted of 20 mM potassium phosphate buffer, 1 M NaCl, and pH 6.0. The elution gradient increased sequentially by 10% (v / v) of solution B. A 20% (v / v) elution intensity of solution B was used to remove weakly bound proteins; a 30% (v / v) linear gradient elution with solution B was used to separate medium-bound impurities; and isocratic elutions with 40% and 50% (v / v) solutions were 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.

[0059] SDS-PAGE electrophoresis detection of protein purification, such as Figure 3 As shown.

[0060] Depend on Figure 3 It can be seen that during the gradient elution process, two UV absorption peaks appeared in the 30% (v / v) B solution, which were denoted as the 30% (1) peak and the 30% (2) peak, respectively. SDS-PAGE analysis showed that both peaks contained impurity proteins. In the elution peak of the 40% (v / v) B solution, the content of impurity proteins was significantly reduced. When the integral of B solution increased to 50% (v / v), a specific elution peak of the target protein appeared. SDS-PAGE analysis confirmed that the peak was the purified recombinant type III collagen, and the impurity proteins were effectively removed.

[0061] 3. Desalting and Lyophilization: The collected solution containing high-purity target protein was desalted and ultrafiltered using an ultrafiltration membrane. The resulting product was then frozen overnight at -40 °C and subsequently vacuum-dried. Lyophilization yielded solid-state recombinant collagen, preserving its native conformation and activity. This solid-state recombinant collagen is specifically recombinant type III collagen (rCOLIII-C4).

[0062] Example 2: Bioactivity of recombinant type III collagen (rCOLIII)

[0063] I. Determination of cell viability by rCOLIII

[0064] The CCK-8 assay was used to detect protein-induced endothelial cell viability. The study was divided into experimental groups (containing 0.5, 1.0, 3.0, and 5.0 mg / mL recombinant type III collagen rCOLIII-C4, respectively), control groups (without collagen), and blank groups (complete culture medium without cells and collagen). The culture medium was prepared at a density of 5.0 × 10⁻⁶ μL per well. 4Cell suspensions at a concentration of [cells / mL] were inoculated into 96-well plates and cultured for 24 h. The culture medium was then replaced with medium containing different concentrations of the sample. After another 24 h of culture, cell proliferation was verified using the CCK8 assay, and absorbance was measured at 450 nm to calculate relative cell viability. The formula for calculating relative cell viability is as follows:

[0065] Relative cell viability (%) = [(OD)] test -OD blank )] / [(OD control -OD blank ))]×100%

[0066] Meanwhile, the cells incubated for 24 hours were stained with AO / EB and their growth status after being cultured in collagen medium was observed under a fluorescence microscope.

[0067] Quantitative analysis of cell viability, such as Figure 4 As shown.

[0068] Depend on Figure 4 It was found that C4 protein promoted endothelial cell proliferation at concentrations ranging from 0.5 to 5 mg / mL, with the most significant effect observed at a dose of 3 mg / mL. Based on AO / EB staining results, the experimental group exhibited a more pronounced green fluorescence intensity, while the cells did not show any abnormal morphological characteristics. These experimental data demonstrate the safety of the recombinant collagen as a biomaterial and its biological efficacy in promoting endothelial cell proliferation.

[0069] II. Determination of endothelial cell adhesion by rCOLIII

[0070] BSA, full-length type III collagen, and recombinant type III collagen (rCOLIII-C4) were uniformly coated onto well plates and incubated overnight at 4°C. The wells were then blocked for 1 hour at room temperature with 1% (v / v) heat-denatured BSA, and washed twice with PBS to complete the coating. The full-length type III collagen, composed of the full-length α1 chain sequence of human type III collagen, was derived from Xi'an Giant Biogene Technology Co., Ltd.

