Human-derived recombinant III-type collagen as well as preparation method and application thereof

Abstract

The embodiment of the invention discloses human-derived recombinant III-type collagen as well as a preparation method and application thereof. The amino acid sequence of the human-derived recombinant III-type collagen comprises a basic repetitive unit, and the amino acid sequence of the basic repetitive unit is as shown in SEQ ID NO.4. The amino acid sequence of the basic repetitive unit is as shown in SEQ ID NO.3. The amino acid sequence of the basic repetitive unit is as shown in SEQ ID NO.4. The amino acid sequence of the human recombinant III type collagen is shown as SEQ ID NO.3. The invention also discloses a preparation method of the human recombinant III type collagen. According to the invention, a human recombinant III-type collagen gene with the coding molecular weight of 60kD is designed, an escherichia coli expression strain is constructed and expressed, the yield of the human recombinant III-type collagen is increased through amplification culture on a fermentation tank, and the protein purity is improved by virtue of strategies such as ultrafiltration primary purification and affinity chromatography. The human-derived recombinant III-type collagen prepared by the invention avoids potential hidden dangers such as animal diseases or viruses and immunological rejection caused by a traditional extraction method, has excellent medical performance, can effectively control the production cost and the product quality by virtue of a fermentation preparation process, and is also easy to realize large-scale production.

Description

Technical Field

[0001] The present application relates to the field of biological genetic engineering technology, and in particular to a human recombinant type III collagen protein and a preparation method and application thereof. Background Art

[0002] Collagen has the characteristics of biodegradability, good biocompatibility, low immunogenicity, etc. It can promote cell proliferation and adhesion, and has the functions of promoting tissue repair and hemostasis. In addition, collagen can form collagen fibers and has certain mechanical properties. It is a good biomaterial and has been widely used in food, cosmetics, biomedical materials, pharmaceuticals and other fields.

[0003] At present, 28 types of collagen have been found, and different types of collagen have different functions. Generally, they are divided into two categories: one is fibroblastic collagen; the other is non-fibroblastic collagen; fibroblastic collagen includes type I, II, III, V, VI and XXVI collagen, and the rest are non-fibroblastic collagen. The most abundant type of collagen in the human body is type I collagen, accounting for more than 85%. Type I collagen is high in bone, skin, tendon and cornea, type II exists in cartilage, intervertebral disc and vitreous body, and type III exists in blood vessels, new skin and scar tissue.

[0004] At present, the sources of collagen mainly include extraction from animal tissues and expression through genetic engineering technology. Naturally extracted collagen is a mixture of collagens of various molecular weights. It is insoluble in water and has poor biocompatibility. Moreover, since it comes from animal tissues, it may have the risk of immunogenicity and potential pathogen infection. In addition, because type III collagen coexists with other types of collagen and has a low content, there are severe problems such as difficulty, low purity, and high cost in extracting type III collagen from animal tissues. The differences between individual animals will also lead to poor batch stability of collagen. The recombinant collagen obtained by genetic engineering technology is similar to natural collagen, with the advantages of good water solubility, no risk of virus transmission, low immune rejection reaction, and good batch consistency. Therefore, the design and development of excellent recombinant collagen is a problem that needs to be solved urgently. Summary of the invention

[0005] In order to solve the existing technical problems, the present application provides a human recombinant type III collagen and a preparation method and application thereof. The technical solution is as follows: In a first aspect, a human recombinant type III collagen is provided, wherein the amino acid sequence of the human recombinant type III collagen comprises a basic repeating unit, and the amino acid sequence of the basic repeating unit is shown in SEQ ID NO.4.

[0006] Furthermore, the amino acid sequence of the human recombinant type III collagen is shown in SEQ ID NO.3.

[0007] In a second aspect, a polynucleotide sequence encoding the human recombinant type III collagen described in the first aspect is provided, and the polynucleotide sequence is shown as SEQ ID NO.5.

[0008] In a third aspect, an expression vector comprising the polynucleotide sequence described in the second aspect is provided.

[0009] Furthermore, the expression vector is pET30a.

[0010] In a fourth aspect, a host cell comprising the expression vector described in the third aspect is provided.

[0011] Furthermore, the host cell includes: Escherichia coli, Pichia pastoris or Corynebacterium glutamicum.

