Recombinant III-type human-like collagen as well as coding gene and expression method thereof

Abstract

The invention discloses a recombinant III-type human-like collagen as well as a coding gene and an expression method thereof, and the method comprises the following steps: S1, carrying out sequence optimization by taking a human III-type collagen sequence as a template to obtain a recombinant III-type human-like collagen gene sequence; designing PCR (Polymerase Chain Reaction) amplification upstream and downstream primers according to the gene sequence, and amplifying the gene sequence of the recombinant III type human-like collagen; s2, carrying out double enzyme digestion on the recombinant III type human-like collagen gene and a plasmid, and connecting to obtain a recombinant plasmid; s3, linearizing the recombinant plasmid, transforming the linearized recombinant plasmid into agrobacterium, and screening; and S4, infecting lettuce with agrobacterium, culturing to express protein, freezing the infected lettuce with liquid nitrogen, grinding, extracting total plant protein, and purifying to obtain the recombinant III-type human-like collagen. The recombinant III type human-like collagen is expressed through lettuce, so that the method is safe and efficient, and has sufficient market value and important practical significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of recombinant collagen, and in particular, relates to a recombinant type III human collagen and a coding gene and an expression method thereof. Background Art

[0002] Collagen is an extremely important structural protein in connective tissue. Its biological activity enables it to participate in cell migration, differentiation and reproduction, giving connective tissue mechanical strength. In addition, it can promote cell growth and has properties such as hemostasis, biocompatibility, and biodegradability. Collagen is widely used in medical materials and drugs, beauty cosmetics, health products, and various practical industries. So far, more than 30 types of collagen chain coding genes have been discovered, which can form more than 16 types of collagen molecules, and the common types are I, II, III, V, and XI. Type III collagen (CLO3A1) has a strong synthesis ability and can differentiate stem cells from bone marrow into fibroblasts that help repair in wound repair. Therefore, in biomedical engineering, type III collagen is more widely used in repairing tissue wounds, supporting cell reorganization, improving skin condition, and enhancing skin elasticity and tenderness.

[0003] At present, the main method for preparing collagen in industry is the traditional extraction method, that is, using animal tissues such as pig skin, cow skin, donkey skin, etc. to treat with acid, alkali, salt, and enzyme to obtain collagen. Although the collagen prepared from animal sources is simple in operation and has a high recovery rate, due to the differences in individual animals, the stability of collagen in the same batch is very poor, the solubility is poor, and the processability is weak. The prepared protein is water-insoluble collagen, and some collagen activity is lost, which has safety hazards in application. The process fails to completely remove non-collagen donor proteins and biological factors, and the extracted products also have the risk of carrying animal-derived viruses. Direct application to the human body will produce immune rejection reactions, which greatly limits its application range in the biomedical field. Natural collagen has a high molecular weight and has certain post-translational modifications, catalytic cross-linking, and self-assembly processes. As a result, the cost of large-scale preparation of complete recombinant collagen is very high, so the current recombinant human-like collagen products have been modified to a certain extent in the original collagen sequence to retain some functions of natural collagen. Microorganisms are often used in the prior art to obtain collagen, but they have the following shortcomings: the DNA sequences expressing collagen are all natural coding sequences in the human genome (generally obtained by reverse transcription), but there are certain differences between microorganisms and humans in the transcription and translation of genes, especially in the preference of codons. Using the natural coding DNA sequence of human collagen to express it in microorganisms will result in low translation efficiency due to codon preference, mRNA structure, etc.

[0004] Therefore, it is of great value and significance to provide a recombinant type III human collagen protein that can be expressed safely and efficiently and a method for expressing the same. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant type III human collagen protein and its encoding gene and expression method.

[0006] To solve the above problems, the technical solution adopted by the present invention is: Technical theme 1: The present invention provides a recombinant type III human collagen, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] Technical theme 2: The present invention provides a gene encoding the recombinant type III human collagen as described in the first technical subject, and the gene sequence is shown in SEQ ID NO.1.

