Recombinant VI-type collagen as well as preparation method and application thereof

Abstract

The invention discloses a recombinant VI-type collagen as well as a preparation method and application thereof. The recombinant VI-type collagen comprises a protein gene segment derived from a natural VI-type collagen alpha2 chain, and proteins P0607, P060702 and / or P060703 capable of promoting cartilage cell proliferation activity are expressed. Gene segments derived from a natural type VI collagen alpha2 chain are integrated through a genetic engineering means, sequence optimization and repetition are carried out on the gene segments to enhance the biological activity of the collagen, the recombinant type VI collagen P0607, P060702 and P060703 can be stably and efficiently produced, the recombinant collagen has the effect of promoting chondrocyte proliferation, and the recombinant collagen can be applied to the field of cartilage cell proliferation. An efficient and reliable biological manufacturing way is provided for research, development and production of osteoarthritis medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a recombinant type VI collagen and a preparation method and application thereof. Background Art

[0002] Osteoarthritis (OA) is a common chronic joint disease characterized by articular cartilage damage, reduced synovial fluid lubrication, and increased joint friction, leading to joint pain, swelling, and dysfunction. Currently, the treatments for OA mainly include medication, physical therapy, and surgery, but these methods all have certain limitations. Therefore, finding a safe and effective treatment for osteoarthritis is of great clinical significance.

[0003] Collagen is a biopolymer that is widely present in animal connective tissue, accounting for about 25% to 30% of the total protein in the body. It is the most widely distributed and most functional protein in mammals. The use of genetic engineering technology to produce collagen has advantages that the extraction method does not have, and the product is stable and safe. Protein drugs have the following advantages over traditional chemical drugs: 1. More significant efficacy: Protein drugs usually have stronger efficacy because they have a smaller molecular weight and can penetrate the cell membrane more effectively, thereby improving their bioavailability. And they can be regulated by specific molecules or receptors in the disease process to achieve precision treatment. 2. Stronger specificity: Protein drugs have a high degree of specificity and can act more accurately on target molecules or cells, reducing adverse effects on non-target tissues or cells. 3. Lower toxicity and fewer side effects: Due to their high specificity, protein drugs often have lower toxicity and fewer side effects. 4. Clearer biological functions: Protein drugs simulate or interfere with natural biological processes, and their mechanisms of action are usually clearer, which helps to optimize treatment plans. These advantages result in fewer side effects on patients during treatment, improving the quality of life of patients and the safety of treatment. Therefore, protein drugs have great potential and application prospects in the treatment of osteoarthritis and are an important direction for current and future OA drug research.

[0004] Type VI collagen (Col VI) is a milky connective tissue protein composed of three different α chains that form a characteristic beaded microfilament network in the extracellular matrix (ECM). Col VI plays different roles in the tissues in which it is expressed, ranging from the mechanical effects of typical ECM collagen components to more specific cytoprotective functions, including inhibition of apoptosis and oxidative damage, regulation of cell differentiation and autophagy mechanisms, and maintenance of cell stemness. Type VI collagen is a component of the extracellular matrix and is essential for maintaining cartilage structure and function. However, no studies have been able to show that the Col VI α2 chain fragment selected by the present invention is associated with promoting chondrocyte proliferation.

[0005] Pichia pastoris is a single-cell eukaryotic organism. It has the characteristics of easy cultivation, fast reproduction and convenient genetic engineering operation of prokaryotes, and at the same time has the ability of eukaryotic organisms to correctly fold gene products and post-translation processing and modification. Therefore, Pichia pastoris is often used to express foreign proteins. Moreover, the substrate is methanol, and the fermentation cost is low. Therefore, based on synthetic biology technology, using Pichia pastoris as a recombinant protein expression system, the development of recombinant strains capable of efficiently expressing recombinant type VI collagen shows greater application prospects. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a recombinant type VI collagen and its preparation method and application in view of the above-mentioned deficiencies of the prior art. The recombinant collagen has the effect of promoting chondrocyte proliferation, and provides an efficient and reliable biomanufacturing approach for the research and development and production of osteoarthritis drugs.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: a recombinant type VI collagen, characterized in that it includes a protein gene fragment derived from the α2 chain of natural type VI collagen, and expresses proteins P0607, P060702 and / or P060703 with chondrocyte proliferation promoting activity; the amino acid sequence of P0607 is shown in SEQ ID NO: 1, the amino acid sequence of P060702 is shown in SEQ ID NO: 3, and the amino acid sequence of P060703 is shown in SEQ ID NO: 5.

