Recombinant human XVII type collagen with hemostasis function and application of recombinant human XVII type collagen

Abstract

The invention discloses recombinant human XVII type collagen with a hemostasis function and application of the recombinant human XVII type collagen. The recombinant human XVII type collagen is a truncated protein of the human XVII type collagen, and the amino acid sequence of the recombinant human XVII type collagen is shown as SEQ ID NO: 1. The recombinant human XVII type collagen has efficient and rapid hemostatic performance, meanwhile, the recombinant collagen is easy to separate and purify, a pure product with the purity reaching 97% or above can be prepared through simple membrane separation and chromatographic separation, and the production process is easy to amplify. In the aspect of hemostasis performance, compared with commercially available products, the recombinant human XVII type collagen has the advantages that the hemostasis time is obviously shortened, and the hemostasis performance is obviously improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a novel genetically engineered recombinant collagen, a preparation method and use thereof. Background Art

[0002] Collagen is a biopolymer protein, the most widely distributed, richest and most diverse fibrous protein family in animals, accounting for 25%-30% of the total protein. As the main component of the extracellular matrix, it has the characteristics of low antigenicity, biodegradability, easy absorption, non-toxicity and good biocompatibility. It is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging. It is one of the most critical raw materials in the biotechnology industry and is widely used in medical materials, cosmetics and food industries.

[0003] Hemostasis refers to the act of stopping blood from flowing outwards quickly through physiological spontaneous or external intervention during bleeding. External intervention mainly involves treating the wound with hemostatic materials, which is a necessary means to control massive bleeding. Hemostasis is an important part of treatment in trauma emergency and surgical operations. Uncontrolled blood loss is still the main cause of death.

[0004] Recombinant collagen has been widely used in clinical fields such as intraoperative collagen hemostasis, wound dressings, and tissue engineering scaffolds due to its weak antigenicity, excellent biocompatibility, absorption by the body, and high cell adhesion and proliferation. For hemostatic products, hemostasis time is the most effective indicator for judging the hemostatic effect. Compared with the sterile gauze compression hemostasis commonly used in surgery, the recombinant collagen hemostatic products currently on the market, such as hemostatic sponges and hemostatic gauze, can effectively shorten the hemostasis time. However, wound hemostasis is a major health issue related to life, especially in the fields of military and emergency rescue, which is directly related to people's life safety. More effective products with longer hemostasis time have always been the goal pursued by people. Summary of the invention

[0005] In view of the above reasons, an object of the present invention is to provide a recombinant collagen hemostatic product that can stop bleeding more quickly and effectively.

[0006] The inventors conducted in-depth research to solve the above technical problems, and found that among many types of collagen, a truncated protein from human type XVII collagen has unexpected properties - it can effectively shorten the hemostasis time, thereby improving the recombinant type XVII collagen for rapid and efficient hemostasis, as well as rapid hemostasis products using the recombinant type XVII collagen as raw material.

[0007] That is, the present invention includes:

[0008] 1. A recombinant human type XVII collagen with hemostatic function, which is a truncated protein of human type XVII collagen, and its amino acid sequence is shown in SEQ ID NO: 1.

[0009] 2. A nucleic acid encoding the recombinant human type XVII collagen described in item 1.

[0010] 3. An expression vector comprising the nucleic acid described in item 2.

[0011] 4. A host cell introduced with the expression vector according to item 3. The host cell may be Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli or Bacillus subtilis, preferably Pichia pastoris.

[0012] 5. A method for producing the recombinant human type XVII collagen described in item 1, comprising the steps of culturing the host cell described in item 4 and collecting the cell culture.

[0013] 6. The method according to item 5 further comprises the step of separating and purifying the cell culture, wherein the separation and purification adopts one or a combination of salting out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, and membrane separation.

[0014] 7. A hemostatic product, which is prepared using the recombinant human type XVII collagen described in item 1 as a raw material. The hemostatic product is preferably a medical device, such as a hemostatic sponge.

[0015] 8. Use of the recombinant human type XVII collagen described in item 1 in the preparation of a hemostatic product. The hemostatic product is preferably a medical device, such as a hemostatic sponge.

[0016] The beneficial effects of the present invention include: providing a new recombinant human type XVII collagen, which has efficient and rapid hemostatic performance, and the recombinant collagen is easy to separate and purify, and a pure product with a purity of more than 97% can be prepared by simple membrane separation and chromatography separation, and its production process is easy to scale up. In terms of hemostatic performance, the recombinant human type XVII collagen significantly shortens the hemostatic time compared with commercially available products, and the hemostatic time is shortened from about 200s of commercially available products to about 148s of the recombinant human type XVII collagen product, and the hemostatic performance is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The electrophoretic diagrams show the fermentation results and purification results of type XVII recombinant collagen 1-4. DETAILED DESCRIPTION

[0018] Example 1 Preparation of different truncated type XVII recombinant collagen in yeast expression system

[0019] 1. Preparation of shuttle plasmid

[0020] According to the amino acid sequences shown in SEQ ID No: 1 to 4 (all truncated proteins of natural human XVII collagen), codon optimization of the yeast expression system was performed to obtain the target gene sequence XVII (1-4). The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized genes were respectively connected to the pPicZαA plasmid to obtain pPicZαA-XVII (1-4) plasmids.

