Recombinant III-type humanized collagen expressed by hydroponic plants as well as coding gene and expression method of recombinant III-type humanized collagen

Abstract

The invention belongs to the technical field of genes, and particularly relates to a recombinant III-type humanized collagen expressed by a hydroponic plant as well as a coding gene and an expression method of the recombinant III-type humanized collagen, the amino acid sequence of the obtained recombinant III-type humanized collagen is shown as SEQ ID NO.1, and the sequence of the coding gene is shown as SEQ ID NO.2. The technical content of the recombinant III-type humanized collagen expressed by the hydroponic plant disclosed by the invention provides a new scheme for expressing the recombinant III-type humanized collagen by the hydroponic plant. A hydroponic plant lettuce is used as a receptor plant, and the obtained recombinant III-type humanized collagen is safe and non-toxic; meanwhile, the thermal denaturation temperature of the collagen is higher than that of natural collagen extracted from certain animals, so that the collagen has certain thermal stability and shows important practical value and practical significance.

Description

Technical Field

[0001] The present invention belongs to the field of gene technology, and in particular, relates to a recombinant humanized type III collagen expressed by a hydroponic plant, a coding gene thereof 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 found, which can form more than 16 types of collagen molecules. The common types are I, II, III, V and XI. Among them, type III collagen is mainly found in infants' skin or vascular endothelium, intestines and adult cartilage, vitreous body, intervertebral disc, etc. 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 trauma, supporting cell reorganization, improving skin condition, and enhancing skin elasticity and tenderness.

[0003] At present, the main method for preparing collagen in industry is traditional extraction or extraction from microorganisms. Collagen is obtained by acid, alkali, salt and enzyme treatment of animal tissues such as pig skin, cowhide, donkey skin, etc., and collagen prepared by animal sources, although the operation method is simple and the recovery rate is high, there are safety hazards in application, the non-collagen donor protein and biological factor residues cannot be completely removed in the process, 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. Human collagen fragments are obtained using microorganisms, and a lot of metabolic waste is generated during the production process, which needs to be processed, which is time-consuming and labor-intensive. At present, plant expression systems have been regarded as promising alternatives to animal cells and microbial cultures for large-scale production of recombinant proteins. The production method of using plants as chassis to produce recombinant proteins can be found in, for example: Chinese patents CN107827975 and CN106554971.

[0004] At present, how to use plant systems to better express recombinant humanized type III collagen is still an issue that needs to be further explored, and it is also a problem that this research team is committed to solving. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant type III humanized collagen expressed in hydroponic plants, a coding gene thereof and an expression method thereof.

[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 humanized collagen expressed by a hydroponic plant, the amino acid sequence of which is shown as SEQ ID NO.1.

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

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

[0009] Technical Theme 3: The present invention provides a method for expressing recombinant humanized type III collagen expressed in hydroponic plants, the steps of which are as follows: S1. Sequence design: Select the coding region (CDS region) gene sequence of human type III collagen, optimize it according to the codon characteristics of the receptor plant, add restriction sites, and obtain the recombinant type III humanized collagen gene sequence; S2. Construction of recombinant expression vector: The strain containing the recombinant type III humanized collagen gene sequence and the strain containing the plasmid are expanded and cultured, and the plasmid is extracted and then digested to obtain the target gene fragment and the linearized plasmid, which are then connected with a ligase and transformed into Escherichia coli, and the transformed Escherichia coli are screened and expanded and cultured, and the recombinant expression vector is obtained by plasmid extraction; S3, transformation: transforming the obtained recombinant expression vector into Agrobacterium; S4. Infection: hydroponically cultivate recipient plants, obtain recipient plants, immerse them in the infection solution of Agrobacterium containing the recombinant plasmid, and treat them under negative pressure to complete the infection; S5. Protein extraction and purification: The recipient plants infected with Agrobacterium are frozen and ground with liquid nitrogen to obtain plant powder, and a protein extract is added to extract the total plant protein to obtain a total plant protein extract, which is purified to obtain recombinant type III humanized collagen.