[0008] Furthermore, the present invention provides a nucleic acid encoding the above-mentioned recombinant type VI collagen, characterized in that the nucleic acid sequence encoding protein P0607 is shown as SEQ ID NO:2, the nucleic acid sequence encoding protein P060702 is shown as SEQ ID NO:4, and the nucleic acid sequence encoding protein P060703 is shown as SEQ ID NO:6.

[0009] Furthermore, the present invention provides an expression vector comprising the above nucleic acid.

[0010] The above-mentioned expression vector is characterized in that the expression vector is pPICzαA.

[0011] Furthermore, the present invention provides a host cell into which the above expression vector is introduced, characterized in that the host cell is Escherichia coli, yeast or Bacillus subtilis, preferably Pichia pastoris.

[0012] Furthermore, the present invention provides a genetically engineered bacterium for producing the above-mentioned recombinant type VI collagen, characterized in that the host cell of P0607 is Pichia pastoris X33, the recombinant plasmid pPICZαA-P0607 is obtained by linearization with Sal I enzyme, and the recombinant plasmid pPICZαA-P0607 is obtained by transfer into the host cell

[0013] The host cells of P060702 and P060703 were both Pichia pastoris GS115. The recombinant plasmid pPIC9K-P060702 was obtained by linearization with Sal I enzyme and then transferred into the host cells to obtain GS115-pPIC9K-P060702 genetic engineering bacteria. The recombinant plasmid pPIC9K-P060703 was obtained by linearization with Sal I enzyme and then transferred into the host cells to obtain

[0014] GS115-pPIC9K-P060703 genetically engineered bacteria.

[0015] The above-mentioned genetically engineered bacteria are characterized in that when the recombinant plasmid pPICZαA-P0607 is synthesized, Xho I and XBa I recognition sites and a signal peptide recognition site are added to the 5' end and 3' end respectively; when the recombinant plasmid pPIC9K-P060702 is synthesized, Xho I and EcoR I recognition sites and a signal peptide recognition site are added to the 5' end and 3' end respectively; when the recombinant plasmid pPIC9K-P060703 is synthesized, Xho I and EcoR I recognition sites and a signal peptide recognition site are added to the 5' end and 3' end respectively.

[0016] The above-mentioned genetically engineered bacteria is characterized in that the shake flask fermentation conditions of the genetically engineered bacteria are: induction temperature 30°C, induction time 48h, and methanol concentration 1%.

[0017] Furthermore, the present invention provides a method for producing the above-mentioned recombinant type VI collagen, characterized in that it includes culturing host cells and separating and purifying the cell culture, wherein the separation and purification adopts one or a combination of salting-out, ultrafiltration and gel filtration chromatography.

[0018] Furthermore, the present invention provides a use of the above-mentioned recombinant type VI collagen in the preparation of food, health food, medicine or medical device for treating or preventing diseases caused by OA.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. The recombinant type VI collagen of the present invention has the characteristics of high affinity, high safety, low immunogenicity, high purity, good moisture retention and water solubility, suitable pH value and the like.

[0021] 2. The recombinant type VI collagen of the present invention can stimulate the proliferation of chondrocytes. The soluble recombinant type VI collagen fragment can expand the scarce source of chondrocytes, promote the proliferation of chondrocytes, and may be used for autologous chondrocyte implantation, providing an efficient and reliable biomanufacturing approach for the research and development and production of osteoarthritis drugs.

[0022] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the plasmid map of pPICZαA-P0607.

[0024] Figure 2 This is the plasmid map of pPIC9K-P060702.

[0025] Figure 3 This is the plasmid map of pPIC9K-P060703.

[0026] Figure 4 This is the SDS-PAGE image of recombinant type VI collagen P0607, P060702 and P060703.

[0027] Figure 5 This is the purification diagram of 0607 protein.

[0028] Figure 6 This is the purification diagram of 060702 protein.

[0029] Figure 7 This is the purification diagram of 060703 protein.

[0030] Figure 8 This graph shows the promoting effect of recombinant type VI collagen P06070, P060702 and P060703 with different sequence repetition times on the cell proliferation rate of chondrocytes. DETAILED DESCRIPTION

[0031] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0032] The present invention provides three recombinant collagen proteins capable of promoting chondrocyte proliferation, namely P0607, P060702 and P060703 proteins, whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5.