[0021] 2. Preparation of yeast expression strains

[0022] pPicZαA-XVII(1-4) was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Bleomycin resistance was used as a selection marker to select transformants to obtain yeast expression strains.

[0023] 3. Induced expression of target protein

[0024] (1) Pick a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and culture it at 30°C and 200 rpm for 48 h for activation;

[0025] (2) Transfer the inoculum to a 500 ml Erlenmeyer flask (containing 200 ml of YPD culture medium) at a 1% inoculum volume and culture at 30°C and 200 rpm for 24 h to serve as the seed for the tank.

[0026] (3) Prepare 3 L of BSM medium and add it to a 5 L fermenter, sterilize at 121° C. for 20 min, and after cooling to 30° C., adjust the pH to 5.0, and add the seeds prepared in (2) to the fermenter by flame inoculation for fermentation;

[0027] (4) Cultivate until OD 600 When OD was 70, 50% glycerol was added until 600 When the temperature reaches about 120, stop adding, and wait until the dissolved oxygen rebounds to 100%, then start adding methanol for induction;

[0028] (5) During the induction process, the dissolved oxygen was controlled to be not less than 30% and the pH was about 5.0. The induction was continued for 40 h, and the fermentation was terminated. The culture medium was centrifuged at 12,000 rpm for 2 min, and the supernatant was taken for detection of protein yield and purity using the BCA method and SDS-PAGE method.

[0029] 4. Collagen purification

[0030] The collected supernatant was purified by the following steps.

[0031] (1) The obtained supernatant was concentrated by ultrafiltration through a 10 kD membrane and then crudely purified by a hollow fiber column;

[0032] (2) The crude and purified permeate obtained above is then ultrafiltered and concentrated to a certain volume for later use;

[0033] (3) separating and purifying it through an SP cation exchange column, eluting it with a 30% phosphate buffer solution containing 2M NaCl, and collecting the target eluate;

[0034] (4) Ultrafiltration concentration, desalting and freeze-drying are performed. Take 100 mg of freeze-dried powder and dissolve it in 100 ml of physiological saline. After fully dissolving, apply it to SDS-PAGE gel electrophoresis to confirm the molecular weight and protein purity.

[0035] 5. Results

[0036] The recombinant type XVII collagen 1-4 (amino acid sequences are shown in SEQ ID No: 1-4, named Col17-1, Col17-2, Col17-3, Col17-4) prepared by the above method was tested by SDS-PAGE electrophoresis. The results showed that the molecular weight was correct and there was one band without obvious miscellaneous bands. The protein electrophoresis results before and after purification were as follows: Figure 1 shown.

[0037] Example 2 Preparation of recombinant type XVII collagen sponge sample

[0038] The sample preparation process of recombinant type XVII collagen sponge is as follows:

[0039] 1. Accurately weigh 2 g of the above-mentioned recombinant type XVII collagen (1-4) and 98 g of water for injection, and fully dissolve them to obtain 2% by weight recombinant collagen solutions, which are named Col17-1 solution, Col17-2 solution, Col17-3 solution, and Col17-4 solution, respectively.

[0040] 2. Filter the recombinant collagen solution with a 0.22 μm filter membrane to remove insoluble matter and fill the filtered recombinant collagen solution into a mold.

[0041] 3. Set the freeze-drying parameters and use a vacuum freeze dryer to complete the freeze-drying of the recombinant collagen solution.

[0042] 4. The freeze-dried samples were placed in a 2.5% glutaraldehyde solution to complete cross-linking. The samples were ultrasonically cleaned with PBS for more than 10 times and freeze-dried to obtain Col17-1 sponge, Col17-2 sponge, Col17-3 sponge and Col17-4 sponge.

[0043] Example 3 Hemostatic efficacy test of recombinant type XVII collagen sponge

[0044] 1. Materials: Col17-1 sponge, Col17-2 sponge, Col17-3 sponge, Col17-4 sponge (experimental group), commercially available product (control group), non-woven gauze (blank group)

[0045] 2. Experimental animals: New Zealand white rabbits, weighing approximately 1.5 kg, of either sex, 5 rabbits per group.

[0046] 3. Specific steps: After the rabbit is anesthetized, the rabbit hair at the ears is removed, and after disinfection, iodine is removed with 75% alcohol. Fix the rabbit, use a scalpel to make a wound at the central artery of the rabbit ear, absorb the blood with gauze, quickly apply a 1×1×0.5cm collagen sponge to the wound surface, apply pressure vertically with a 20g weight, and start timing immediately. Observe whether the wound is bleeding every 20s, stop timing when there is no bleeding, and get the hemostasis time. The blank group was pressed with gauze and weights, and each group of experiments was repeated 3 times. The specific experimental results are shown in Table 1.

