Recombinant collagen type Ⅲ-like protein expressed based on lettuce base plate and its coding gene and expression method

Abstract

The application belongs to the technical field of genes, and particularly relates to a recombinant type III collagen-like protein based on lettuce bottom plate expression, a coding gene and an expression method, the amino acid sequence of the obtained recombinant type III collagen-like protein is shown as SEQ ID NO. 3, and the gene sequence is shown as SEQ ID NO. 1. The gene sequence is obtained by repeating the human type III collagen conservative region peptide segment gene sequence 8 times, optimizing according to the codon characteristics of a receptor plant, and inserting ASCI and XbaI double enzyme digestion sites at both ends of the gene. The recombinant type III collagen-like protein provided by the application has good activity and stability, the amino acid sequence of the recombinant type III collagen-like protein is 100% identical to the corresponding part of natural collagen, the recombinant type III collagen-like protein has important practical significance and market value when applied to human bodies, and can avoid allergy and immune rejection.

Description

Technical Field

[0001] This invention belongs to the field of gene technology, specifically relating to a recombinant type III human collagen protein expressed on a lettuce tray, its encoding gene, and an expression method. Background Technology

[0002] Collagen is a crucial structural protein in connective tissue. Its biological activity enables it to participate in cell migration, differentiation, and proliferation, giving connective tissue its mechanical strength. Furthermore, it promotes cell growth and possesses hemostatic, biocompatibility, and biodegradability properties. Collagen is widely used in medical materials and pharmaceuticals, cosmetics, health products, and various practical industries.

[0003] To date, over 30 genes encoding collagen chains have been discovered, which can form more than 16 types of collagen molecules. Common types are I, II, III, V, and XI. Type III collagen is mainly found in the skin or vascular endothelium of infants, the intestines, and in adult cartilage, vitreous humor, and intervertebral discs. Type III collagen (CLO3A1) has a strong synthetic capacity and can differentiate bone marrow-derived stem cells into fibroblasts that aid in wound repair. Therefore, in biomedical engineering, type III collagen has a wider range of applications in repairing tissue damage, supporting cell regeneration, improving skin condition, and enhancing skin elasticity and suppleness.

[0004] Currently, the main industrial method for preparing collagen is the traditional extraction method, which uses animal tissues such as pig skin, cow skin, and donkey skin to obtain collagen through acid, alkali, salt, and enzyme treatment. While animal-derived collagen is simple to produce and has a high recovery rate, it poses safety risks in application. The process may not completely remove non-collagen donor proteins and residual biological factors, and the extracted product may carry animal-derived viruses. Direct application to humans can cause immune rejection, significantly limiting its application in the biomedical field. Currently, plant expression systems are considered promising alternatives to animal cell and microbial cultures for large-scale production of recombinant proteins. Production methods using plants as a substrate for recombinant protein production can be found in, for example, Chinese patents CN107827975 and CN106554971.

[0005] However, there are currently no reports on the expression of recombinant type III human collagen using lettuce. Therefore, providing an expression method that can be mass-produced and whose expressed recombinant collagen has good biological activity and can be used in humans without causing immune rejection is of great practical significance and market value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for expressing recombinant type III human collagen based on lettuce tray.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a recombinant type III human-like collagen expressed in lettuce substrate, the amino acid sequence of which is shown in SEQ ID NO.3.

[0009] In another aspect, the present invention provides an AGⅢC8 gene encoding the recombinant type III human collagen, the gene sequence of which is shown in SEQ ID NO.1.

[0010] Preferably, the recipient plant is lettuce.

[0011] In another aspect, this invention provides a method for expressing recombinant type III human-like collagen based on lettuce cartilage, the steps of which are as follows:

[0012] S1: Using the gene with the sequence shown in SEQ ID NO.1, the recombinant type III human collagen AGⅢC8 gene fragment was obtained by PCR amplification and purification;

[0013] S2: The gene fragment and plasmid obtained in S1 were double-digested and ligated, and then transformed into E. coli. The transformed E. coli were screened and cultured on a large scale. After plasmid extraction, the recombinant type III human collagen expression vector was obtained.