[0033] SEQ ID NO: 1:

[0034] GERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNNGAPGSPGVKGAKGGPGPRGPKGEPGRRGDPGTKGSPGSDGPKGEKGDPGPEGPRGLAGEVGNKGAKGDRGLPGPRGPQGALGEPGKQGSRGDPGDAGPRGDSGQPGPKGDPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRGDFGLKGEPGRKGEKGEPADPGPPGEPGPRGPRGVPGPEGEPGPP

[0035] SEQ ID NO:3:

[0036] GERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNNGAPGSPGVKGAKGGPGPRGPKGEPGRRGDPGTKGSPGSDGPKGEKGDPGPEGPRGLAGEVGNKGAKGDRGLPGPRGPQGALGEPGKQGSRGDPGDAGPRGDSGQPGPKGDPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRGDFGLKGEPGRKGEKGEPADPGPPGEPGPRGPRGVPGPEGEPGPPGERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNNGAPGSPGVKGAKGGPGPRGPKGEPGRRGDPGTKGSPGSDGPKGEKGDPGPEGPRGLAGEVGNKGAKGDRGLPGPRGPQGALGEPGKQGSRGDPGDAGPRGDSGQPGPKGDPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRGDFGLKGEPGRKGEKGEPADPGPPGEPGPRGPRGVPGPEGEPGPP

[0037] SEQ ID NO:5:

[0038] GERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNNGAPGSPGVKGAKGGPGPRGPKGEPGRRGDPGTKGSPGSDGPKGEKGDPGPEGPRGLAGEVGNKGAKGDRGLPGPRGPQGALGEPGKQGSRGDPGDAGPRGDSGQPGPKGDPGRPGFSYPGPRGAPGEKG EPGPRGPEGGRGDFGLKGEPGRKGEKGEPADPGPPGEPGPRGPRGVPGPEGEPGPPGERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNNGAPGSPGVKGAKGGPGPRGPKGEPGRRGDPGTKGSPGSDGPKGEKGDPGPEGPRGLAGEVGNKGAKGDRGLPGP RGPQGALGEPGKQGSRGDPGDAGPRGDSGQPGPKGDPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRGDFGLKGEPGRKGEKGEPADPGPPGEPGPRGPRGVPGPEGEPGPPGERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNNGAPGSPGVKGAKGGPGPRG PKGEPGRRGDPGTKGSPGSDGPKGEKGDPGPEGPRGLAGEVGNKGAKGDRGLPGPRGPQGALGEPGKQGSRGDPGDAGPRGDSGQPGPKGDPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRGDFGLKGEPGRKGEKGEPADPGPPGEPGPRGPRGVPGPEGEPGPP

[0039] The present invention provides three recombinant collagens that promote chondrocyte proliferation, and codon optimization is performed for host cell expression. Signal peptide cleavage sites and enzyme cleavage sites are added to both ends during the design process to facilitate later gene manipulation. After the above optimization, the nucleotide sequences are shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

[0040] SEQ ID NO:2:

[0041] CTCGAGAAAAGAGGTGAGAGAGGTGATCAGGGTGGTAAAGGCGATCCAGGCAGACCTGGCAGAAGAGGTCCACCTGGTGAAATTGGTGCCAAGGGTAGTAAGGGTTATCAGGGTAACAACGGTGCACCTGGTTCTCCAGGTGTTAAGGGTGCTAAGGGTGGTCCAGGCCCTAGAGGTCCAAAGGGAGAACCTGGAAGAAGAGGTGATCCAGGTACTAAGGGTTCACCAGGCTCTGATGGACCAAAGGGTGAGAAGGGAGATCCAGGTCCAGAGGGACCAAGAGGTTTGGCTGGAGAAGTTGGTAACAAAGGTGCTAAAGGTGATAGAGGCTTGCCAGGTCCAAGGGGACCACAAGGTGCTTTGGGCGAACCTGGTAAGCAAGGCTCACGTGGTGATCCAGGCGATGCTGGTCCAAGAGGTGACAGTGGTCAACCTGGACCTAAGGGTGATCCAGGTAGACCAGGTTTCAGTTATCCAGGTCCAAGAGGTGCTCCAGGCGAGAAAGGTGAGCCTGGTCCACGAGGTCCAGAAGGCGGAAGAGGTGATTTCGGTTTGAAAGGCGAACCAGGTAGAAAGGGAGAGAAGGGCGAACCAGCTGATCCAGGTCCACCAGGCGAGCCTGGACCTAGAGGACCTAGAGGTGTACCTGGACCAGAAGGTGAACCTGGTCCACCATAATCTAGA

[0042] SEQ ID NO:4:

[0043]

[0044] SEQ ID NO:6:

[0045]

[0046] The present invention provides three expression vectors comprising the above nucleic acid molecules.