[0008] As a further embodiment of the present invention, the recipient plant expressing the gene is lettuce.

[0009] Technical Theme 3: The present invention provides a method for expressing recombinant type III human collagen, the steps of which are as follows: S1: Using the human type III collagen sequence as a template, sequence optimization is performed to obtain a recombinant type III human-like collagen gene sequence; based on the obtained gene sequence, upstream and downstream primers for PCR amplification are designed to amplify the recombinant type III human-like collagen gene sequence to obtain a recombinant type III human-like collagen gene; S2: double-digest the obtained recombinant type III human collagen gene and the vector, and connect them to obtain a recombinant plasmid; S3: The obtained recombinant plasmid is linearized and then transformed into Agrobacterium, and Agrobacterium containing the recombinant plasmid is obtained after screening; S4: Infect lettuce with the Agrobacterium containing the recombinant plasmid obtained in S3, and culture it to express recombinant type III human collagen. The infected lettuce is frozen with liquid nitrogen and then ground to extract total plant protein, and the recombinant type III human collagen is obtained after purification.

[0010] As a further embodiment of the present invention, the sequence optimization is to optimize the human type III collagen sequence according to the codon characteristics of lettuce, and eliminate the restriction sites of EcoR I, Not I, Hind III, and EcoR V in the human type III collagen gene sequence by synonymous conversion, and at the same time remove 2 CAACCAGGCCCTCCTGG splicing sites and 3 CTGCTGGTCCTCCTGG splicing sites.

[0011] As a further embodiment of the present invention, the upstream primer sequence for PCR amplification is shown as SEQ ID NO.2; the downstream primer sequence for PCR amplification is shown as SEQ ID NO.3.

[0012] As a further embodiment of the present invention, the plasmid is pCAMBIA3300; the restriction endonucleases selected for the double enzyme digestion are EcoR I and Hind III.

[0013] As a further embodiment of the present invention, the Agrobacterium is GV3101.

[0014] As a further embodiment of the present invention, the screening in S3 is carried out using kanamycin-resistant LB solid culture medium.

[0015] As a further embodiment of the present invention, the purification operation is: adding the obtained plant total protein to a balanced gravity column, eluting the impurities, and then gradient eluting the recombinant type III human collagen with an eluent having a series of imidazole concentration gradients, collecting the eluent flowing out in sections, and obtaining purified recombinant type III human collagen.

[0016] The beneficial effects of adopting the above technical solution are: 1. The preparation method provided by the present invention uses lettuce as the receptor plant, and the obtained recombinant type III human collagen is safer than the protein extracted from conventional animals, and the toxicity of the obtained collagen is effectively reduced. It can be used in biomedical materials, beauty cosmetics or skin care products, etc.

[0017] 2. The present invention performs sequence optimization without changing the amino acid sequence of human type III collagen to ensure normal translation, and optimizes the rare codons that are not commonly used in the original sequence into common codons with high translation efficiency in the host, thereby increasing the expression level of soluble recombinant type III human collagen.