[0012] In a fifth aspect, a method for preparing human recombinant type III collagen is provided, comprising the following steps: Synthesize the nucleotide sequence SEQ ID NO.5 encoding human recombinant type III collagen; The nucleotide fragment was connected to the pET30a plasmid through the multiple cloning site to obtain the recombinant plasmid pET30a; Take BL21 (DE3) competent cells, place them on ice to melt, add the recombinant plasmid pET30a, gently pipette to mix thoroughly, place on ice for 30-40 minutes, heat shock in a water bath at 42°C for 90 seconds, place on ice for 3-5 minutes, add room temperature LB liquid culture medium, shake and culture in a shaker, mix the bacterial solution and apply it to an ampicillin resistance plate, invert the plate, and culture at 37°C overnight; A single clone was picked from the plate, inoculated into an LB tube containing 100 μg / ml ampicillin, and cultured in a shaker. When OD600 reached 0.6-0.8, IPTG was added to the tube and cultured at 16°C for 20 hours or 37°C for 4 hours.

[0013] In a sixth aspect, a use of the human recombinant type III collagen described in the first aspect in the preparation of food, medicine, cosmetics, health products or medical devices is provided.

[0014] Furthermore, the medical device includes an artificial blood vessel, a hemostatic dressing, a skin wound repair material, a cartilage repair material or a medical cosmetic material.

[0015] The beneficial effects of the technical solution provided in the embodiments of the present application are as follows: the amino acid sequence of the human recombinant type III collagen of the present application includes a basic repeating unit, and the amino acid sequence of the basic repeating unit is shown in SEQ ID NO.4. The present application designs a human recombinant type III collagen gene encoding a molecular weight of 60kD, constructs an Escherichia coli expression strain and expresses it, increases the yield of human recombinant type III collagen by expanding the culture in a fermenter, and improves the protein purity by means of strategies such as ultrafiltration primary purification and affinity chromatography. The expression of human recombinant type III collagen prepared by the present application avoids potential hidden dangers such as animal diseases or viruses and immune rejection reactions brought about by traditional extraction methods, and has excellent medical properties. At the same time, its fermentation preparation process can effectively control production costs and product quality, and is also easy to achieve large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is a hydrophobicity analysis diagram of polypeptide 1 provided in Example 1 of the present application; Figure 2 This is a hydrophobicity analysis diagram of polypeptide 2 provided in Example 1 of the present application; Figure 3 This is a hydrophobicity analysis diagram of polypeptide 3 provided in Example 1 of the present application; Figure 4 This is the optimized recombinant vector map provided in Example 3 of the present application; Figure 5 This is the SDS-PAGE electrophoresis diagram of the optimized human recombinant type III collagen provided in Example 4 of the present application; Figure 6 This is a purification detection diagram of the recombinant bacteria product cultured in a 2L reactor provided in Example 6 of the present application; Figure 7 This is a diagram showing that human recombinant type III collagen peptide promotes cell proliferation and migration rate, as provided in Example 7 of the present application. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present application clearer, the implementation methods of the present application will be further described in detail below with reference to the accompanying drawings.

[0019] It should be clear that the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0020] When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. Instead, they are only examples of devices and methods consistent with some aspects of the present application as detailed in the attached claims.

[0021] In the description of this application, it should be understood that the terms "first", "second", "third", etc. are only used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence, nor can they be understood as indicating or implying relative importance. In the description of this application, unless otherwise specified, "plurality" means two or more.

[0022] Example 1 Design of human recombinant type III collagen peptide fragment Based on the characteristics of the Gly-XY tripeptide repeat sequence of the α1 chain of human type III collagen, the hydrophobicity analysis tool was used to screen out the Gly-XY sequence with low hydrophobicity. The antigen epitope prediction tool was used to screen the amino acids in the low antigenic determinant region to obtain the following three peptide sequences.