[0047] The vector may contain regulatory sequences (such as transcriptional and translational initiation and termination codons) that are specific for the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, and whether the vector is DNA-based or RNA-based, as appropriate.

[0048] The present invention provides three host cells, which contain the above nucleic acid molecules.

[0049] The host cell refers to a cell into which exogenous nucleic acid has been introduced, including the offspring of such cells. At present, microorganisms are often selected as host cells in the production and development system of recombinant collagen, because its fermentation system is simple to operate, has a short culture cycle, is suitable for mass production, has stable product quality and high safety, eliminates problems such as virus hidden dangers and immune rejection, and has become the preferred method for industrial production of recombinant collagen at home and abroad in recent years. In a specific embodiment, the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis. The host cells finally selected are Pichia pastoris X-33 and GS115.

[0050] The present invention provides a method for preparing the aforementioned recombinant collagen, which comprises the following steps:

[0051] The host cell is Pichia pastoris. After obtaining the genetically engineered Pichia pastoris, the host cell is used to express the target protein, and then the protein is separated and purified to obtain the desired protein.

[0052] The present invention does not impose any limitation on the expression mode, which can be confirmed as needed, for example, the expression is induced expression, and the inducer for induced expression can be, for example, methanol.

[0053] In a specific embodiment, methanol is added for induction culture at a temperature of 30° C. and a pH of 6.0, and the fermentation is terminated after 48 hours of induction.

[0054] The present application does not impose any limitation on the separation and purification method, which can be determined according to the method, for example, salting-out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method, or a combination of the above methods can be used.

[0055] The present invention also provides the use of the above-mentioned recombinant collagen, or the recombinant collagen encoded by the above-mentioned nucleic acid molecule, or the recombinant collagen expressed by the above-mentioned expression vector, or the recombinant collagen produced by the above-mentioned host cell in the preparation of a drug for treating diseases caused by abnormal proliferation of chondrocytes.

[0056] Example 1

[0057] Construction and expression of genetically engineered bacteria

[0058] According to the preference of Pichia pastoris codons, without changing the amino acid sequence, the gene encoding the recombinant type VI collagen fragment was codon optimized using codon optimization software, the optimized gene sequence was fully synthesized, and connected to the expression vector. The gene encoding the recombinant type VI collagen P0607 was inserted into the plasmid pPICZαA to obtain the recombinant plasmid pPICZαA-P0607, and the recombinant plasmid pPICZαA-P0607 was transformed into Pichia pastoris X33 to construct the above-mentioned strain X33-pPICZαA-P0607 for producing recombinant type VI collagen. The gene encoding recombinant type VI collagen P060702 was inserted into the plasmid pPIC9K to obtain the recombinant plasmid pPIC9K-P060702, and the recombinant plasmid pPIC9K-P060707 was transformed into Pichia pastoris GS115 to construct the strain GS115-pPIC9K-P060702 for producing the recombinant type VI collagen. The gene encoding recombinant type VI collagen P060703 was inserted into the plasmid pPIC9K to obtain the recombinant plasmid pPIC9K-P060703, and the recombinant plasmid pPIC9K-P060703 was transformed into Pichia pastoris GS115 to construct the strain GS115-pPIC9K-P060703 for producing the recombinant type VI collagen.

[0059] The recombinant plasmid was transformed into competent host cells by electroporation. The strain X33-pPICZαA-P0607 was screened for positive transformants on a YPD plate with Bolet resistance. The strains GS115-pPIC9K-P060702 and GS115-pPIC9K-P060703 were screened for positive transformants on a histidine-deficient MD plate; the positive transformants were then selected on a YPD plate with G418 resistance to screen for high-copy. The transformants on the YPD plate were then used as templates to design primers for PCR identification.