[0018] 3. The recombinant type III human-like collagen obtained by the preparation method provided by the present invention was analyzed by the Bioinformatics Online website (http: / / www.novopro.cn / tools / translate.html). The results showed that the recombinant type III human-like collagen obtained by the present invention had a triple helical structure, and the pyrolysis test results showed that it was stable. The protein immunoblotting results in the protein immunoassay showed that the protein had a complete protein structure and was connected with histidine and Srtep-tagⅡ specific affinity purification tags, so that high-purity products were easy to obtain and industrial large-scale production was easy to achieve. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is a schematic diagram of the structure of the recombinant plasmid pCAMBIA3300-cola3 obtained in Example 1 of the present invention; Figure 2 The protein triple helical structure prediction result diagram obtained in step S1 of Example 1 of the present invention; Figure 3 The agarose electrophoresis gel result diagram of the recombinant plasmid pCAMBIA3300-cola3 and the linearized recombinant plasmid pCAMBIA3300-cola3 obtained in step S2 of Example 1 of the present invention; wherein lane M is a protein molecular weight marker, lanes 1 and 3 are the electrophoresis results of the recombinant plasmid pCAMBIA3300-cola3, and lanes 2 and 4 are the electrophoresis results of the linearized recombinant plasmid pCAMBIA3300-cola3; Figure 4 This is a graph showing the SDS-PAGE electrophoresis detection result of the centrifuged supernatant of the total protein of the transgenic lettuce plant in Test Example 1 of the present invention; wherein lane M is a protein molecular weight marker, and lanes 1-8 correspond to the SDS-PAGE electrophoresis detection results of samples 1, 2, 3, 4, 5, 6, 7, and 8, respectively; Figure 5 The figure is a WB result diagram of the anti-Srtep-tagII antibody and anti-his antibody detection of the recombinant collagen obtained in Example 1 of the present invention; wherein, lane M is a protein molecular weight marker, lane 1 is a positive control and Srtep-tagⅡ antibody, his antibody, lane 2 is recombinant type III collagen and Srtep-tagⅡ antibody, and lane 3 is recombinant type III collagen and his antibody; Figure 6 The figure is a diagram of the SDS-PAGE electrophoresis result of the supernatant purification obtained in step S4 of Example 1 of the present invention; wherein, lane M corresponds to the protein molecular weight marker, lane 1 is the washing solution, lanes 2-10 are 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 50mM, 80mM, and 100mM imidazole eluents, respectively, and lane 11 is a protein concentrated sample; Figure 7 This is a graph showing the stability test results of the recombinant type III human collagen obtained in Example 1 of the present invention; wherein, lane M is a protein molecular weight marker, lane 1 is a non-pyrolyzed control protein sample, and lanes 2, 3, and 4 are pyrolyzed protein samples at 33°C, 38°C, and 42°C, respectively. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is clearly and completely described below in conjunction with specific embodiments.

[0021] Example 1 S1. Acquisition of recombinant collagen gene: (1) Sequence design: The amino acid sequence of NP_000081.2 published in the NCBI database (https: / / www.ncbi.nlm.nih.gov) was used as a template. After comparison with the gene of human type III collagen α3 chain with sequence number NM_000090.4 accessed from Genebank, it was confirmed that the obtained protein sequence was consistent. On the basis of retaining the gene sequence of the amino acid conservative domain in the human type III collagen α3 chain gene, the human type III collagen α3 chain protein sequence was optimized according to the codon characteristics of lettuce. At the same time, the synonymous conversion method was used to eliminate EcoR I (GAATTC), Not I (GCGGCCGC), Hind III (AAGCTT) and EcoR V (GATATC) restriction site was removed, and two CAACCAGGCCCTCCTGG splicing sites and three CTGCTGGTCCTCCTGG splicing sites were removed; the codon adaptation index of the adjusted recombinant type III human collagen gene sequence in lettuce was 0.81, the GC content was 0.509, and the optimal codon frequency was also adjusted, and finally the recombinant type III human collagen gene sequence was obtained: His-cola3 gene sequence, whose nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The adjusted His-cola3 gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd.; the obtained amino acid sequence was analyzed by the Bioinformatics Online website (http: / / www.novopro.cn / tools / translate.html), and the results are as follows Figure 2 The result diagram shows that the amino acid sequence of the obtained recombinant type III human collagen has a triple helical structure.