[0014] S3: The recombinant type III human collagen expression vector obtained in S2 was transformed into Agrobacterium, screened, cultured and preserved;

[0015] S4: Agrobacterium containing a recombinant type III human collagen expression vector transiently infects lettuce leaves;

[0016] S5: The lettuce leaves obtained in S4 were quick-frozen with liquid nitrogen, ground, and the extract was added. After mixing evenly, the mixture was centrifuged, and the supernatant was collected for affinity chromatography purification to obtain recombinant type III human collagen AGⅢC8.

[0017] Preferably, the plasmid in S2 is pBINPLUS.

[0018] Preferably, the restriction endonucleases used for the double digestion in S2 are ASCI and XbaI.

[0019] Preferably, the Agrobacterium in S3 is Agrobacterium EHA105.

[0020] Preferably, the transient infection described in S4 is as follows: transferring overnight cultured Agrobacterium to LB liquid medium, shaking the culture, and monitoring the OD of the bacterial culture. 600 The OD value is 1.5~2.0. Collect the bacteria by centrifugation, resuspend the bacterial cells in resuspending buffer, and dilute the bacterial cells to their corresponding OD values. 600The concentration was 0.4~0.6, and the mixture was left to stand at room temperature for 2~5 hours. The plants were then immersed in the Agrobacterium mixed infection solution and treated for 5~6 minutes under an air pressure of -0.1~-0.05MPa.

[0021] Preferably, the resuspension is composed of 8-12 mM magnesium chloride, 8-12 mM MES and 100-200 μM acetylsylgenone.

[0022] Preferably, the ratio of the extract to lettuce leaves in S5 is 1~3mL:1g.

[0023] The beneficial effects of adopting the above technical solution are as follows:

[0024] 1. This invention uses plant substrate expression, avoiding the risk of viral infection from animal extraction and the expression of endogenous proteins from bacteria; using lettuce as a substrate to produce collagen improves the purity of the target protein, while also having a short production cycle, low production cost, and being easy to achieve large-scale production.

[0025] 2. The recombinant type III human collagen prepared by this invention has good activity and stability. After storage for 90 days, the recombinant type III human collagen obtained showed no significant degradation.

[0026] 3. The recombinant type III human collagen prepared by this invention has an amino acid composition that is 100% identical to the corresponding amino acid sequence of natural collagen. When applied to the human body, it can avoid allergic reactions and immune rejection, and can be widely used in the biopharmaceutical and cosmetic industries. Attached Figure Description

[0027] Figure 1 This is an image showing the electrophoresis verification results of the PCR product of gene AGⅢC8 in Example 1 of this invention;

[0028] Figure 2 This is a schematic diagram of the structure of plasmid pBINPLUS-AGⅢC8 in Example 1 of the present invention;

[0029] Figure 3 This is an electrophoresis verification result of the Escherichia coli DH5α colony PCR product transformed by plasmid pBINPLUS-AGⅢC8 in Example 1 of this invention;

[0030] Figure 4 This is a PAGE gel electrophoresis image of the supernatant of the lettuce protein extract obtained in Example 1 of this invention;

[0031] Figure 5The image shows the PAGE gel electrophoresis results of the recombinant type III human collagen purified sample obtained in Example 1 of this invention; where (a) and (b) are the recombinant type III human collagen purified samples at 0d; (c) and (d) are the recombinant type III human collagen purified samples after being stored at -20℃ for 90 days.

[0032] Figure 6 This is a graph showing the cell adhesion rate of the recombinant type III human collagen obtained in Example 1 of this invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below with reference to specific embodiments. This description is only for the purpose of facilitating the description of this invention and should not be construed as a limitation thereof. Unless otherwise specified, the experimental methods and operations in this embodiment are conventional methods and operations; the raw materials, reagents, and other materials used are commercially available products unless otherwise specified.

[0034] It should be noted that the plasmid extraction kit was purchased from Sangon Biotech; Agrobacterium EHA105 was purchased from Weidi Biotechnology; the BCA protein concentration assay kit (enhanced version) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., model number 20201ES76; mouse fibroblasts 3T3 were purchased from Shanghai Jihe Biotechnology Co., Ltd., catalog number JH-M2003; human collagen standard was purchased from Shanghai Sangon Biotech Engineering Technology Service Co., Ltd., catalog number A001654; and cell adhesion detection... The reagent kits were purchased from Beyotime Biotechnology, catalog number BB-48120; the SDS-PAGE gel preparation kit was purchased from Beyotime Biotechnology, catalog number P0012A; the Coomassie Brilliant Blue R-250 was purchased from Beyotime Biotechnology, catalog number P0017; the ALB solid medium (40.0 g / L) was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number A507003; and the LB liquid medium (25.0 g / L) was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number A507002.