[0023] Polypeptide 1 (SEQ ID NO.1) GARGNDGARG SDGQPGPPGP PGPKGNDGAP GKNGERGGPG GPGPQGPPGK NGETGPQGPP GPTGPGGDKG DTGPPGPQGP RGSPGERGET GPPGPAGAPG QNGEPGGKGE RGAPGEKGEG GPPGPPGKDG TSGHPGPIGP PGPRGNRGER GSEGSPGHPG QPGPPGPPGA P Peptide 2 (SEQ ID NO.2) GARGNDGARG SDGQPGPPGP PGAKGEVGPA GSPGSNGAPG QRGEPGPQGH AGAQGPPGPP GINGSPGGKG EMGAAGERGA PGFRGPAGPN GIPGEKGPAG ERGAPGPAGP RGAAGEPGRD GVPGGPGMRG MPGSPGGPGS DGKPGPPGSQ GESGRPGPPG PSGPRGQPGP KGNDGAPGKN GERGGPGGPG PQGPPGKNGE TGPQGPPGPT GPGGDKGDTG PPGPQGTGGP PGENGKPGEP GPKGDAGAPG AKGDAGAPGE RGPPGPEGGK GAAGPPGLQG MPGERGGLGS PGPKGDKGEP GGPGADGVPG KDGPRGPTGP IGPPGPAGQP GDKGEGGAPG PRGSPGERGE TGPPGPAGAP GQNGEPGGKG ERGAPGEKGE GGPPGEPGRD GNPGSDGLPG RDGSPGGKGD RGENGSPGAP GAPGHPGPPG PVGPAGKSGD RGESGPAGSR GAPGPQGPRG DKGETGERGA AGIKGHRGFP GNPGAPGSPG PAGQQGPPGK DGTSGHPGPI GPPGPRGNRG ERGSEGSPGH PGQPGPPGPP GAP Polypeptide 3 (SEQ ID NO.3) GERGAPGFRG PAGPNGIPGE KGPAGERGAP GPAGPRGERG APGFRGPAGP NGIPGEKGPA GERGAPGPAG PRGERGAPGF RGPAGPNGIP GEKGPAGERG APGPAGPRGE RGAPGFRGPA GPNGIPGEKG PAGERGAPGP AGPRGERGAP GFRGPAGPNG IPGEKGPAGE RGAPGPAGPR GERGAPGFRG PAGPNGIPGE KGPAGERGAP GPAGPRGERG APGFRGPAGP NGIPGEKGPA GERGAPGPAG PRGERGAPGF RGPAGPNGIP GEKGPAGERG APGPAGPRGE RGAPGFRGPA GPNGIPGEKG PAGERGAPGP AGPRGERGAP GFRGPAGPNG IPGEKGPAGE RGAPGPAGPR GERGAPGFRG PAGPNGIPGE KGPAGERGAP GPAGPRGERG APGFRGPAGP NGIPGEKGPA GERGAPGPAG PRGERGAPGF RGPAGPNGIP GEKGPAGERG APGPAGPRGE RGAPGFRGPA GPNGIPGEKG PAGERGAPGP AGPRGERGAP GFRGPAGPNG IPGEKGPAGE RGAPGPAGPR GERGAPGFRG PAGPNGIPGE KGPAGERGAP GPAGPR The hydrophobicity of the three peptide sequences was analyzed using the ProtScale tool software. The results of the hydrophobicity analysis are as follows: Figure 1 , Figure 2 and Figure 3 As shown in Table 1, the hydrophilicity of the three amino acid sequences is significantly different. The protein properties were analyzed using the Expasy-ProtParam tool software, and the results are shown in Table 1.

[0024] Table 1 Protein characteristics of three amino acid sequences

[0025] As can be seen from Table 1 above, the protein of SEQ ID NO.1 has the highest polarity and the lowest hydrophobicity; the protein of SEQ ID NO.2 is the most unstable; and the protein of SEQ ID NO.3 has high stability, low polarity and high hydrophobicity.

[0026] The above three sequences were transformed into Escherichia coli respectively, and small-scale expression was carried out using conventional technology. The results were detected by SDS-PAGE and Western blot. The results are shown in Table 2.

[0027] Table 2 Expression results of three amino acid sequences

[0028] It can be seen from Table 2 above that under the same inoculation amount and culture conditions, the three proteins were all induced at 37°C for 4 hours, and SDS-PAGE detection showed that SEQ ID NO.3 had the highest expression level.

[0029] Finally, SEQ ID NO.3 was selected as the amino acid sequence of this application for recombinant vector construction. This application uses 36 amino acids (repeating unit SEQ ID NO.4: GERGAPGFRG PAGPNGIPGE KGPAGERGAP GPAGPR) for multiple repetitions, totaling 576 amino acids, and uses Expasy-ProtParam tool software and ProtScale tool software to analyze protein properties and hydrophobicity. The calculated molecular weight is 53324.59, the aliphatic index is 24.72, the hydrophilicity average value is -1.033, and the calculated instability index is 4.66, and the protein of SEQ ID NO.3 is classified as a stable protein.

[0030] The nucleotide sequence was determined using the online codon optimization tool https: / / sg.idtdna.com / pages / tools / codon-optimization-tool. The protein sequence of polypeptide 3 was input into the webpage; the desired expression host was selected; the restriction sites to be avoided were selected; after optimization, the nucleotide sequence encoding polypeptide 3, SEQ ID NO.5, was obtained, as shown below.