[0010] As a further embodiment of the present invention, the plasmid in S2 is PEAQ-HT.

[0011] As a further embodiment of the present invention, the endonucleases used for the digestion in S2 are AgeI and Xhol; and the ligase in S2 is T4 ligase.

[0012] As a further embodiment of the present invention, the Agrobacterium is Agrobacterium LBA4404.

[0013] As a further embodiment of the present invention, the negative pressure treatment is placed under -0.10~-0.09MPa conditions for 3~10 minutes.

[0014] As a further embodiment of the present invention, the ratio of the plant powder to the protein extract is 1 g: 1-3 mL.

[0015] As a further embodiment of the present invention, the purification is as follows: adding the plant total protein extract to a balanced gravity column, retaining it for 2 to 5 minutes, and repeatedly loading it 2 to 5 times; washing away impurity proteins with a washing solution containing 3 to 10 mM imidazole; adding an elution solution containing 150 to 450 mM imidazole to the gravity column to elute the recombinant humanized type III collagen.

[0016] The principle and beneficial effects of the above technical solution are: 1. The present invention uses hydroponic lettuce as a receptor, avoiding the risk of viral infection extracted from animals and the expression of endogenous proteins from bacteria; plants have a higher level of physiological structure than microorganisms, and are more likely to obtain human-like structures, components and tissues; using hydroponic lettuce as a receptor plant to produce recombinant type III humanized collagen improves the purity of the target protein, while having a short production cycle, low production costs and easy large-scale production.

[0017] 2. The gene sequence of the recombinant type III collagen obtained in the present invention contains a his tag, which is easy to purify and is conducive to obtaining high-purity recombinant type III humanized collagen.