[0035] Example 1

[0036] S1. Obtaining the recombinant type III human collagen AGⅢC8 gene:

[0037] (1) Select the conserved region peptide of human type III collagen, whose amino acid sequence is shown in SEQ ID NO.2. Repeat the sequence 8 times to obtain the amino acid sequence of recombinant type III human collagen as shown in SEQ ID NO.3. Optimize according to the characteristics of lettuce codon. At the same time, add ASCI and XbaI restriction sites to both ends of the gene to obtain the nucleotide sequence shown in SEQ ID NO.1, which is denoted as gene AGⅢC8. Gene was synthesized by Genscript Biotech Co., Ltd.

[0038] (2) Amplification of gene AGⅢC8 using high-fidelity PCR: Mix 2×PrimeSTAR GC Buffer (Mg 2+ The following PCR products were prepared: 10 μL of dNTP Mixture (2.5 mM each), 1.6 μL of Primer F (sequence shown in SEQ ID NO. 4), 0.4 μL of Primer R (sequence shown in SEQ ID NO. 5), 0.1 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), and 1 μL of AGⅢC8 gene DNA. Finally, ddH2O was added to a final volume of 20 μL. The mixture was placed in a PCR instrument, and the reaction conditions were set. After the reaction was complete, the PCR product of the AGⅢC8 gene was obtained. The reaction conditions are shown in Table 1 below.

[0039] Table 1. High-fidelity PCR reaction conditions for gene AGⅢC8 fragment

[0040] ;

[0041] The PCR product of gene AGⅢC8 was verified by electrophoresis. The electrophoresis verification results are as follows: Figure 1 As shown;

[0042] (3) Purification of PCR products: The PCR products were subjected to agarose gel electrophoresis using the DiaSpin column DNA gel extraction kit. The agarose gel containing the target fragment was cut off under UV light and weighed. Buffer B2 was added at 3-6 times the weight of the gel block and the gel was dissolved in water at 50℃ for 10 min. The solution was transferred into the adsorption column and centrifuged at 8000g for 30 s. The liquid in the collection tube was discarded. 500uL Wash Solution was added and centrifuged at 9000g for 30 s. The liquid in the collection tube was discarded. 500uL Wash Solution was added again and centrifuged at 9000g for 30 s. The liquid in the collection tube was discarded. The empty adsorption column was centrifuged at 9000g for 1 min. The adsorption column was placed in a clean 1.5mL centrifuge tube. 40uL Elution Buffer was added to the center of the adsorption membrane. After standing at room temperature for 1 min, the column was centrifuged for 1 min to obtain the purified gene AGⅢC8 fragment.

[0043] S2. Construction of the recombinant expression vector pBINPLUS-AGⅢC8:

[0044] (1) Construction of plasmid pBINPLUS-AGⅢC8:

[0045] The PCR product of gene AGⅢC8 was double-digested using ASCI and XbaI: 1 μg of gene AGⅢC8 DNA, 5 μL of 10×NEBuffer, and 1 μL of Restriction Enzyme were added, and finally, Nuclease-free water was added to a final volume of 50 μL. The mixture was incubated at 37°C for 15 min and then at 80°C for 20 min to obtain fragment COL1446(NB). Simultaneously, the empty vector pBINPLUS was double-digested using ASCI and XbaI: 1 μg of plasmid pBINPLUS, 5 μL of 10×NEBuffer, and 1 μL of Restriction Enzyme were added, and finally, Nuclease-free water was added to a final volume of 50 μL. The mixture was incubated at 37°C for 15 min and then at 80°C for 20 min to obtain fragment pBINPLUS(NB).

[0046] Mix 50 ng of fragment pBINPLUS (NB), 150 ng of fragment COL1446 (NB), 5 uL of rapid ligation buffer (2×), 0.5 uL of rapid T4 DNA ligase, and finally add ddH2O to 10 uL. Ligate at 25℃ for 4 hours to obtain recombinant plasmid pBINPLUS-AGⅢC8.