[0031] Example 2 Construction of human recombinant type III collagen recombinant plasmid There are three main types of plasmid vectors used: one is the pET-30a vector, which is used to construct the expression plasmid of Escherichia coli BL21 (DE3) recombinant protein; the second is the pPIC9K vector, which is used to construct the expression plasmid of Pichia pastoris recombinant protein; the third is the p19 series vector of the pBL1 replicon, which is used to construct the expression plasmid of Corynebacterium glutamicum recombinant protein; each plasmid is constructed according to the following process: A nucleotide sequence encoding human recombinant type III collagen (as shown in SEQ ID NO.5) was synthesized, with NdeI and XhoI multiple cloning sites at both ends of the sequence; the synthetic products were separated by agarose gel electrophoresis, and the target DNA fragment was recovered from them using Gel Extraction Kit D2500 (Omega Bio-Tek, USA); the synthetic fragment and pET-30a vector were digested with NdeI and XhoI restriction endonucleases, and the synthetic fragment and vector were purified by gel recovery; the synthetic fragment and vector were assembled using T4 ligase, and the reaction conditions were all 37°C for 90 min; the ligated products were transformed into DH5α or TOP10F competent cells, and plated and cultured overnight; after 24 hours, a single clone was picked for culture, and the plasmid was extracted using Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA), and verified by Sanger sequencing.

[0032] In the construction of pET-30a and p19 vectors, E. coli DH5α was used as the cloning host for plasmid construction. LB medium (containing 1% peptone, 0.5% yeast extract, and 1% sodium chloride per liter of medium) was used for transformation and bacterial amplification. When necessary, the antibiotic concentration added was 30μg / mL chloramphenicol or 50μg / mL kanamycin. E. coli TOP10F was used as the cloning host for plasmid construction in the construction of pPIC9K vector, and the transformation method was the same as that of E. coli DH5α.

[0033] Example 3 Construction of recombinant bacteria producing human recombinant type III collagen Construction of recombinant E. coli: Take out BL21 (DE3) competent cells from the ultra-low temperature refrigerator, thaw them on ice, and add 100 ng of plasmid ( Figure 4), gently pipette to mix thoroughly, place on ice for 30-40 minutes, heat shock in a water bath at 42°C for 90 seconds, place on ice for 3-5 minutes, add 100μl room temperature LB liquid culture medium, shake and culture in a shaker at 37°C, 200-250rpm for 60 minutes, mix the bacterial solution and apply it to the ampicillin resistance plate, turn the plate upside down, and culture at 37°C overnight. Pick a single clone for culture, use Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA) to extract the plasmid, and verify it by Sanger sequencing. Strains with correct plasmid sequencing are positive recombinant bacteria.

[0034] Construction of Pichia recombinant bacteria: Boil 1mL salmon sperm DNA 2mg / L for 5min and quickly cool on ice; centrifuge competent GS115 cells at 12000rpmin for 15s and discard the upper LiCl layer; add 240μL of 500g / L PEG-3350, 36μL of 1mol / L LiCl, 25μL salmon sperm DNA and 50μL linearized recombinant plasmid DNA (5-10μg) in sequence, mix vigorously in a vortex mixer for 1min to fully mix the cell pellet and the added solution; incubate at 30℃ for 30min. After heat shock in a 42℃ water bath for 20min, collect the cells by centrifugation at 8000rpmin for 10min, resuspend the cells in 200μl YPD medium, and shake culture at 30℃; add 20μL directly to the MD plate after resuspension, and culture at 30℃ for 2-4d until the colony diameter is 1mm. Single clones were selected for culture, and plasmids were extracted using Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA) and verified by Sanger sequencing. Strains with correct plasmid sequencing were considered positive recombinant bacteria.

[0035] Construction of recombinant bacteria of Corynebacterium glutamicum: Take out the competent cells of Corynebacterium glutamicum ATCC13032 from the ultra-low temperature freezer, place them on ice to melt, add 100 ng of plasmid, gently pipette to mix thoroughly, let stand on ice for 15 minutes, transfer the competent cells to an electroporation cup (0.1 cm gap, Bio-Rad) frozen at -20°C, and use a BTX electroporator for electroporation with parameters of 1.8 kv, 200 Ω, 25 μF (5 ms). The electroporated cells were transferred to an Eppendorf tube containing 1 mL of LBHis liquid culture medium (preheated at 46°C), incubated at 46°C for 6 minutes, and then renatured at 30°C for 1.5 hours at 200 rpm. Take an appropriate amount of bacterial solution (the electroporation efficiency is about 10 5cfu / μg DNA), evenly spread on LBHis agar (15g / L) plates containing the corresponding antibiotics, cultured overnight in a 30°C incubator, and colonies formed on plates containing chloramphenicol after 36 hours. Pick a single clone for culture, extract the plasmid using Plasmid Mini Kit ID6943 (Omega Bio-Tek, USA), and verify by Sanger sequencing. Strains with correct plasmid sequencing are positive recombinant bacteria.