[0060] The correct transformant identified by PCR was inoculated into BMGY liquid culture medium, cultured at 30°C, 220rpm for about 24h until OD600 = 10-15, and collected by centrifugation at 8000×g, 4°C for 5min. The collected cells were then resuspended in BMMY culture medium until the initial OD600 was close to 1.0, and cultured at 30°C, 200rpm with shaking, and methanol was added every day with a final concentration of 1%; after fermentation for 72h, the fermentation supernatant was recovered by centrifugation at 8000×g, 4°C for 5min; SDS-PAGE analysis showed obvious specific bands, and the recombinant type VI collagen was collected.

[0061] Example 2

[0062] Purification of recombinant collagen

[0063] The supernatant collected by centrifugation was filtered through a hollow fiber filter to remove impurities, and then ultrafiltered to 10% of the initial volume.

[0064] Prepare the equilibrium buffer according to the isoelectric point of the protein: 20mmol / LPBS buffer (liquid A, pH 6.0), and use 20mmol / LPBS buffer + 1.0mol / L NaCl (liquid B, pH 6.0) as the eluent. Dilute the protein solution in the previous step with liquid A to prepare the loading solution, and load it on a 30mL MMC cation exchange chromatography column after filtration. Equilibrate the column with equilibrium buffer before loading. After loading, rinse 3 column volumes with liquid A, and then use liquid B for gradient elution at a flow rate of 8mL / min. Collect the eluted components and detect them with SDS-PAGE. The electrophoresis diagram of the purified protein is shown below. Figure 2 shown.

[0065] Ultrafiltration desalting: Concentrate to 20-30% of the initial volume through ultrafiltration, desalt, and then place in a -20℃ refrigerator for pre-freezing for 4 hours, and then transfer to a vacuum freeze dryer for freeze-drying. Collect the freeze-dried protein after 72 hours, and store the freeze-dried protein sample in a -80℃ refrigerator for later use.

[0066] Example 3

[0067] Mass spectrometry detection

[0068] Mass spectrometry analysis was performed to verify the recombinant type VI collagen P0607, P060702, and P060703. The target protein bands with apparent molecular weights of 31 kDa, 55 kDa, and 80 kDa were digested with trypsin, and then the peptide samples after digestion were analyzed using LC-MS / MS mass spectrometer. Finally, Thermo BioPharma Finder software was used to analyze the LCMS / MS data, and the algorithm results were used to determine whether the amino acid sequence of the sample was consistent with the theoretical sequence. The results show that the P0607 protein sequence matching rate is 99.1%, indicating that the protein with an apparent molecular weight of 31kDa is the P0607 protein to be expressed by the present invention; the P060702 protein sequence matching rate is 97.3%, indicating that the protein with an apparent molecular weight of 55kDa is the P060702 protein to be expressed by the present invention; the P060703 protein sequence matching rate is 97.3%, indicating that the protein with an apparent molecular weight of 80kDa is the P060703 protein to be expressed by the present invention. After the enzyme-cut samples were analyzed by LC-MS / MS, it can be seen from the mass spectrometry results that the enzyme cutting is relatively sufficient.

[0069] Example 4

[0070] In vitro cell proliferation assay

[0071] Immortalized human articular chondrocytes SV40T can be used as materials for osteoarthritis research. Immortalized cells can be cultured indefinitely in vitro, which enables researchers to conduct long-term experiments and observe changes in cells under different conditions, providing a continuous source of cells for the study of the chronic development process of osteoarthritis.

[0072] SV40T cells in logarithmic growth phase were cultured at 1×10 4 100 μL / well were inoculated in a 96-well tissue culture plate and cultured in a 5% CO2 biochemical incubator at 37°C for 1 day. The purified P0607, P060702 and P060703 proteins were dissolved in DMEM culture medium and sterilized by filtration. After incubation for 24 hours, the old culture medium was discarded, and 100 μL of fresh complete culture medium was added to the control group, while the experimental group was 100 μL of equal volumes of P0607 protein, P060702 and P060703 protein solution. Subsequently, the CCK8 method was used to verify cell proliferation, and the absorbance was measured at 450 nm. Figure 5 By applying different repetitive recombinant type VI collagen fragments to chondrocytes at different concentrations, the results showed that the P060703 protein at a concentration of 0.5 mg / mL had the best effect in promoting cell proliferation.

[0073] The relative cell viability was calculated as follows:

[0074]

[0075] Values ​​are mean ± SD (n = 3), compared with the model group * P<0.05, compared with the model group ** P<0.01, compared with the model group *** P<0.001, compared with the control group *** P<0.001.