[0071] Endothelial cells in good growth condition were collected, counted using a counting chamber, and prepared to a cell density of 2.0 × 10⁶ cells / year. 5Cell suspensions of [cell density] / mL were seeded into 48-well plates coated with different proteins. The plates were then incubated in a CO2 incubator for 4 h. After incubation, the culture medium was removed, and the cells were washed three times with PBS. Next, the cells were fixed with 4% (v / v) paraformaldehyde solution for 15 min. After fixation, the paraformaldehyde solution was discarded, and 0.1% (v / v) crystal violet solution was added for staining for 20 min. After staining, the crystal violet solution was removed, and the morphological characteristics of the cells were observed and recorded using an optical microscope. Subsequently, 1% SDS solution was added, and finally, the absorbance (OD) was measured at 590 nm using a microplate reader. 590 Cell adhesion rate is calculated using the following formula:

[0072] Cell adhesion rate (%) = [(OD)] test -OD blank )] / [(OD control -OD blank ))]×100%

[0073] Quantitative analysis of cell adhesion, such as Figure 5 As shown.

[0074] Depend on Figure 5 Quantitative analysis of adhesion showed that the adhesion rate of C4 protein to endothelial cells was higher than that of natural full-length type III collagen, indicating that the recombinant type III collagen of this invention has a good effect on promoting endothelial cell adhesion.

[0075] III. Determination of rCOLIII's effect on platelet adhesion

[0076] BSA, full-length type III collagen, and recombinant type III collagen (rCOLIII-C4) were uniformly coated into wells of a plate and incubated overnight at 4°C. Then, at room temperature, the wells were blocked with 1% (v / v) heat-denatured BSA for 1 hour, followed by two washes with PBS to complete the coating. Subsequently, 100 μL of platelet-rich plasma was added to the protein-coated wells, incubated at 37°C for 1 hour, and the wells were washed with PBS. Adhering platelets were then lysed with 1% (v / v) Triton X-100 to release lactate dehydrogenase. A control group was prepared by directly adding 1% (v / v) Triton X-100 lysis buffer to 100 μL of platelet-rich plasma. Finally, the LDH ELISA kit was used according to the manufacturer's instructions, and absorbance was measured at 450 nm to calculate the platelet adhesion rate. The specific calculation formula is as follows:

[0077] Platelet adhesion rate = OD test / ODreference value ×100%; OD test OD reference value These represent the absorbance of the sample and the absorbance of the reference value, respectively.

[0078] Platelet adhesion assay showed that... Figure 6 As shown.

[0079] Depend on Figure 6 Quantitative analysis of adhesion showed that the adhesion rate of C4 protein to platelets was significantly lower than that of natural full-length type III collagen, indicating that the recombinant type III collagen-C4 of this invention has certain antithrombotic properties.

[0080] The above results indicate that the recombinant type III collagen of the present invention has good biocompatibility, can significantly promote the adhesion and proliferation of endothelial cells, and at the same time reduce the adhesion level of platelets, showing good endothelialization and antithrombotic properties.

[0081] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0082] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

Claims

1. A recombinant type III collagen protein that promotes endothelialization and has low platelet adhesion, characterized in that, The recombinant collagen is sequentially connected by four repeating units, and the amino acid sequence of each repeating unit is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant collagen III of claim 1.

3. An expression vector, characterized by, The expression vector contains the nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that, The recombinant cell contains the expression vector of claim 3.

5. The recombinant cell of claim 4, wherein, The host cell of the recombinant cell is selected from one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

6. The method for preparing recombinant type III collagen according to claim 1, characterized in that, The method comprises the following steps: Culturing the recombinant cell of claim 4; Purifying the recombinant collagen III after inducing the expression of the recombinant collagen III to obtain the recombinant collagen III.

7. Use of the recombinant collagen III of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3 or the recombinant cell of claim 4 in the preparation of a product for blood contact device.

8. Use according to claim 7, characterized in that, The product is a cardiovascular stent, an artificial blood vessel or a dialysis device.

9. Use of the recombinant collagen III of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3 or the recombinant cell of claim 4 in the preparation of a blood vessel repair or an anti-thrombosis drug.

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