[0018] 3. The recombinant type III humanized collagen obtained by the present invention has thermal stability, and its thermal denaturation temperature is about 18°C, which is higher than the thermal denaturation temperature (15.9°C) of natural collagen directly extracted from deep-sea red salmon skin; at the same time, its degradation rate is slow after reaching the thermal denaturation temperature, and it can still maintain the basic structure of part of the protein within a certain thermal denaturation temperature range, thereby extending the effective time of the protein after reaching the thermal denaturation temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the electrophoresis verification of the PCR product of the Escherichia coli colony containing the recombinant plasmid obtained in Example 1 of the present invention; wherein lane M is the 15000 marker, and lanes 1-4 are the electrophoresis results of the PCR product of the Escherichia coli colonies at different positions on the same plate; Figure 2 The figure is a graph showing the SDS-PAGE protein gel electrophoresis results of the lettuce total protein extract and the purified recombinant type III humanized collagen obtained in Example 1 of the present invention; wherein lane M is 180Marker, lanes 1 and 2 are the electrophoresis results of the lettuce total protein extract, and lanes 3 and 4 are the electrophoresis results of the purified recombinant type III humanized collagen; Figure 3 This is a graph showing the gradient temperature degradation results of the recombinant humanized type III collagen obtained in Example 1 of the present invention. 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 Culture medium configuration: LB liquid medium: Add 4 g of LB beef extract medium powder to 100 mL of purified water, adjust the pH with 5% to 20% sodium hydroxide solution to a pH between 6.8 and 7.3, and sterilize by high pressure at 121°C for 30 minutes. LB solid medium: add 4 g LB beef extract medium powder and 4 g agar powder to 100 mL purified water, adjust the pH with 5% to 20% sodium hydroxide solution to make the pH of the prepared solution between 6.8 and 7.3, and sterilize by high pressure at 121°C for 30 minutes; LB solid medium containing streptomycin sulfate (50 μg / mL): add 4 g of LB beef extract medium powder and 4 g of agar powder to 100 mL of purified water, adjust the pH with 5% to 20% sodium hydroxide solution to make the pH of the prepared solution between 6.8 and 7.3, and sterilize it by high pressure at 121°C for 30 minutes; after sterilization, wait until the temperature drops to 60°C, and add 0.1 mL of 50 mg / mL streptomycin sulfate solution filtered through a 0.22 µm filter membrane in a clean bench; LB liquid medium containing streptomycin sulfate (50 μg / mL): add 4 g of LB beef extract medium powder to 100 mL of purified water, adjust the pH with 5% to 20% sodium hydroxide solution to make the pH of the prepared solution between 6.8 and 7.3, and sterilize by high pressure at 121°C for 30 minutes; after sterilization, wait until the temperature drops to 60°C, and add 0.1 mL of 50 mg / mL streptomycin sulfate solution filtered through a 0.22 μm filter membrane in a clean bench; LB solid medium containing kanamycin sulfate (50 μg / mL): add 4 g of LB beef extract medium powder and 4 g of agar powder to 100 mL of purified water, adjust the pH with 5% to 20% sodium hydroxide solution to make the pH of the prepared solution between 6.8 and 7.3, and sterilize by high pressure at 121°C for 30 minutes; after sterilization, wait until the temperature drops to 60°C, and add 0.1 mL of 50 mg / mL kanamycin sulfate solution filtered through a 0.22 µm filter membrane in a clean bench; LB liquid medium containing kanamycin sulfate (50 μg / mL): add 4 g of LB beef extract medium powder to 100 mL of purified water, adjust the pH with 5% to 20% sodium hydroxide solution to make the pH of the prepared solution between 6.8 and 7.3, and sterilize by high pressure at 121°C for 30 minutes; after sterilization, wait until the temperature drops to 60°C, and add 0.1 mL of 50 mg / mL kanamycin sulfate solution filtered through a 0.22 µm filter membrane in a clean bench; LB solid medium containing kanamycin sulfate (50 μg / mL) and rifampicin (20 μg / mL): add 4 g of LB beef extract medium powder and 4 g of agar powder to 100 mL of purified water, adjust the pH with 5% to 20% sodium hydroxide solution to make the pH of the prepared solution between 6.8 and 7.3, and sterilize by high pressure at 121°C for 30 minutes; after sterilization, wait until the temperature drops to 60°C, add 0.1 mL of 50 mg / mL kanamycin sulfate solution filtered through a 0.22 μm filter membrane and 0.1 mL of 20 mg / mL rifampicin solution filtered through a 0.22 μm filter membrane in a clean bench; LB liquid culture medium containing kanamycin sulfate (50 μg / mL) and rifampicin (20 μg / mL): add 4 g of LB beef extract culture medium powder to 100 mL of purified water, adjust the pH with 5%~20% sodium hydroxide solution to make the pH of the prepared solution between 6.8 and 7.3, and sterilize by high pressure at 121°C for 30 minutes; after sterilization, wait until the temperature drops to 60°C, add 0.1 mL of 50 mg / mL kanamycin sulfate solution filtered through a 0.22µm filter membrane and 0.1 mL of 20 mg / mL rifampicin solution filtered through a 0.22µm filter membrane in the clean bench.

[0022] S1. Sequence design: The corresponding gene sequence of the coding region (CDS region) fragment of human type III collagen was selected, optimized according to the codon characteristics of the recipient plant (lettuce), and AgeI, Xhol restriction sites and his tag were added to obtain the gene sequence shown in SEQ ID NO.2; at the same time, the universal plant vector PEAQ-HT plasmid was selected; the target gene and plasmid PEAQ-HT were synthesized by GenScript Biotechnology Co., Ltd.; glycerol bacteria containing the target gene and plasmid PEAQ-HT were obtained; S2. Obtaining the expression vector recombinant plasmid: 1. Expansion culture of plasmid PEAQ-HT and target gene: (1) Dip the glycerol bacteria containing the target gene on LB solid medium containing streptomycin sulfate (50 μg / mL) and streak the glycerol bacteria containing the plasmid PEAQ-HT on LB solid medium containing kanamycin sulfate (50 μg / mL). Incubate the culture medium upside down at 37°C in an incubator for 12 hours until colonies grow; (2) Pick the glycerol bacteria containing the target gene into LB liquid culture medium containing streptomycin sulfate (50 μg / mL), and pick the glycerol bacteria containing the plasmid PEAQ-HT into LB liquid culture medium containing kanamycin sulfate (50 μg / mL); culture in an incubator at 37°C and 200 rpm for 12 hours; then inoculate the glycerol bacteria containing the target gene into LB liquid culture medium containing Streptomyces sulfate (50 μg / mL) at a ratio of 2%, and inoculate the glycerol bacteria containing PEAQ-HT into another LB liquid culture medium containing kanamycin sulfate (50 μg / mL) at a ratio of 2%, and culture in an incubator at 37°C and 200 rpm for 12 hours; add the sterilized glycerol solution to the bacterial solution, mix and package, and freeze at -80°C; (3) Use a plasmid extraction kit to extract plasmids from the cultured bacterial solution; obtain the PEAQ-HT plasmid and the plasmid containing the target gene respectively.