[0076] The recombinant type VI collagen of the present invention has high affinity: recombinant collagen is a homogeneous collagen with high affinity to the human body; high safety: recombinant collagen is produced by genetic engineering technology and can be fermented in virus-free yeast, so there is no risk of carrying animal viruses; low immunogenicity: because it is human-derived collagen, its protein is homogeneous and is not easy to cause allergic reactions; high purity: recombinant collagen is a single collagen with fixed ingredients and a purity of up to 95%; good moisture retention and water solubility: recombinant collagen has high moisture retention and is soluble in water; suitable pH value: the pH value of recombinant collagen is between 6.7-7.5, which is close to neutral, while the pH value of animal-derived collagen is between 3.0-4.5, which is slightly acidic.

[0077] In addition, human cartilage regeneration is inherently inefficient, and current cell-based cartilage repair methods have the following problems: lack of chondrocytes, high donor site morbidity, and the production of poorly functional fibrocartilage rather than articular cartilage. The recombinant type VI collagen of the present invention can stimulate chondrocyte proliferation, and the soluble recombinant type VI collagen fragment can expand the scarce source of chondrocytes and may be used for autologous chondrocyte implantation.

[0078] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A recombinant type VI collagen, characterized in that: It comprises a protein gene fragment derived from the α2 chain of natural type VI collagen, and expresses proteins P0607, P060702 and / or P060703 having the activity of promoting chondrocyte proliferation; the amino acid sequence of P0607 is shown in SEQ ID NO:1, the amino acid sequence of P060702 is shown in SEQ ID NO:3, and the amino acid sequence of P060703 is shown in SEQ ID NO:

5.

2. A nucleic acid encoding the recombinant type VI collagen according to claim 1, characterized in that: The nucleic acid sequence encoding protein P0607 is shown in SEQ ID NO:2, the nucleic acid sequence encoding protein P060702 is shown in SEQ ID NO:4, and the nucleic acid sequence encoding protein P060703 is shown in SEQ ID NO:

6.

3. An expression vector comprising the nucleic acid according to claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector is pPICzαA.

5. A host cell introduced with the expression vector according to claim 3, characterized in that: The host cell is Escherichia coli, yeast or Bacillus subtilis, preferably Pichia pastoris.

6. A genetically engineered bacterium for producing the recombinant type VI collagen as claimed in claim 1, characterized in that: The host cell of P0607 is Pichia pastoris X33. The recombinant plasmid pPICZαA-P0607 was obtained by linearization with SalI enzyme, and then transferred into the host cells to obtain the X33-pPICZαA-P0607 genetically engineered bacteria; the host cells of P060702 and P060703 are both Pichia pastoris GS115. The recombinant plasmid pPIC9K-P060702 was obtained by linearization with SalI enzyme, and then transferred into the host cells to obtain the GS115-pPIC9K-P060702 genetically engineered bacteria; the recombinant plasmid pPIC9K-P060703 was obtained by linearization with SalI enzyme, and then transferred into the host cells to obtain the GS115-pPIC9K-P060703 genetically engineered bacteria.

7. The genetically engineered bacterium according to claim 6, characterized in that: When the recombinant plasmid pPICZαA-P0607 was synthesized, XhoI and XBaI recognition sites and a signal peptide recognition site were added to the 5' and 3' ends, respectively; When the recombinant plasmid pPIC9K-P060702 was synthesized, Xho I and EcoRI recognition sites and a signal peptide recognition site were added to the 5' and 3' ends, respectively; When the recombinant plasmid pPIC9K-P060703 was synthesized, Xho I and EcoR I recognition sites and a signal peptide recognition site were added to the 5' end and 3' end, respectively.

8. The genetically engineered bacterium according to claim 6, characterized in that: The shake flask fermentation conditions of the genetically engineered bacteria were as follows: induction temperature 30°C, induction time 48h, and methanol concentration 1%.

9. A method for producing the recombinant type VI collagen as claimed in claim 1, characterized in that: The method comprises culturing host cells and separating and purifying the cell culture, wherein the separation and purification adopts one or a combination of salting-out method, ultrafiltration method and gel filtration chromatography method.

10. Use of the recombinant type VI collagen as claimed in claim 1 in the preparation of food, health food, medicine or medical device for treating or preventing diseases caused by OA.

Images