A nucleic acid preparation encoding human full-length collagen type III mRNA, in vivo delivery and use

Abstract

The present application provides a sequence-optimized human collagen type III gene sequence, and an mRNA nucleic acid preparation encoding human collagen type III is prepared. The protein expression amount of the nucleic acid preparation in mammalian cells is increased by about 30%, and the generated collagen type III has a full-length amino acid sequence, a triple helix structure and high biological activity. Compared with animal-derived collagen, the mRNA nucleic acid preparation of the present application has clear composition, high purity and no risk of virus transmission. The LNP encapsulating the full-length collagen type III mRNA prepared by microfluidic technology has good mRNA delivery capacity and can be translated and expressed by mouse intradermal fibroblasts. The mRNA nucleic acid preparation and the LNP lipid nanoparticles encapsulating the same can efficiently express collagen type III in mammalian cells, and are suitable for the fields of skin repair, tissue engineering, biomaterials, medical devices and health foods.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and provides a nucleic acid preparation encoding full-length human type III collagen, its in vivo delivery and application. Background Technology

[0002] Collagen is a key structural protein of the extracellular matrix, widely distributed in animal bodies, and plays a vital role in maintaining tissue strength and stability. Type III collagen is mainly found in tissues such as skin, blood vessels, and tendons, and plays important biological functions in wound repair, angiogenesis, and tissue regeneration. Type III collagen is a homotrimer composed of three identical α1(III) chains, and its unique triple helix structure gives it excellent flexibility and tensile strength. In skin tissue, the proportion of type III collagen is higher in infancy and gradually decreases with age. This change in proportion is closely related to decreased skin elasticity and weakened tissue repair capacity. Therefore, type III collagen has broad application potential in tissue engineering and regenerative medicine.

[0003] Currently, type III collagen is mainly produced through animal tissue extraction or recombinant technology. However, extracting type III collagen from animal tissues presents several challenges, including immunogenicity, risk of viral transmission, significant batch-to-batch variability, and a complex extraction process. Meanwhile, due to the complex triple-helix structure of type III collagen, recombinant type III collagen prepared using expression systems such as E. coli and yeast generally only represents a portion of the natural protein sequence and struggles to form a stable triple-helix structure, resulting in significantly reduced bioactivity. Therefore, preparing full-length, highly bioactive type III collagen remains a significant challenge.

[0004] With the development of molecular biology and genetic engineering, mRNA technology has gradually become an emerging protein replacement therapy. mRNA molecules obtained through in vitro transcription can be delivered to target cells, directly synthesizing the desired functional proteins using the host cell's translation mechanism. Compared with traditional gene therapy technologies, mRNA technology has the following significant advantages: (1) it does not require genome integration, avoiding the potential risks caused by gene insertion; (2) its expression level is controllable and can achieve efficient expression in a short time; (3) its production process is simple and suitable for large-scale industrial production. The successful application of mRNA technology in vaccine and therapeutic protein development demonstrates its development potential in the biomedical field.

[0005] Lipid nanoparticles (LNPs) are among the most widely used delivery systems today. They deliver mRNA into target cells via electrostatic interactions and liposome fusion with the cell membrane. In vitro synthesized mRNA needs to cross the cell membrane to enter the cytoplasm and be translated into proteins to perform its function. However, mRNA is a negatively charged hydrophilic macromolecule with limited autonomous transmembrane capacity and is easily degraded by RNases in vivo. Therefore, microfluidic technology, with its advantages of simplicity, speed, and mild conditions, is attracting increasing attention in the preparation of mRNA-encapsulated LNP lipid nanoparticles.