[0022] (2) Primer design: Based on the His-cola3 gene sequence and in accordance with the primer design principles, snapgene 7.0 software was used to design gene PCR amplification primers. A 6×His tag and a Srtep-tagII purification tag were added to the reverse amplification primer. An EcoR I restriction site was added to the 3' end of the upstream primer, and a Hind III restriction site was added to the 5' end of the downstream primer. The resulting upstream and downstream primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0023] (3) Mix 10 μL 2×Fid Cycle Fast high-fidelity PCR Mix enzyme, 0.5 μL upstream primer, 0.5 μL downstream primer, and 0.5 μL His-cola3 DNA, add double distilled water to make up to 20 μL, put it into the PCR instrument, set the reaction conditions and run the program. The reaction conditions are shown in Table 1 below to obtain the His-cola3 gene PCR product.

[0024] Table 1 High-fidelity PCR reaction conditions for His-cola3 gene sequence S2. Construction of recombinant plasmid: (1) Double digestion of His-cola3 gene: Add 250U EcoR I, 300U Hind III and 5μL 10×rCutSmart™ buffer to 1μg His-cola3 gene PCR product, add sterile water to a total volume of 50μL; react at 37℃ for 15 minutes; react at 65℃ for 20 minutes to inactivate EcoR I enzyme, and react at 80℃ for 20 minutes to inactivate Hind III enzyme. The resulting product is the His-cola3 gene fragment with sticky ends; (2) Linearization of vector plasmid pCAMBIA3300 by double enzyme digestion: Add 250U EcoR I, 300U Hind III and 5μL 10×rCutSmart™ buffer to 1μg plasmid pCAMBIA3300, add sterile water to a total volume of 50μL; react at 37°C for 15 minutes to obtain the linearized vector plasmid pCAMBIA3300; (3) 25 ng of linearized vector plasmid pCAMBIA3300 and 75 ng of His-cola3 gene fragment were connected by T4 DNA enzyme and SolutionI ligation kit (Shanghai Biotech Co., Ltd., D7009S) to obtain the recombinant collagen expression vector: recombinant plasmid pCAMBIA3300-cola3. The results were verified by agarose gel electrophoresis. Figure 3 As shown; (4) Add 250U EcoR I and 300U Hind III to 10μL 10×rCutSmart™ buffer and 10μg recombinant plasmid pCAMBIA3300-cola3, and finally add sterile water to a total volume of 100μL; react at 37℃ for 1 hour to obtain linearized recombinant plasmid pCAMBIA3300-cola3; verify by 1.0% agarose gel electrophoresis, the results are as follows Figure 3 As shown, the linearized recombinant plasmid was recovered by gel recovery kit; (5) Add 10 μL of linearized pCAMBIA3300-cola3 recombinant plasmid to 100 μL of E. coli competent cells, mix gently in a clean bench, and continue to pre-cool on ice for 30 min; heat shock at 42°C for 1 min and pre-cool on ice for 2 min to seal the recombinant plasmid in the competent cells, and then inoculate into 100 mL of kanamycin-resistant LB liquid culture medium to obtain recombinant bacterial liquid; place the bacterial liquid at 37°C and 200 rpm for 16 h, and then use a plasmid extraction kit to extract the plasmid of the recombinant bacteria to complete the plasmid expansion culture; Kanamycin-resistant LB liquid medium: add 4 g of LB beef extract medium powder to 100 mL of water, sterilize at 121°C for 20 minutes, and after the temperature drops to 60°C, add 100 µL of kanamycin antibiotics with a final concentration of 50 mg / L in a clean bench; S3. Preparation of engineered bacteria expressing recombinant collagen: (1) Double enzyme digestion: Add 250U EcoR I and 300U Hind III to 10μL 10×rCutSmart™ buffer and 10μg recombinant plasmid pCAMBIA3300-cola3, and finally add sterile water to a total volume of 100μL; react at 37℃ for 1 hour to obtain linearized recombinant plasmid pCAMBIA3300-cola3; verify by 1.0% agarose gel electrophoresis, the results are as follows Figure 3 , gel recovery kit to recover the linearized recombinant plasmid; (2) Transformation: Add 10 μL of linearized pCAMBIA3300-cola3 recombinant plasmid to 100 μL of Agrobacterium GV3101 competent cells, mix gently in a clean bench, and continue to pre-cool on ice for 30 min; heat shock at 42°C for 1 min, and pre-cool on ice for 2 min to block the recombinant plasmid in the competent cells; (3) Add 800 μL of LB liquid medium to 100 μL of transformed Agrobacterium, gently blow and mix, transfer to a 1.5 mL centrifuge tube, and culture at 30°C for 3 h; centrifuge at 4000 rpm for 3 min, spread the suspension on kanamycin-resistant LB solid medium, spread 100 μL of bacterial suspension on each plate, let stand at room temperature for 10 min, and culture inverted at 30°C for 2 days until single colonies appear; LB liquid medium: add 4 g of LB beef extract medium powder to 100 mL of water and sterilize at 121°C for 20 minutes; Kanamycin-resistant LB solid medium: add 4g of LB beef extract medium powder and 4g of agar powder (40g / L) to 100mL of water, sterilize at 121℃ for 20 minutes, and after the temperature drops to 60℃, add 100µL of kanamycin antibiotic with a final concentration of 50mg / L in a clean bench; S4. Obtaining recombinant collagen: (1) Take 100 μL of the single colony obtained from S3 and add it to 5 mL of LB liquid