[0023] 2. Enzyme digestion: (1) Prepare agarose gel: add water to 10×TAE solution to obtain 1×TAE solution; weigh agarose powder and dissolve it in 1×TAE solution to prepare 1% agarose gel solution. Heat the agarose powder in a microwave oven to melt it. Take it out and when the liquid is placed at 55±5℃, add 1 / 10000 volume of nucleic acid colorimetric solution, mix well, and pour onto a gel plate and let it stand to solidify; (2) Establishment of enzyme digestion system: Add 1 μL Xhol enzyme, 1 μL Agel enzyme, and 2 μL enzyme digestion buffer solution to 1 μg of the plasmid containing the target gene, and add ddH2O to 20 μL; add 1 μL Xhol enzyme, 1 μL Agel enzyme, and 2 μL enzyme digestion buffer solution to 1 μg of the plasmid PEAQ-HT, and add ddH2O to 20 μL; react the two enzyme digestion systems at 37°C for 30 min respectively; finally obtain the target gene fragment and linearized plasmid PEAQ-HT; (3) Gel excision and recovery: The target gene fragment obtained after the enzyme digestion in step (2) and the linearized plasmid PEAQ-HT are subjected to agarose gel electrophoresis. Under the ultraviolet light of a gel imager, a band appears at a specific length on the agarose gel. The desired band is cut off with a scalpel; the gel is recovered using a gel recovery kit to obtain the linearized plasmid PEAQ-HT and the target gene fragment.

[0024] 3. Enzyme ligation: Add 2 μL 10× Ligation Buffer and 1 μL T4 DNA ligase to 50 mol of linearized plasmid PEAQ-HT and 150 mol of target gene fragment, and finally add ddH2O to 20 μL, incubate the ligation at 4°C overnight; obtain the recombinant plasmid PEAQ-HT, and store it in a -20°C refrigerator for use.

[0025] 4. Screening: 100uL of E. coli DH5α competent cells were taken out from the -80℃ storage environment and quickly inserted into ice. After the bacterial block melted, 10uL of recombinant plasmid PEAQ-HT was added and gently mixed by hand on the bottom of the EP tube. It was allowed to stand on ice for 25 minutes, heat-shocked in a 42℃ water bath for 45 seconds, and quickly returned to ice and allowed to stand for 2 minutes. 700μL of LB liquid culture medium without antibiotics was added to the tube, and after mixing, it was revived at 37℃, 200 rpm for 60 minutes. The bacteria were collected by centrifugation at 5000rpm for 1min, and about 100μL of supernatant was retained. The bacterial block was gently blown to resuspend and spread on LB solid culture medium containing kanamycin sulfate (50μg / mL). The culture medium was inverted and placed in a 37℃ incubator for 72h. After the colonies grew out, single colonies were randomly picked for colony PCR, and the colony PCR products were verified by electrophoresis. The results were as follows: Figure 1 As shown; 5. Expand the culture: inoculate the colony into 5 mL of LB liquid medium containing kanamycin sulfate (50 μg / mL), culture overnight at 28°C and 200 rpm, and extract the plasmid using a plasmid extraction kit to obtain the recombinant plasmid PEAQ-HT; S3. Transformation: Take out 100 μL of Agrobacterium LBA4404 competent cells stored at -80°C, insert into ice, wait for the bacterial block to melt, add 1 μg of recombinant plasmid, and gently mix by tapping the bottom of the EP tube with your hand to avoid using a pipette, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes; add 700 μL of antibiotic-free LB liquid culture medium, shake and culture at 28°C for 3 hours; centrifuge at 6000 rpm for 1 minute to collect the bacteria. , keep about 100 μL of supernatant and gently blow to resuspend the bacterial block, spread it on LB solid culture medium containing kanamycin sulfate (50 μg / mL) and rifampicin (20 μg / mL), invert it and culture it in a 28°C incubator for 72 hours. After the colonies grow, inoculate the colonies into 5 mL of LB liquid culture medium containing kanamycin sulfate (50 μg / mL) and rifampicin (20 μg / mL), culture it at 28°C and 200 rpm for 24 hours, and then use it to prepare the infection solution.