[0047] (2) Obtaining the recombinant plasmid pBINPLUS-AGⅢC8:

[0048] 100 μL of *E. coli* DH5α competent cells were removed from storage at -80°C and immediately placed on ice. After 5 minutes, once the bacterial block had thawed, 10 μL of recombinant plasmid pBINPLUS-AGⅢC8 was added and gently mixed by tapping the bottom of the EP tube. The mixture was then incubated on ice for 25 minutes. The cells were then heat-shocked in a 42°C water bath for 45 seconds, immediately returned to ice, and incubated for 2 minutes. 700 μL of antibiotic-free sterile LB liquid medium was added to the tube, mixed, and the cells were thawed at 37°C and 200 rpm for 60 minutes. The cells were collected by centrifugation at 5000 rpm for 1 minute. Approximately 100 μL of the supernatant was collected, gently resuspended by pipetting, and spread onto LB solid medium containing 50 μg / mL kanamycin. The medium was then incubated inverted at 37°C overnight.

[0049] Single colony clones were selected using LB solid medium containing 50 μg / mL kanamycin, PCR was performed, and the PCR products were electrophoresed to identify positive clones. The identification results are as follows: Figure 3 As shown; positive clones were selected for sequencing to determine whether the recombinant plasmid was successfully used to construct the recombinant expression vector pBINPLUS-AGⅢC8;

[0050] The correct positive clone colonies were inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. The plasmid was extracted using a plasmid extraction kit to obtain the recombinant plasmid pBINPLUS-AGⅢC8.

[0051] S3. Agrobacterium-mediated transformation of recombinant type III human collagen-like carrier:

[0052] (1) Take Agrobacterium EHA105 competent cells stored at -80℃ and hold them in your palm for a moment until they partially thaw. When they are in an ice-water mixed state, insert them into ice. Add 0.5 μg of recombinant plasmid pBINPLUS-AGⅢC8 DNA to every 100 μL of competent cells, shake the bottom of the tube to mix, and place them on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37℃ water bath for 5 minutes, and in an ice bath for 5 minutes in sequence. Add 700 μL of antibiotic-free LB liquid medium and culture at 28℃ with shaking for 3 hours. Centrifuge at 6000 rpm for 1 min to collect the bacteria. Take about 100 μL of supernatant, gently pipette it, resuspend the bacterial block and spread it on LB solid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin. Invert the medium and incubate at 28℃ for 3 days.

[0053] (2) Shaking: Screen and pick positive clones into 1 mL of LB liquid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin, and shake at 30℃ and 200 rpm for 48 h.

[0054] (3) Large shaking: Take the small shaking bacterial solution and inoculate it into 20 mL of LB liquid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin at a ratio of 2%, and shake it at 30℃ and 200 rpm for 48 h;

[0055] (4) The obtained bacterial culture was inoculated into LB solid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampicin and cultured at 28°C for 60 h to screen out positive clones; the positive colonies were cultured overnight at 28°C in 5 mL of LB liquid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampicin, and transferred to LB liquid medium at a volume ratio of 1:100. The culture was cultured in a shake flask at 28°C until the OD600 was 0.6, and cultured at 28°C for 60 h. The culture was centrifuged at 5000g for 5 minutes to collect the bacterial precipitate and stored at -80°C.

[0056] S4. Agrobacterium containing recombinant type III human collagen-like carrier transiently infects lettuce leaves:

[0057] Resuspension: 10 mM magnesium chloride, 10 mM MES and 150 μM acetylsuccinone;

[0058] A single colony of Agrobacterium EHA105 transformed from the recombinant plasmid pBINPLUS-AGⅢC8 obtained from S3 was picked with an inoculation loop and placed in 5 mL of LB broth. The culture was incubated overnight at 28°C with shaking at 200 rpm. 1 mL of the overnight cultured Agrobacterium was then transferred to 50 mL of LB broth and incubated at 28°C with shaking at 200 rpm until the OD of the culture was monitored. 600 The bacterial cells were collected by centrifugation at 5000g, 15min, and 4℃, with an OD value of 1.8. The cells were then resuspended in resuspension buffer and diluted to their corresponding OD values. 600 The concentration was 0.4, and the mixture was left to stand at room temperature for 3 hours. Lettuce was then immersed in the Agrobacterium mixed infection solution and placed under negative pressure for 6 minutes at a pressure of -0.09 MPa to complete the instantaneous infection of lettuce.