medium. Incubate at 30°C for 48 hours. The next day, inoculate 1 mL of the bacteria into 50 mL of kanamycin-resistant solid medium and add 100 μM acetosyringone to obtain the infection solution. (2) Adjust the OD of the bacteria in the UV spectrophotometer 600 value, making it OD 600 The value is between 1.0 and 1.5. Collect the bacterial precipitate in a low-temperature centrifuge at 5000 g for 15 minutes, resuspend the bacterial cells in a resuspension solution (10 mM magnesium chloride, 10 mM MES and 100 μM acetosyringone), and let it stand at room temperature for 3 hours to completely dissolve; (3) Healthy lettuce plant materials were immersed in the Agrobacterium resuspension solution, and the air pressure was set to -0.1 MPa as the infection condition, and the negative pressure treatment was performed for 5 minutes; after the treatment, the plant materials were treated in the dark for 24 hours, and then cultured under normal light to obtain transgenic lettuce; the transgenic lettuce was collected, wrapped in tin foil, and quickly frozen in liquid nitrogen and temporarily stored in a -80°C freezer; the software was used to predict the properties and state of the protein expressed by pCAMBIA3300-cola3, and it was found that the protein was mainly a soluble recombinant protein, which was present in the supernatant of the solution. Therefore, the supernatant needs to be collected when extracting protein; take out the frozen transgenic lettuce leaves, pour liquid nitrogen into a mortar, add the lettuce leaves to grind, control the grinding speed during the grinding process to prevent liquid nitrogen from splashing, if the liquid nitrogen evaporates during the grinding process, re-add liquid nitrogen, continue grinding until the leaves are powdery, use a plant protein extractant (Bebo Bio, BB-3183) to extract plant total protein, then centrifuge at 4°C and 5000rpm for 15min, collect the supernatant, and obtain the transgenic lettuce total protein extract; (4) Ni NTA Beads gravity method was used to purify proteins. The imidazole concentration gradient was set to 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 50mM, 80mM, and 100mM Elution Buffer. The imidazole concentration was adjusted according to the protein purity and elution state. The principle of low-concentration imidazole buffer for loading and high-concentration imidazole buffer for elution was followed. Preparation of lysis buffer: 50 mM NaH per liter 2 PO 4 , 300 mM NaCl, 10 mM imidazole, adjust pH to 8.0 with NaOH, filter with 0.45 μM filter membrane, and store at 4 °C; Prepare Wash Buffer: 50 mM NaH per liter 2 PO 4, 300 mM NaCl, 20 mM imidazole, adjust pH to 8.0 with NaOH, filter with 0.45 μM filter membrane, and store at 4 °C; Prepare Elution Buffer: 50 mM NaH per liter 2 PO 4 , 300 mM NaCl, imidazole (the concentrations were set to 25 mM, 30 mM, 40 mM, 50 mM, 80 mM, and 100 mM, respectively), adjust the pH to 8.0 with NaOH, filter with a 0.45 μM filter membrane, and store at 4 °C; The pre-filled gravity column (Ni NTA Beads 6FF gravity column, Tiandirenhe) is balanced with 5 times the volume of lysate so that the filler and the protein are in the same buffer system, and the process is repeated 3 times; after the transgenic lettuce total protein extract obtained in step (3) is added to the gravity column, it is kept in the column for at least 2 minutes to fully contact with the medium and enhance the binding efficiency of the column; then, it is washed with 15 times the volume of Wash Buffer to remove non-specifically adsorbed impurities; 10 times the volume of Elution Buffer with different imidazole concentrations is used for elution from low concentration to high concentration to obtain purified recombinant type III human-like collagen, and the eluate is collected in sections, and each eluate is collected separately and detected separately, and the optimal imidazole concentration for elution of the plant total protein extract obtained by the present invention is 50 mM; after the above operation, a purified sample of recombinant type III human-like collagen is obtained; (5) The 50 mM imidazole eluate obtained in step (4) was purified by Thermo Scientific TM Pierce TM The protein was concentrated using a protein concentrator tube (Thermo Fisher Shanghai Instrument Co., Ltd., model 88536) to obtain a concentrated solution of recombinant type III human collagen; (6) The washing solution of step (4), the elution solution of step (4) with different imidazole concentration gradients and the concentrated solution of step (5) were subjected to SDS-PAGE electrophoresis. The results were as follows: Figure 6 As shown in the figure, it can be seen that in the electrophoresis result diagram, the band width of the elution solution with a concentration of 50 mM imidazole is wider than the band width of the elution solution with other imidazole concentrations, indicating that the protein purification effect of the elution solution with 50 mM imidazole is the best; after concentration, the electrophoresis result of the concentrated solution in step (5) shows a specific band.