[0026] S4. Infection: (1) Hydroponic plants: Soak the plant seeds in water for 30 minutes, absorb excess water and set aside; fill the culture equipment with water, turn on the faucet to circulate the water in the equipment, and add nutrient solution. After the equipment circulates for 24 hours, use a small hanging basket to plant lettuce. Add expanded clay and plant seeds to the small hanging basket. Cultivate in the dark before the plant buds grow. After the seedlings grow out, separate the seedlings. Cultivate with 12 hours of light and 12 hours of darkness at a temperature between 15-25°C; wait until the plants grow to a medium size (6-8 leaves, no obvious bolting and good growth) before use.

[0027] (2) Prepare the infection solution: 100 mL of 1M KOH solution, 500 mL of 100 mM MgCl2, 100 mL of 500 mM MES (adjust the pH to 5.6 with KOH), and 7.5 mL of 100 mM acetosyringone (dissolve in DMSO and filter with a 0.22 µm filter membrane). Prepare 5 L of infection solution according to the proportions. (3) Agrobacterium LBA4404 containing the recombinant plasmid PEAQ-HT was inoculated into LB liquid culture medium containing kanamycin sulfate (50 μg / mL) and rifampicin (20 μg / mL) at a ratio of 2%, and cultured in an incubator at 28°C and 200 rpm for 24 hours; the OD value was monitored in real time. 600 Value, to OD 600 to between 1.5 and 3; (4) Centrifuge the bacterial solution at 5000g for 15 min at 4°C to collect the bacteria, resuspend the bacteria in the infection solution, and dilute the bacteria to the corresponding OD 600 The concentration gradient is 0.2, 0.4, 0.6, 0.8, 1.0, and after adjustment, it is placed at room temperature for 3 hours; (5) The plants were immersed in mixed infection solutions of Agrobacterium with different concentration gradients, and placed under negative pressure of -0.09 MPa for 10 minutes, then slowly restored to atmospheric pressure and cultured in the dark for 24 hours; S5. Protein extraction and purification: (1) Grinding plants: Wrap the mortar with tin foil for sterilization, put the sterilized mortar into a drying oven, dry it at 60°C for 48 hours, and cool it to room temperature for use; pour liquid nitrogen into the mortar, wait for it to completely evaporate, then add 1 / 2 of the volume of the mortar, add lettuce into the mortar, grind it, control the grinding rate to prevent liquid nitrogen from splashing, add liquid nitrogen again after the liquid nitrogen evaporates, and continue to grind the lettuce until there are no obvious particles; (2) Extraction of total plant protein solution: Prepare protein extract: 20 mM Tris-HCl, adjust pH to 7.5 with HCl, 150 mM potassium chloride, 7 mM magnesium acetate, 150 mM glucose, 30 mM mercaptoethanol, 1 mM EDTA, 100 μM PMSF, 10% glycerol, and make up the rest with pure water; Add 2 mL of extract to every 1 g of plant tissue, mix well, centrifuge twice at 15000 × g, 4 ° C, 30 minutes / time, take the supernatant, filter with 0.22 μm fiber filter, add to the concentration tube, 5000 rpm, 30 minutes to concentrate, obtain lettuce total protein concentrated extract, -80 ° C frozen; electrophoresis to determine the infection solution of this example dilute the Agrobacterium solution to OD 600 When the value is 0.6, the protein extraction amount of the lettuce total protein extract is the largest, and the subsequent experiments are conducted using the lettuce total protein extract infected with the mixed infection solution of Agrobacterium at this dilution concentration; (3) Protein purification: Prepare Wash Buffer: 20 mM NaH2PO4, 50 mM NaCl, 5 mM imidazole per liter, adjust pH to 8.0 with NaOH, filter with 0.45 µm filter membrane, and store at 4°C; Prepare Elution Buffer: Each liter contains 20mM NaH2PO4, 50mM NaCl, and imidazole (the concentrations are 20mM, 50mM, 100mM, 200mM, 250mM, or 300mM, respectively). Adjust the pH to 8.0 with NaOH, filter with a 0.45µm filter membrane, and store at 4°C. A gravity column (Tian Di Ren He Gravity Column, Ni-Smart Beads 6FF) was used for protein purification. The loaded gravity column was balanced with 5 column volumes of protein extract to place the filler in the same buffer system as the target protein, and the process was repeated 3 times. The obtained lettuce total protein extract was added to the balanced gravity column, and the retention time was at least 2 minutes to ensure full contact with the medium. The effluent was collected and the sample was repeatedly loaded 3 times to increase the binding efficiency. The impurities were washed with 10 column volumes of Wash Buffer to remove non-specifically adsorbed impurities (the concentration of imidazole can be adjusted if necessary), and the washing solution was collected. The recombinant type III humanized collagen was eluted with 10 column volumes of Elution Buffer in order from low to high concentrations, and the purified recombinant type III humanized collagen was obtained. The optimal imidazole concentration of the Elution Buffer in this example was determined to be 250 mM for electrophoresis verification. (5) Concentrate the purified recombinant type III humanized collagen eluted with Elution Buffer (imidazole concentration: 250 mM): Use Millipore UFC901008 to concentrate the purified recombinant type III humanized collagen by centrifugation (5000 rpm, 60 minutes). Collect the recombinant type III humanized collagen and store it in a -80°C refrigerator.