[0006] This invention provides a nucleic acid formulation encoding full-length human type III collagen. Through the rational design of the 5'UTR, 3'UTR, and Poly(A) tail sequences, the stability and translation efficiency of the mRNA molecule are ensured. In mammalian cells, this nucleic acid formulation can efficiently express type III collagen, avoiding the immunogenicity and pathogen transmission risks associated with animal-derived collagen. Simultaneously, this invention utilizes microfluidic technology to prepare LNPs using a formulation of SM102, DSPC, PEG2000, and cholesterol to encapsulate full-length type III collagen mRNA. The ability of LNPs to deliver mRNA was verified through intradermal injection in mice, further confirming that LNP-CoL III eGFP can be translated and expressed by mouse dermal fibroblasts. The full-length type III collagen nucleic acid formulation prepared by this invention is applicable to multiple fields such as skin repair dressings, implants, artificial skin, medical devices, and anti-aging cosmetic products. This nucleic acid formulation will provide a more efficient, safe, and economical solution for the clinical application of type III collagen, promoting technological innovation and industrialization in the field of regenerative medicine. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide a nucleic acid preparation encoding full-length human type III collagen, its in vivo delivery, and its application. Specifically, it includes the following:

[0008] In a first aspect, the present invention provides a gene encoding full-length human type III collagen, the sequence of which is shown in SEQ ID NO.1.

[0009] Secondly, the present invention provides a nucleic acid encoding full-length human type III collagen, wherein the nucleic acid comprises, from the 5' to the 3' end, the following in sequence: 5'UTR, the gene shown in SEQ ID NO.1, 3'UTR, and Poly(A) structure.

[0010] Preferably, the 5'UTR is a β-globulin sequence containing an enhancing sequence;

[0011] Preferably, the 3'UTR is an AES sequence.

[0012] Preferably, the Poly(A) is a Poly(A) tail separated by a spacer sequence.

[0013] Preferably, the gene sequence of the nucleic acid is shown in SEQ ID NO.2.

[0014] Thirdly, the present invention provides that the nucleic acid described in the second aspect above has any of the following uses:

[0015] (1) Application in the preparation of biomaterials;

[0016] (2) Applications in the preparation of skin repair dressings, implants, artificial skin, and medical devices;

[0017] (3) Application in the preparation of health food products;

[0018] (4) Application in the preparation of antitumor drugs;

[0019] (5) Application in the preparation of drugs for treating photodamage.

[0020] Fourthly, the present invention provides a nucleic acid preparation encoding full-length human type III collagen, wherein the nucleic acid preparation is obtained by adding a 5' cap structure after nucleic acid transcription as described in the second aspect above.

[0021] Preferably, the 5' cap structure is a Cap1 structure.

[0022] Fifthly, the present invention provides the nucleic acid preparation described in the fourth aspect above to have any of the following uses:

[0023] (1) Application in the preparation of biomaterials;

[0024] (2) Applications in the preparation of skin repair dressings, implants, artificial skin, and medical devices;

[0025] (3) Application in the preparation of health food products;

[0026] (4) Application in the preparation of antitumor drugs;

[0027] (5) Application in the preparation of drugs for treating photodamage.

[0028] Sixthly, the present invention provides a method for preparing the nucleic acid preparation described in the fourth aspect above, the method comprising:

[0029] (1) Add 5'UTR, 3'UTR and Poly(A) tail to the gene sequence shown in SEQ ID NO.1 to form a complete nucleic acid sequence;

[0030] (2) Construct recombinant plasmids containing the above nucleic acid sequences;

[0031] (3) Transcription is performed after linearization of the recombinant plasmid;

[0032] (4) After the transcription product undergoes a capping reaction, it is purified to obtain a nucleic acid preparation encoding full-length human type III collagen.

[0033] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.2.

[0034] Preferably, a tag protein sequence is introduced into the nucleic acid to facilitate screening.

[0035] Preferably, the tag protein sequence is located at the 3' end of the gene shown in SEQ ID NO.1.

[0036] Preferably, the tag protein is linked to the gene shown in SEQ ID NO.1 via a linker.

[0037] Preferably, the tag protein is eGFP.

[0038] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.3.

[0039] In a seventh aspect, the present invention provides LNP lipid nanoparticles that encapsulate the nucleic acid preparation encoding full-length human type III collagen as described in the fourth aspect above.