[0059] S5. Extraction and purification of recombinant type III human collagen AGⅢC8:

[0060] Extraction solution: 20 mM Tris-HCl (pH=7.5), 150 mM potassium chloride, 7 mM magnesium acetate, 150 mM glucose, 30 mM mercaptoethanol, 1 mM EDTA, 100 μM PMSF and 10% glycerol;

[0061] After infection, the cells were cultured daily at 50% humidity and 28℃ for 14 hours, and then at 50% humidity and 18℃ for 10 hours. After 7 days of cyclic culture, lettuce leaves were collected, flash-frozen in liquid nitrogen, and then ground. 2 mL of extraction solution was added to every 1 g of lettuce leaves, and the mixture was thoroughly mixed. The mixture was centrifuged twice at 15000 g, 4℃, and 30 min. The supernatant was collected and subjected to PAGE gel electrophoresis to determine protein size. The results are as follows: Figure 4 As shown, the supernatant contains a large amount of recombinant type III human collagen AGⅢC8;

[0062] Prepare protein buffer: 20 mM NaH2PO4, 500 mM NaCl, adjust pH to 8.0 with NaOH solution, and filter using a 0.45 μm filter membrane; wash the nickel ion affinity column with protein buffer, then gently shake the affinity column and supernatant on ice for 30 min and load them onto the column; wash off contaminating proteins with 5 mL of PBS solution containing 5 mM imidazole; finally, release the AGⅢC8 protein from the column with 10 mL of PBS solution containing 200 mM imidazole; collect the eluent and dialyze overnight to obtain purified recombinant type III human collagen AGⅢC8.

[0063] Test Example 1: SDS-PAGE Protein Gel Detection:

[0064] Following the method described in J. Sambrook et al., "Molecular Cloning: A Laboratory Manual, Third Edition", the purified recombinant type III human collagen sample obtained in Example 1 was sealed and stored at -20°C. PAGE gel electrophoresis was performed at 0 days and 90 days to detect the protein size and purity.

[0065] (1) Sample preparation: Add recombinant type III human collagen AGⅢC8 to the sample loading buffer, mix well, boil in a water bath for 10 min, cool naturally, and set aside.

[0066] (2) Prepare a gel with a concentration of 10% using an SDS-PAGE gel preparation kit, load the sample, and electrophores at 80V for 30min, 120V for 50min until the bromophenol blue band reaches the bottom of the gel;

[0067] (3) Staining: After electrophoresis, pry open the gel plate and take out the gel, then put it into a staining container containing 100mL of staining solution; place it on a horizontal shaker and shake it slowly at room temperature for 1 hour; pour out the staining solution, add an appropriate amount of Coomassie brilliant blue staining destaining solution to 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, changing the destaining solution 3 times during the period, until the blue background is basically completely removed and the background is clear;

[0068] The results are as follows Figure 5 As shown, there is a single, clear target protein band at a marker close to 55 kDa; and the detection results contain almost no impurities, proving that the obtained recombinant type III human collagen has high purity; the purified recombinant type III human collagen sample was tested after 90 days of storage, and no impurities were found, indicating that the obtained recombinant type III human collagen sample has no significant degradation and has good stability.

[0069] Test Example 2: Detection of the activity of recombinant type III human collagen AGⅢC8:

[0070] (1) Protein concentration detection:

[0071] Protein concentration was determined using the BCA Protein Quantification Kit (Enhanced Version).

[0072] BCA working solution: Prepare BCA working solution by mixing BCA reagent A and BCA reagent B at a volume ratio of 50:1 and mix thoroughly.

[0073] The purified recombinant type III human collagen AGⅢC8 obtained in Example 1 was prepared into a 0.5 mg / mL sample using PBS. 20 μL of the sample was added to the wells of a 96-well plate, and 200 μL of BCA working solution was added to each well. The plate was incubated at 37°C for 30 minutes, and A562 was measured. The concentration of recombinant type III human collagen in the sample was calculated to be 2 mg / mL.