[0028] Test Example 1: SDS-PAGE protein gel detection: According to the fifth method SDS-polyacrylamide gel electrophoresis (SDS-PAGE method) in 0514 electrophoresis method of the Pharmacopoeia of the People's Republic of China: 2020 Edition. Part IV, the purified recombinant type III humanized collagen obtained in Example 1 was subjected to SDS-PAGE gel electrophoresis; (1) Sample treatment: add the purified recombinant humanized type III collagen obtained in Example 1 to the SDS-PAGE protein loading buffer, mix well, boil in a boiling water bath for 10 min, cool naturally, and collect by centrifugation to obtain a protein mixture for later use; use an SDS-PAGE gel preparation kit to prepare a separation gel concentration of 12% and a concentrated gel concentration of 5%; (2) Load the protein mixture and the colored pre-stained protein molecular weight standard with an initial voltage of 80 V and an initial current of 20 mA. When entering the separation gel, adjust the voltage to 120 V and the current to 50 mA. Stop electrophoresis when bromophenol blue migrates to the bottom of the gel. (3) Staining: After electrophoresis is completed, pry open the gel sheet to remove the gel, and then place it in a staining container containing 100 mL of staining solution; place it on a horizontal shaker and shake it slowly, and stain it at room temperature for 1 hour; pour out the staining solution, add Coomassie Brilliant Blue staining destaining solution, and ensure that the destaining solution can fully cover the gel; pour out the destaining solution, add new destaining solution, and destain at room temperature for 24 hours, during which time the destaining solution is replaced 3 times until the blue background is basically completely removed and the background is clear; (5) Use a gel imaging instrument to verify the size of the destained gel and the results are as follows: Figure 2 As shown, a band can be seen at about 60 kd, which is consistent with the molecular weight of the recombinant type III humanized collagen obtained in the present invention, proving that the present invention successfully obtains the target protein.