[0040] Preferably, the LNP lipid nanoparticles comprise an aqueous phase and an organic phase; the aqueous phase comprises the nucleic acid preparation encoding full-length human type III collagen as described in the fourth aspect above.

[0041] Preferably, the organic phase is a lipid.

[0042] Preferably, the organic phase is selected from SM102, DSPC, PEG2000, and cholesterol.

[0043] Preferably, the concentration of the organic phase is 12mM-16mM.

[0044] Preferably, the N / P ratio of the lipids in the organic phase and the nucleic acid preparation in the aqueous phase is 6-8.

[0045] Eighthly, the present invention provides an application of the LNP lipid nanoparticles described in the seventh aspect above, having any of the following uses:

[0046] (1) Application in the preparation of biomaterials;

[0047] (2) Applications in the preparation of skin repair dressings, implants, artificial skin, and medical devices;

[0048] (3) Application in the preparation of health food products;

[0049] (4) Application in the preparation of antitumor drugs;

[0050] (5) Application in the preparation of drugs for treating photodamage.

[0051] The beneficial effects of this invention are:

[0052] (1) This invention provides an optimized gene sequence encoding full-length human type III collagen and prepares a nucleic acid preparation encoding full-length human type III collagen; compared with the nucleic acid reagent before sequence optimization, the optimized type III collagen nucleic acid reagent is delivered to cells for expression, and its expression level is increased by about 30%; (2) Compared with animal-derived type III collagen therapy, the nucleic acid preparation encoding full-length human type III collagen prepared in this invention has a single component, high purity, small batch-to-batch differences, no immunogenicity, and no risk of viral transmission; (3) The nucleic acid preparation encoding full-length human type III collagen prepared in this invention is encapsulated with LNP lipid nanoparticles. After delivery to cells, the intracellular translation mechanism can be used to fully translate and modify the collagen to produce a complete triple helix type III collagen; (4) The nucleic acid preparation encoding full-length human type III collagen prepared in this invention is relatively simple to synthesize and can be efficiently produced by an in vitro transcription system, making it suitable for large-scale production; (5) The nucleic acid preparation encoding full-length human type III collagen prepared in this invention can be widely used in skin repair dressings, implants, artificial skin, biomaterials, medical devices, health foods and other fields; (6) The nucleic acid preparation encoding full-length human type III collagen prepared in this invention can be widely used in tumor treatment, photodamage treatment and other diseases. Attached Figure Description

[0053] The above and / or additional aspects and advantages of the present invention will be described in conjunction with the following drawings and embodiments.

[0054] Figure 1 A schematic diagram of the molecular structure of a nucleic acid preparation encoding full-length human type III collagen;

[0055] Figure 2 Plasmid map of nucleic acid preparation encoding full-length human type III collagen (sequence not optimized);

[0056] Figure 3 The plasmid map of the nucleic acid preparation encoding the optimized human full-length type III collagen of this invention.

[0057] Figure 4The image shows the plasmid restriction enzyme digestion process during the preparation of the nucleic acid preparation encoding the optimized human full-length type III collagen of this invention. From left to right, the first band is the DNA marker; the second band is the control group eGFP nucleic acid plasmid; the third band is the EcoR V linearized eGFP nucleic acid plasmid; the fourth band is the CoL III(O)-eGFP collagen nucleic acid plasmid; the fifth band is the EcoR V linearized CoL III-eGFP collagen protein plasmid; and the sixth band is the EcoR V linearized CoL III(O)-eGFP collagen protein plasmid.

[0058] Figure 5 The image shows the mRNA integrity detection process during the preparation of the nucleic acid preparation of the optimized human full-length type III collagen of this invention; wherein, from left to right, the first band is the RNA marker; the second band is the eGFP mRNA of the control group after transcription capping; the third band is the CoL III-eGFP collagen mRNA after transcription capping; and the fourth band is the CoL III(O)-eGFP collagen mRNA after transcription capping.