[0074] Protein concentration = (A562 value - 0.16) / 0.712.

[0075] (2) Cell adhesion rate test:

[0076] Culture of 3T3 cells: Mouse fibroblasts (3T3) were seeded into DMEM medium containing 10% FBS and cultured at 37°C for 6 hours. Cell adhesion was observed. The culture medium was changed every two days until the cells reached 80% cell growth, at which point 3T3 cells were obtained.

[0077] The purified recombinant type III human collagen AGⅢC8 and human collagen standard prepared in Example 1 were respectively prepared into protein solutions of 0.5 mg / mL using PBS;

[0078] 100 μL of 0.5 mg / mL recombinant type III human collagen AGⅢC8 solution and human collagen standard solution were added to a 96-well plate as test cells and control cells, respectively, with PBS as a blank control. The plates were incubated at room temperature for 60 min. 10⁵ 3T3 cells were added to each well and incubated at 37°C for 60 min. Each well was washed four times with PBS. The absorbance at OD450 nm was measured using a cell adhesion assay kit. Based on the values ​​of the blank control, the degree of cell adhesion could be calculated. The cell adhesion rate reflects the activity of collagen: the higher the protein activity, the better it can provide a high-quality external environment for cells in a short time, helping cells adhere.

[0079] Using the adhesion rate of human collagen standard as 1, the relative cell adhesion activity of AGⅢC8 sample can be calculated, and the results are as follows: Figure 6 As shown, compared to the 40% cell adhesion efficiency of human collagen, the cell adhesion efficiency of recombinant type III human collagen AGⅢC8 can reach 92%.

[0080] Cell adhesion rate % = ((OD of test cells - OD of blank cells) / (OD of control cells - OD of blank cells)) * 100.

[0081] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; 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 collagen type III based on lettuce disc expression, characterized in that, The amino acid sequence is shown as SEQ ID NO.

3.

2. A method for expressing the recombinant collagen type III-like protein of claim 1 based on a lettuce base plate, characterized by, The steps are as follows: S1: optimizing according to the codon characteristics of lettuce, adding ASCI and XbaI enzyme cutting sites at both ends of the gene respectively, obtaining the nucleotide sequence encoding the recombinant type III collagen-like protein of claim 1, and obtaining the recombinant type III collagen-like protein gene fragment through PCR amplification and purification; S2: double enzyme cutting of the gene fragment obtained in S1 and the plasmid and then connecting, transforming into E. coli, screening and expanding the culture of the transformed E. coli, extracting the plasmid to obtain the recombinant type III collagen-like protein expression vector; S3: transforming the recombinant type III collagen-like protein expression vector obtained in S2 into Agrobacterium, screening, expanding and culturing, and saving; S4: transiently infecting the lettuce leaves containing the recombinant type III collagen-like protein expression vector with Agrobacterium, and then culturing; S5: grinding the lettuce leaves obtained by culturing in S4 after quick freezing in liquid nitrogen, adding an extraction solution, mixing uniformly, centrifuging, collecting the supernatant, and purifying by affinity chromatography to obtain the recombinant type III collagen-like protein; S4 said transient infection is: the overnight culture of Agrobacterium transfer to LB liquid medium, shake bacteria, to monitor the OD of bacteria liquid 600 The value is 1.5~2.0, centrifugal set bacteria, resuspend the bacteria with resuspension liquid, dilute the bacteria to its corresponding OD 600 0.4~0.6, room temperature for 2~5h;The lettuce is immersed in the mixed infection liquid of Agrobacterium, and is placed under the condition that the air pressure is-0.1~-0.05MPa, and is treated for 5~6min.

3. The method of claim 2, wherein, The plasmid in S2 is pBINPLUS.

4. The method of claim 2, wherein, The restriction endonuclease used in the double enzyme cutting in S2 is ASCI and XbaI.

5. The method of claim 2, wherein, The Agrobacterium in S3 is Agrobacterium EHA105.

6. The method of claim 2, wherein, The resuspension solution is composed of 8-12 mM magnesium chloride, 8-12 mM MES and 100-200 μM acetyl-syringone.

7. The method of claim 2, wherein, The ratio of the use amount of the extraction solution to the lettuce leaves in S5 is 1-3 mL:1 g.

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