[0025] Test Example 1: Molecular Weight Test of Recombinant Type III Human Collagen The total protein extract of transgenic lettuce collected in step S4 of Example 1 was divided into samples 1, 2, 3, 4, 5, 6, 7, and 8 for SDS-PAGE detection. The detection method was carried out according to the method of J. Sambrook et al., Molecular Cloning Experiment Guide 3rd Edition. The results are as follows: Figure 4 As shown; (1) Sample processing: Collect the total protein extract of transgenic lettuce prepared in step S4 of Example 1, add loading buffer (18 mol / L urea, 50% glycerol, 100 nmol / L Tris-HCl (pH 8.3), 0.02% bromophenol blue, sodium dodecyl sulfate), mix well, boil in a boiling water bath for 10 min, centrifuge at 12000 rpm at room temperature, and cool naturally before use; (2) Prepare SDS-PAGE gel using the SDS-PAGE Gel Preparation Kit (12%) from Yisheng Biotechnology Co., Ltd. Load the sample after solidification and run electrophoresis at 120 V for 120 min until the bromophenol blue band reaches the bottom of the gel.

[0026] The experimental results showed that a specific protein band appeared at around 200 KDa in samples 1-8, which was in line with expectations, indicating that recombinant type III human collagen was successfully expressed in the transgenic lettuce. It can be seen that the total protein extract prepared by step S4 contains recombinant type III human collagen with a molecular weight of about 200 KDa.

[0027] Test Example 2: Recombinant human type III collagen immunoblot test The purified samples of recombinant type III human collagen obtained in Example 1 were tested with Srtep-tagII antibody and his antibody, respectively, and human type III collagen (extracted from human placenta) (Sigma, 9007345) was used as a positive control. The results are as follows: Figure 5 As shown, lanes 2 and 3 are WB images of Srtep-tagII antibody and his antibody, respectively. The band of about 180 kDa can be recognized by both antibodies, proving that it is a complete recombinant protein with 6×His and Srtep-tagII double tags.