[0059] Figure 6 Fluorescence images of plasmid-transfected cells before and after sequence optimization of the nucleic acid preparation encoding full-length human type III collagen;

[0060] Figure 7 Fluorescence images of mRNA expression levels in transfected cells before and after sequence optimization of the nucleic acid preparation encoding full-length human type III collagen;

[0061] Figure 8 Western blot images of the expression levels of type III collagen nucleic acid preparation transfected into cells before and after sequence optimization.

[0062] Figure 9 In vivo fluorescence images of mice at 1, 3, 4, 6 and 8 days after intradermal injection of LNP-CoL III eGFP mRNA. Detailed Implementation

[0063] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0064] Unless otherwise specified, all experimental materials used in the following examples are commercially available.

[0065] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0066] Example 1: Sequence optimization and plasmid construction of nucleic acid preparation encoding full-length human type III collagen

[0067] The human type III collagen gene sequence was downloaded from the NCBI website as shown in SEQ ID NO.4, and codon optimization was performed to obtain the optimized sequence shown in SEQ ID NO.1. A 5' UTR, 3' UTR, and Poly(A) tail were added to both sequences (the optimized nucleic acid reagent sequence is shown in SEQ ID NO.2). To facilitate screening, a tag protein sequence was introduced, and the sequences of the resulting nucleic acid reagents are shown in SEQ ID NO.3 and 5, respectively). Transcription was completed in vitro, and a Cap 1 cap structure was added to the 5' end to form the complete nucleic acid reagent, the structure of which is shown in [image missing]. Figure 1 As shown. The two constructed nucleic acid formulation sequences were respectively constructed into the pPCDNA3.1(+) plasmid vector, resulting in recombinant plasmids pPCDNA3.1-CoL III-eGFP (unoptimized) and pPCDNA3.1-CoL III(O)-eGFP (optimized), as shown. Figure 2 , Figure 3 As shown.

[0068] Example 2: Preparation of mRNA Encoding Full-Length Human Type III Collagen

[0069] 1. Linearization of recombinant plasmids

[0070] Linearization and purification of recombinant plasmids: Recombinant plasmids pPCDNA3.1-eGFP (control), pPCDNA3.1-CoLIII-eGFP, and pPCDNA3.1-CoL III(O)-eGFP were amplified, and their concentration and purity were determined using an ultra-micro spectrophotometer. Then, they were linearized using EcoR V restriction enzyme. The reaction system is shown in Table 1 below.

[0071] Table 1 Linearized reaction system

[0072]

[0073] Gently mix the above system, incubate at 37°C for 30 min, then incubate at 80°C for 20 min to inactivate the enzyme, and stop the reaction. Purify the linearized plasmid using a PCR product purification kit.

[0074] The purified and recovered linearized plasmid was subjected to DNA gel electrophoresis to verify whether the enzyme digestion was complete. The results are as follows: Figure 4 As shown, all three plasmids were completely digested with enzymes.

[0075] 2. Transcription

[0076] The T7 High Yield RNA Transcription Kit was used for in vitro transcription. When preparing the in vitro transcription reaction mixture, UTP was replaced with N1-Methylpseudouridine-5'-Triphosphate. The experimental procedure is as follows:

[0077] (1) Shake well all components except T7 RNA Polymerase Mix, briefly centrifuge to collect the contents at the bottom of the tube, and store on ice for later use.

[0078] (2) Prepare the reaction system according to Table 2 below:

[0079] Table 2 Transcription reaction system

[0080]

[0081] (3) Gently mix each component with a pipette, collect by short centrifugation, and incubate at 37°C for 2 hours.

[0082] (4) Add 1 μL of DNase I to the reaction system and incubate at 37°C for 15 min to digest the transcribed template DNA.