[0036] Example 4 Expression of human recombinant type III collagen in Escherichia coli The positive recombinant bacteria were inoculated into 4-6 ml LB test tubes containing 100 μg / ml ampicillin, and cultured in a shaker at 37°C and 250 rpm. When OD600 reached 0.6-0.8, 0.5-1 mM IPTG was added to two test tubes, respectively, and cultured at 16°C for 20 hours or 37°C for 4 hours. The last test tube was used as a negative reference. The expression and solubility of the protein were detected by SDS-PAGE. The results are shown in Figure 5 In the figure, 1 is protein marker, 2 is BSA (1μg), 3 is BSA (2μg), 4 is uninduced whole cells, 5 is whole cells induced at 16℃ for 20h, 6 is whole cells induced at 37℃ for 4h, 7 is uninduced cell lysis supernatant, 8 is cell lysis supernatant induced at 16℃ for 20h, 9 is cell lysis supernatant induced at 37℃ for 4h, 10 is uninduced cell lysis precipitate, 11 is cell lysis precipitate induced at 16℃ for 20h, and 12 is cell lysis precipitate induced at 37℃ for 4h. Figure 5 It can be seen that this human recombinant type III collagen protein only expresses recombinant collagen protein after adding IPTG inducer. This human recombinant type III collagen protein is mainly expressed in the form of inclusion bodies when induced at 16°C, and is expressed in both the supernatant and inclusion bodies of the cell lysate when induced at 37°C, and the protein expression level after 4h of induction at 37°C is significantly higher than that after 20h of induction at 16°C.

[0037] Example 5 Fermentation of human recombinant type III collagen peptide Seed liquid culture: 1-2% of the glycerol bacteria was inoculated into 10ml LB, cultured for 8-10h, and 5% was inoculated into 3 bottles of 100ml LB overnight. The OD600 was 8-10 the next day.

[0038] The 2L fermentation system (medium composition see Table 3) was inoculated at 10% inoculation volume, and the specific fermentation parameters were 37°C, initial speed 700-750rpm, ventilation 5L / h, pH7.0-7.2. DO% was controlled at 50-60% before induction and 30-40% at the beginning of induction. When the OD value was between 1-2, nitrogen source peptone was added at 400-500ml / h (see Table 4), and a small amount of carbon source glycerol was added in the late fermentation period (see Table 4). 0.5-1mM IPTG was induced at 37°C for 2.5-4h after inoculation for 4-5h, and the bacteria were harvested when the OD value was about 34-40.

[0039] Table 3 Fermentation medium composition

[0040] Table 4 Feed medium / nitrogen source-carbon source

[0041] Example 6 Purification of human recombinant type III collagen peptide After the cells were subjected to low-temperature ultrasonic disruption, the supernatant of the disrupted liquid was initially purified by ultrafiltration centrifuge tube and filtered through a 0.22μm fiber filter membrane. Affinity chromatography AKTA (mixed buffer: 0.5mol / L NaCl, 20-30mmol / L phosphate buffer, 40mmol / L imidazole, pH7.4; eluent: 0.5mol / LNaCl, 20-30mmol / L phosphate buffer, 0.5mol / L imidazole, pH7.4) was performed using a HisTrap FF11.2mL nickel column by linear elution, and the eluent was collected. The elution yield was approximately 80.64OD, and the expression level detected by electrophoresis was approximately 94.5mg / L, with a purity of approximately 88-92%. Results are shown in Figure 6 , where 1 is protein marker, 2 is sample (10μl), 3 is flow-through (10μl), 4 is elution (10μl), 5 is blank, 6 is sample (20μl), 7 is flow-through (20μl), 8 is elution (20μl), 9 is BSA (1μl), and 10 is BSA (2μl). Figure 6 It can be seen that the protein concentration in the sample of lane 2 is low, and the target protein cannot be observed with a sample load of 10μl; no target protein is seen in the flow-through of lane 3, while the target protein is shown in the eluted sample of lane 4, indicating that the collagen column has a good effect and the sample is concentrated and purified after passing through the nickel column; lanes 6, 7 and 8 have a sample load of 20μl, which well repeats the experimental results of lanes 2, 3 and 4; the protein amount of BSA in lanes 9 and 10 can roughly quantify the protein content of the eluted samples in lanes 4 and 8.