[0029] Test Example 2: Stability Test The purified recombinant type III humanized collagen obtained in Example 1 was identified by circular dichroism, and the thermal curve was obtained by monitoring the CD signal at 225 nm; the temperature was increased from 5°C to 40°C at a heating rate of 5°C / h, and each time the temperature was increased by 1°C, it was equilibrated at this temperature for 8s.

[0030] The results are as follows Figure 3As shown, circular dichroism was used to monitor the thermal change curve from 5°C to 40°C at 225nm. The results showed that the thermal denaturation temperature of the obtained recombinant type III humanized collagen was around 18°C, which was higher than the thermal denaturation temperature (15.9°C) of the natural collagen directly extracted from deep-sea red salmon skin, proving that the recombinant type III humanized collagen obtained by the present invention has good thermal stability and has sufficient prospects for practical application. At the same time, the degradation rate of the obtained recombinant type III humanized collagen within the thermal denaturation temperature range is slower than that of conventional recombinant type III humanized collagen, and the temperature tolerance range is wider. The basic structure of some proteins can be maintained within a certain thermal denaturation temperature range, and it has a certain structural stability.

[0031] 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 humanized type III collagen expressed in a hydroponic plant, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding recombinant humanized type III collagen expressed by a hydroponic plant as claimed in claim 1, characterized in that: The gene sequence is shown in SEQ ID NO.

2.

3. The gene encoding recombinant humanized type III collagen expressed by hydroponic plants according to claim 2, characterized in that: The recipient plant expressing the gene is hydroponic lettuce.

4. A method for expressing recombinant humanized type III collagen expressed in hydroponic plants, characterized in that: Here are the steps: S1. Sequence design: Select the coding region gene sequence of human type III collagen, optimize it according to the codon characteristics of the receptor plant, add restriction sites, and obtain the recombinant type III humanized collagen gene sequence; S2. Construction of recombinant expression vector: The strain containing the recombinant type III humanized collagen gene and the strain containing the plasmid are expanded and cultured, and the plasmid is extracted and then digested to obtain the target gene fragment and the linearized plasmid, which are then connected with a ligase and transformed into Escherichia coli, and the transformed Escherichia coli are screened and expanded, and the recombinant expression vector is obtained by plasmid extraction; S3, transformation: transforming the obtained recombinant expression vector into Agrobacterium; S4. Infection: hydroponically cultivate recipient plants, obtain recipient plants, immerse them in the infection solution of Agrobacterium containing the recombinant expression vector, and treat them under negative pressure to complete the infection; S5. Protein extraction and purification: The recipient plants infected with Agrobacterium are frozen and ground with liquid nitrogen to obtain plant powder, and a protein extract is added to extract the total plant protein to obtain a total plant protein extract, which is purified to obtain recombinant humanized type III collagen.

5. The method for expressing recombinant humanized type III collagen expressed in hydroponic plants according to claim 4, characterized in that: S2 The plasmid is PEAQ-HT.

6. The method for expressing recombinant humanized type III collagen expressed in hydroponic plants according to claim 4, characterized in that: The endonucleases used in the digestion described in S2 are AgeI and Xhol; the ligase described in S2 is T4 ligase.

7. The method for expressing recombinant humanized type III collagen expressed in hydroponic plants according to claim 4, characterized in that: The Agrobacterium is Agrobacterium LBA4404.

8. The method for expressing recombinant humanized type III collagen expressed in hydroponic plants according to claim 4, characterized in that: The negative pressure treatment is to place it under -0.10~-0.09MPa conditions for 3~10 minutes.

9. The method for expressing recombinant humanized type III collagen expressed in hydroponic plants according to claim 4, characterized in that: The ratio of the plant powder to the protein extract is 1g:1-3mL.

10. The method for expressing recombinant humanized type III collagen expressed in hydroponic plants according to claim 4, characterized in that: The purification is as follows: adding the plant total protein extract to a balanced gravity column, retaining it for 2-5 minutes, and repeatedly loading it 2-5 times; washing away impurity proteins with a washing solution containing 3-10 mM imidazole; adding an elution solution containing 150-450 mM imidazole to the gravity column to elute the recombinant type III humanized collagen.

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