[0083] (5) Lithium chloride precipitation purification: Add 30 μL of LiCl precipitation buffer (7.5 mol / L LiCl, 50 mmol / L EDTA) and 30 μL of RNase-free water (the final concentration of LiCl should be maintained at 2.5-2.8 mol / L) to 20 μL of water. Mix well and incubate at -20℃ for at least 30 min. Centrifuge at 12,000 rpm for 15 min, discard the supernatant, and collect the precipitate. Wash three times with pre-cooled 70% ethanol. Reconstitute with RNase-free water and determine the concentration and purity using NanoDrop for subsequent capping reactions.

[0084] 3. Add a hat

[0085] The Cap1 Capping System kit was used for the reaction, and the procedure was as follows: 50 μg mRNA was diluted to 67 μL with RNase-free water. The mRNA was heated at 65°C for 5 min, and then immediately placed on ice for 5 min. The components listed in Table 3 were added sequentially, and the reaction was carried out at 37°C for 30 min. The mRNA was then capped and ready for further purification.

[0086] Table 3 Capped Components

[0087]

[0088] 4. Purification of Cap-mRNA

[0089] According to MEGAclear Kit TM Purification of capped mRNA was performed according to the instructions of the Purification for Large Scale Transcription Reaction Kit. The mRNA sample was diluted to 100 μL with Elution solution and gently mixed. 350 μL of Binding Solution Concentrate was added and gently mixed with a pipette. 250 μL of 100% ethanol was added and gently mixed. The mixed sample was transferred to a purification column and centrifuged at 12,000 g for 1 min, discarding the filtrate. 500 μL of Wash Solution was added and centrifuged at 12,000 g for 1 min, discarding the filtrate. Step 5 was repeated. The empty adsorption column and collection tube were placed in a centrifuge and centrifuged at 12,000 g for 1 min to completely remove the Wash Buffer. The purification column was transferred to a clean, RNase-free collection tube, and 50 μL of Elution Buffer was added. The tube was incubated at 65-70 °C for 5-10 min. Centrifuged at 12,000 g for 1 min at room temperature. To increase the amount of mRNA recovered, you can add 50 μL of Elution Buffer for elution, collect the elution in the same tube, and store at -80°C.

[0090] Gel electrophoresis was used to detect the integrity of purified eGFP mRNA, CoL III-eGFP mRNA, and CoL III(O)-eGFP mRNA, such as... Figure 5 As shown, all its bands are intact.

[0091] Example 3: Detection of cell expression by transfection with nucleic acid preparation plasmid encoding full-length human type III collagen.

[0092] Lipofectamine was selected TM The pPCDNA3.1-CoLIII-eGFP and pPCDNA3.1-CoL III(O)-eGFP plasmids were transfected with 3000Reagent transfection reagent before and after sequence optimization, and the changes in expression levels after sequence optimization were compared. The specific procedures are as follows: Mammalian 293T cells were transfected at 2.5 x 10⁻⁶ m² / h². 4Cells were seeded per well in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum. The plates were then incubated at 37°C in a 5% CO2 incubator until the cell density reached approximately 70-90%. Following the transfection reagent instructions, 3 μg of pPCDNA3.1-CoLIII-eGFP and pPCDNA3.1-CoL III(O)-eGFP plasmids were transfected into two wells of each 6-well plate containing 293T cells. After 24 hours of culture, the expression of the target protein was observed using a fluorescence microscope.

[0093] like Figure 6 As shown, after transfection of the sequence-optimized pPCDNA3.1-CoL III(O)-eGFP plasmid into 293T cells, its expression level increased by 30% compared to the pPCDNA3.1-CoL III-eGFP plasmid.

[0094] Example 4: Detection of mRNA transfection cells encoding full-length human type III collagen.