[0047] Table 1 Hemostatic efficacy test of various recombinant type XVII collagen sponges

[0048] Serial number Sample name Bleeding time 1 Blank control 440±48s 2 Commercially available products <![CDATA[200±43s ** ]]> 3 Col17-1 <![CDATA[148±39s **△ ]]> 4 Col17-2 392±35s 5 Col17-3 <![CDATA[336±46s * ]]> 6 Col17-4 <![CDATA[308±47s ** ]]>

[0049] Note: Compared with the blank group: * P<0.05, ** P<0.01; compared with commercially available products: △ P<0.05, △△ P < 0.01;

[0050] From the results in Table 1, it can be seen that different truncated recombinant collagen samples exhibited different hemostatic effects. Compared with the blank group, Col17-1 and the commercially available product both showed good hemostatic effects, with significant differences (**P<0.01). Compared with the commercially available product, the hemostatic time of Col17-1 was shorter, showing a significant difference ( △ P<0.05). In summary, the recombinant type XVII collagen sponge of the present invention has good hemostatic function.

[0051] Sequence information

[0052] SEQ ID No: 1 (Col17-1)

[0053] gspgpkgdmgspgpkgdrgfpgtpgipgplghpgpqgpkgqkgsvgdpgmegpmgqrgregpmgprgeagppgsgekgergaagepgphgppgvpgsvgpkgssgspgpqgppgpvglqglrgevglpgvkgdkgpmgppgpkgdqgekgprgltgepgmrglpgavgepgakgamgpagpdghqgprgeqgltgmpgirgppgpsgdpgkpgltgpqgpqglpgtpgrpgikgepgapgkivtsegssmltvpgppgppgamgppgppgapgpagpaglpghqevlnlqgppgppgprgppgpsipgppgprgppgeglpgppgppgsflsnsetflsgppgppgppgpkgdqgppgprghqgeqglpgfstsgsssfglnlqgppgppgpqgpkgdkgdpgvpgalgipSEQ ID No:2(Col17-2)

[0054] gspgpkgdmgspgpkgdrgfpgtpgipgplghpgpqgpkgqkgsvgdpgmegpmgqrgregpmgprg

[0055] eagppgsgekgergaagepgphgppgvpgsvgpkgssgspgpqgppgpvglqglrgevglpgvkgdkgp

[0056] mgppgpkgdqgekgprgltgepgmrglpgavgepgakgamgpagpdghqgprgeqgltgmpgirgppg

[0057] psgdpgkpgltgpqgpqglpgtpgrpgikgepgapgki

[0058] SEQ ID No:3(Col17-3)

[0059] gspgpkgdmgspgpkgdrgfpgtpgipgplghpgpqgpkgqkgsvgdpgmegpmgqrgregpmgprg

[0060] eagppgsgekgergaagepgphgppgvpgsvgpkgssgspgpqgppgpvglqglrgevglpgvkgdkgp

[0061] mgppgpkgdqgekgprgltgepgmrglpgavgepgakgamgpagpdghqgprgeqgltgmpgirgppg

[0062] psgdpgkpgltgpqgpqglpgtpgrpgikgepgapgkivtsegssmltvpgppgppgamgppgppgapgp

[0063] agpaglpghq

[0064] SEQ ID No:4(Col17-4)

[0065] gspgpkgdmgspgpkgdrgfpgtpgipgplghpgpqgpkgqkgsvgdpgmegpmgqrgregpmgprg

[0066] eagppgsgekgergaagepgphgppgvpgsvgpkgssgspgpqgppgpvglqglrgevglpgvkgdkgp

[0067] mgppgpkgdqgekgprgltgepgmrglpgavgepgakgamgpagpdghqgprgeqgltgmpgirgppg

[0068] psgdpgkpgltgpqgpqglpgtpgrpgikgepgapgkivtsegssmltvpgppgppgamgppgppgapgp

[0069] agpaglpghqevlnlqgppgppgprgppgpsipgppgprgppgeglpgppgppgsflsnsetflsgppgppg

[0070] ppgpkgdqgppgprghqgeqglpgfstsgsssfglnlqgppgppgpqgpkgdkgdpgvpgalgipsgpseg

[0071] gssstmyvsgppgppgppgppgsi

Claims

1. A recombinant human type XVII collagen with hemostatic function, which is a truncated protein of human type XVII collagen, and its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid encoding the recombinant human type XVII collagen according to claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell into which the expression vector according to claim 3 has been introduced; the host cell may be Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli or Bacillus subtilis, preferably Pichia pastoris.

5. A method for producing the recombinant human type XVII collagen according to claim 1, comprising: The step of culturing the host cell according to claim 4 and collecting the cell culture.

6. The method according to claim 5, further comprising the step of separating and purifying the cell culture, wherein the separation and purification adopts one or a combination of salting out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, and membrane separation. 7 . A hemostatic product comprising the recombinant human type XVII collagen according to claim 1 .

8. Use of the recombinant human type XVII collagen according to claim 1 in the preparation of hemostatic products.

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