[0028] Test Example 3 Stability test of recombinant type III human collagen (1) Extraction of total soluble protein from lettuce: 400 mg of the transgenic lettuce leaves obtained in Example 1 were ground in 2 mL of 50 mM Tris-HCl buffer (pH = 7.5), centrifuged at 13,000 rpm for 10 minutes, and the supernatant was collected; the total soluble protein from the transgenic lettuce obtained in Example 1 was extracted to prepare three groups of samples, which were labeled as samples 1, 2, and 3 respectively; (2) Pyrolysis treatment: 100 μL of the centrifugal supernatant of samples 1, 2, and 3 were heat treated at 33°C, 38°C, and 42°C for 20 min, respectively, and then immediately placed on ice; (3) Load the treated protein samples 1, 2, and 3 into the wells of a 12% polyacrylamide gel, and use SDS-PAGE to detect the decomposition of enzyme treatment and heat treatment; The electrophoresis results are as follows Figure 7 As shown, it can be seen that after heat treatment at 33°C, 38°C and 42°C, there is still a band with a molecular weight of about 200KDa. It can be seen that the recombinant type III human collagen prepared by the present invention has a certain heat resistance and can maintain the original basic structure within a suitable temperature range.

[0029] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant type III human collagen, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

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

1.

3. The gene according to claim 2, characterized in that The recipient plant expressing the gene was lettuce.

4. A method for expressing recombinant type III human collagen, characterized in that: Here are the steps: S1: Using the human type III collagen sequence as a template, sequence optimization is performed to obtain a recombinant type III human-like collagen gene sequence; based on the obtained gene sequence, upstream and downstream primers for PCR amplification are designed to amplify the recombinant type III human-like collagen gene sequence to obtain a recombinant type III human-like collagen gene; S2: double-digest the obtained recombinant type III human collagen gene and the vector, and connect them to obtain a recombinant plasmid; S3: The obtained recombinant plasmid is linearized and then transformed into Agrobacterium, and Agrobacterium containing the recombinant plasmid is obtained after screening; S4: Infect lettuce with the Agrobacterium containing the recombinant plasmid obtained in S3, and culture it to express recombinant type III human collagen. The infected lettuce is frozen with liquid nitrogen and then ground to extract total plant protein, and the recombinant type III human collagen is obtained after purification.

5. The method for expressing recombinant type III human collagen according to claim 4, characterized in that: The sequence optimization is to optimize the human type III collagen sequence according to the codon characteristics of lettuce, eliminate the restriction sites of EcoR I, Not I, Hind III, and EcoR V in the human type III collagen gene sequence by using the synonymous conversion method, and remove two CAACCAGGCCCTCCTGG splicing sites and three CTGCTGGTCCTCCTGG splicing sites.

6. The method for expressing recombinant type III human collagen according to claim 4, characterized in that: The upstream primer sequence for PCR amplification is shown in SEQ ID NO.2; the downstream primer sequence for PCR amplification is shown in SEQ ID NO.

3.

7. The method for expressing recombinant type III human collagen according to claim 4, characterized in that: The plasmid is pCAMBIA3300; the restriction endonucleases selected for double enzyme digestion are EcoR I and Hind III.

8. The method for expressing recombinant type III human collagen according to claim 4, characterized in that: The Agrobacterium is GV3101.

9. The method for expressing recombinant type III human collagen according to claim 4, characterized in that: The screening described in S3 was performed using kanamycin-resistant LB solid medium.

10. The method for expressing recombinant type III human collagen according to claim 4, characterized in that: The purification operation is as follows: adding the obtained plant total protein to a balanced gravity column, eluting the impurities, gradient eluting the recombinant type III human collagen with an eluent having a series of imidazole concentration gradients, and collecting the eluent flowing out in sections to obtain purified recombinant type III human collagen.

Images