[0095] Messenger MAX transfection reagent was used for Cap-mRNA transfection experiments. The specific procedures are as follows: Mammalian 293T cells were transfected at 2.5 × 10⁻⁶ cells / year. 4 Cells were seeded per well in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum. The plates were then incubated at 37°C in a 5% CO2 incubator until the cell density reached approximately 70-90%. 3 μg of eGFP mRNA (control), CoL III-eGFP mRNA, and CoL III(O)-eGFP mRNA were transfected into three wells of the 6-well plates containing 293T cells, respectively. After 24 hours of culture, the expression of the target protein was observed using a fluorescence microscope, and the cells transfected with the target mRNA were harvested for Western blotting (WB).

[0096] like Figure 7 As shown, after the in vitro transcribed mRNA is transfected into mammalian cells, the CoLIII-eGFP mRNA can be translated using the translation system within the mammalian cells. Furthermore, after the sequence-optimized CoL III(O)-eGFP mRNA is transfected into 293T cells, its expression level increases by 30% compared to CoL III-eGFP mRNA.

[0097] Example 5: Western blot detection of mRNA transfected into cells containing nucleic acid preparations encoding full-length human type III collagen.

[0098] Three days after mRNA transfection, cells were collected and counted using a cell counter. Cells were treated with RIPA lysis buffer at the same dilution ratio to prepare protein samples for polyacrylamide gel electrophoresis. After electrophoresis, the protein samples were transferred to a 0.45 μm PVDF membrane. Western blots of the corresponding proteins were detected using rabbit-derived GFP antibody and rabbit anti-human β-turblin antibody. An anti-rabbit IgG-HRP conjugate antibody was used as a secondary antibody, and signal acquisition was performed using an imaging system.

[0099] like Figure 8 As shown, the expression level of the sequence-optimized CoLIII(O)-eGFP mRNA is much higher than that of the unoptimized CoLIII-eGFP mRNA.

[0100] Example 6: Preparation method of LNP-encapsulated nucleic acid preparation mRNA encoding full-length human type III collagen.

[0101] (1) Preparation of lipid-ethanol solution: Ionizable lipid SM102, neutral lipid DSPC, PEG2000 and cholesterol were prepared to 12mM and 16mM respectively.

[0102] (2) Preparation of mRNA citrate buffer: Prepare 50.0 mL each of a 100 mmol / L citric acid solution and a sodium citrate solution using deionized water. Then, mix 33.0 mL of the citric acid solution with 17.0 mL of the sodium citrate solution and add 0.1% DEPC. After the DEPC is completely dissolved, remove it by autoclaving. After sterilization, bring the solution to a final volume of 100 mL with DEPC-treated water to obtain a 50 mmol / L citrate buffer with a pH of 4. Calculate the required RNA concentration based on a nitrogen-to-phosphorus ratio of 6 or 8 (N / P = 6 or 8) and an aqueous-to-organic phase flow rate ratio of FRR = 3. Measure an appropriate amount of citrate buffer to prepare the mRNA citrate buffer.

[0103] Example 7: Intradermal injection of LNP-encapsulated human full-length type III collagen nucleic acid preparation into mouse skin.

[0104] LNP-encapsulated human full-length type III collagen nucleic acid was injected intradermally into the skin of mice, and human full-length type III collagen was synthesized by translation by dermal fibroblasts. Female Kunming mice aged 6-8 weeks were selected, and the hair on their backs was removed, followed by treatment with depilatory cream. 50 μL of LNP-encapsulated human full-length type III collagen nucleic acid was injected intradermally into the mouse skin. Fluorescence images of the mouse backs were acquired on days 1, 3, and 4.

[0105] The results are as follows Figure 9As shown, under the conditions of a lipid concentration of 12 mM, an N / P ratio of 8, and a flow rate ratio of 3, and a lipid concentration of 16 mM, an N / P ratio of 8, and a flow rate ratio of 3, LNP-encapsulated human full-length type III collagen nucleic acid was successfully delivered to fibroblasts in the dermal layer of mouse skin after injection, and the translation of human full-length type III collagen mRNA was completed. Fluorescence imaging of mouse skin showed that the delivered CoL III(O)-eGFP mRNA was successfully translated into CoL III(O)-eGFP protein. CoL III(O)-eGFP protein was detectable from day 1 to day 4 post-injection.