[0042] Example 7 Biological Activity Detection of Human Recombinant Type III Collagen Peptide Take out the cultured L929 cells from the incubator, discard the old solution, add 1.5 ml of trypsin to digest, discard the trypsin after the cells shrink and become round, add 5 ml of complete culture medium to resuspend the cells, and adjust the cell density to 1×10 after counting. 5 cell / ml, inoculate into a 6-well plate, 2ml / well, so that the 6-well plate can be 100% covered overnight. The next day, use the tip of the gun to scratch against a ruler, wash the cells 3 times with PBS, and remove the scratched cells. Dilute the human recombinant type III collagen peptide to 10mg / ml with serum-free culture medium and add it to the 6-well plate. The blank control group only needs to add an equal amount of serum-free culture medium and culture in a 37℃, 5% CO2 incubator. Take points every 24 hours, observe and take pictures. See the results. Figure 7 .Depend on Figure 7 It can be seen that compared with 0h, cells in both the blank control group and the experimental group proliferated at 24h; and cells in the experimental group added with this human recombinant type III collagen proliferated significantly more than the blank control group at 24h. This experimental result shows that the addition of this human recombinant type III collagen enhances cell activity.

[0043] The amino acid sequence of the human recombinant type III collagen of the present application includes a basic repeating unit, and the amino acid sequence of the basic repeating unit is shown in SEQ ID NO.4. The present application designs a human recombinant type III collagen gene encoding a molecular weight of 60kD, constructs an Escherichia coli expression strain and expresses it, increases the yield of human recombinant type III collagen by expanding the culture in a fermenter, and improves the protein purity by means of strategies such as ultrafiltration primary purification and affinity chromatography. The expression of human recombinant type III collagen prepared by the present application avoids potential hidden dangers such as animal diseases or viruses and immune rejection reactions brought about by traditional extraction methods, and has excellent medical properties. At the same time, its fermentation preparation process can effectively control production costs and product quality, and is also easy to achieve large-scale production.

[0044] The above is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A human recombinant type III collagen, characterized in that: The amino acid sequence of the human recombinant type III collagen includes a basic repeating unit, and the amino acid sequence of the basic repeating unit is shown in SEQ ID NO.4; the amino acid sequence of the human recombinant type III collagen is shown in SEQ ID NO.

3.

2. A polynucleotide sequence encoding the human recombinant type III collagen as claimed in claim 1, characterized in that: The polynucleotide sequence is shown as SEQ ID NO.

5.

3. An expression vector comprising the polynucleotide sequence according to claim 2, characterized in that: The expression vector is pET30a.

4. A host cell comprising the expression vector according to claim 3, characterized in that: The host cell is: Escherichia coli, Pichia pastoris or Corynebacterium glutamicum.

5. A method for preparing human recombinant type III collagen, characterized in that: The following steps are involved: Synthesize the nucleotide sequence SEQ ID NO.5 encoding human recombinant type III collagen; The nucleotide fragment was connected to the pET30a plasmid through the multiple cloning site to obtain the recombinant plasmid pET30a; Take BL21 (DE3) competent cells, place them on ice to melt, add the recombinant plasmid pET30a, gently pipette to mix thoroughly, place on ice for 30-40 minutes, heat shock in a water bath at 42°C for 90 seconds, place on ice for 3-5 minutes, add room temperature LB liquid culture medium, shake and culture in a shaker, mix the bacterial solution and apply it to an ampicillin resistance plate, invert the plate, and culture at 37°C overnight; A single clone was picked from the plate, inoculated into an LB tube containing 100 μg / ml ampicillin, and cultured in a shaker. When OD600 reached 0.6-0.8, IPTG was added to the tube and cultured at 16°C for 20 hours or 37°C for 4 hours.

6. Use of the human recombinant type III collagen as claimed in claim 1 in the preparation of food, medicine, cosmetics, health products or medical devices.

7. The use according to claim 6, characterized in that: The medical devices include artificial blood vessels, hemostatic dressings, skin wound repair materials, cartilage repair materials or medical cosmetic materials.

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