[0106] In summary, this invention provides a nucleic acid preparation encoding full-length human type III collagen, its preparation, and its application. The prepared type III collagen nucleic acid preparation, when delivered to mammalian eukaryotic cells, can utilize intracellular translation mechanisms to produce type III collagen. The sequence-optimized type III collagen nucleic acid reagent, when delivered to mammalian eukaryotic cells for expression, showed a 30% increase in expression levels. Compared to animal-derived type III collagen therapies, it has a single component, high purity, minimal batch-to-batch variation, no immunogenicity, and no risk of viral transmission. The type III collagen nucleic acid preparation encapsulated with LNP lipid nanoparticles can be delivered in vivo for translational synthesis of type III collagen. The synthesis of the LNP lipid nanoparticle-encapsulated type III collagen nucleic acid preparation is relatively simple and can be efficiently produced using an in vitro transcription system, making it suitable for large-scale production. It can be widely applied in skin repair dressings, implants, artificial skin, biomaterials, medical devices, and health foods.

Claims

1. A gene encoding full-length human type III collagen, characterized in that, The sequence of the gene is shown in SEQ ID NO.

1.

2. A nucleic acid encoding full-length human type III collagen, characterized in that, The nucleic acid comprises, from 5' to 3', the following structures in sequence: 5'UTR, the gene shown in SEQ ID NO.1, 3'UTR, and Poly(A) structure.

3. The nucleic acid as described in claim 2, characterized in that, The 5'UTR is a β-globulin sequence containing an enhancement sequence; the 3'UTR is an AES sequence; the gene sequence of the nucleic acid is shown in SEQ ID NO.

2.

4. A nucleic acid preparation encoding full-length human type III collagen, characterized in that, The nucleic acid preparation is obtained by adding a 5' cap structure after nucleic acid transcription as described in claim 2 or 3.

5. The nucleic acid preparation according to claim 4, characterized in that, The 5' cap structure is a Cap1 structure.

6. The method for preparing the nucleic acid preparation as described in claim 5, characterized in that, The method includes: (1) Add a 5'UTR, a 3'UTR and a Poly(A) tail to the gene sequence shown in SEQ ID NO.1 to form a nucleic acid sequence as shown in SEQ ID NO.2; (2) Construct recombinant plasmids containing the above nucleic acid sequences; (3) Transcription is performed after linearization of the recombinant plasmid; (4) After the transcription product undergoes a capping reaction, it is purified to obtain a nucleic acid preparation encoding full-length human type III collagen.

7. An LNP lipid nanoparticle encapsulating a nucleic acid formulation encoding full-length human type III collagen as described in claim 4 or 5, characterized in that, The LNP lipid nanoparticles comprise an aqueous phase and an organic phase; the aqueous phase comprises a nucleic acid preparation of full-length type III collagen as described in claim 4 or 5; the organic phase is selected from SM102, DSPC, PEG2000, and cholesterol.

8. The LNP lipid nanoparticles as described in claim 7, characterized in that, The concentration of the organic phase is 12mM-16mM; the N / P ratio of the lipids in the organic phase and the nucleic acid preparation in the aqueous phase is 6-8.

9. The LNP lipid nanoparticles as described in claim 8, characterized in that, The concentration of the nucleic acid preparation of full-length type III collagen as described in claim 4 or 5 in the aqueous phase is 80-120 ng / uL.

10. The nucleic acid as described in claim 2 or 3, or the nucleic acid preparation as described in claim 4 or 5, or the LNP lipid nanoparticles as described in any of claims 7-9, have any of the following uses: (1) Application in the preparation of biomaterials; (2) Applications in the preparation of skin repair dressings, implants, artificial skin, and medical devices; (3) Application in the preparation of health food products; (4) Application in the preparation of antitumor drugs; (5) Application in the preparation of drugs for treating photodamage.

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