Human collagen type xvii mRNA based on antigen sequence screening and preparation method thereof

Abstract

This invention discloses a human type XVII collagen mRNA screened based on antigen sequence and its preparation method, relating to the field of biomedical technology. The human type XVII collagen mRNA comprises, from the 5′ end to the 3′ end, a 5′ untranslated region, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2, and a 3′ untranslated region; the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2 has undergone codon optimization, and its nucleotide sequence is shown in SEQ ID NO:3. This invention also provides a method for preparing this type XVII collagen mRNA. The advantageous antigen sequence screened by this invention (only 234 nucleotides) is significantly shorter, improving the utilization rate of the mRNA's coding capacity, while avoiding interference from non-functional regions on translation efficiency and protein folding.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a human type XVII collagen mRNA based on antigen sequence screening and its preparation method. Background Technology

[0002] Type XVII collagen (COL17A1), also known as BP180 or BPAG2, is a type II transmembrane glycoprotein with a molecular weight of approximately 180 kDa. It is enriched in the hemidesmosomes of basal keratinocytes and is a key component in maintaining the structural integrity of the skin's basement membrane. Type XVII collagen plays an important biological role and has significant application value in maintaining hair follicle stem cell function, regulating the hair follicle cycle, and promoting skin regeneration. Currently, a considerable technological landscape has been established surrounding Type XVII collagen, primarily involving the following technological routes: 1. Recombinant protein technology route Several methods for preparing recombinant type XVII collagen and their applications have been disclosed. One method for preparing human type XVII collagen fragments (CN122081372A) discloses a multidimensional synergistic expression system based on Pichia pastoris, involving "molecular chaperone co-expression-precursor supply-osmolarity adaptation regulation," for stable and efficient secretory expression of collagen fragments. Existing technologies also involve co-transforming two expression vectors into Pichia pastoris to screen for recombinant expression strains capable of co-expressing human collagen and hydroxylase. ZL202410154497.6 discloses recombinant human type XVII collagen, its preparation method, and its applications; other technologies disclose recombinant type XVII human collagen COL-1012-A and COL-1012-B and their preparation methods. These technologies produce type XVII collagen through gene recombinant expression systems (such as Escherichia coli, Pichia pastoris, or CHO cells), which, after purification, are used as active ingredients in cosmetics or medical devices. However, recombinant protein technology has the following inherent limitations: As an exogenous macromolecular protein, recombinant type XVII collagen suffers from high production costs, complex purification processes, poor batch stability, and easy degradation and inactivation during storage, which limits its large-scale application. Due to the lack of hydroxylases, it is difficult to simultaneously achieve proline hydroxylation and high yield of macromolecular collagen in commonly used exogenous expression systems such as Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris, resulting in products that are difficult to form the correct triple helix structure and have low biological activity.

[0003] 2. mRNA technology route CN122038410A discloses an mRNA molecule and its application in the preparation of cosmetic skincare products. This technical solution achieves skin repair by applying mRNA encoding type XVII collagen to the skin, thereby inducing skin cells to produce collagen themselves. However, this prior art solution has the following shortcomings: (1) No antigen epitope screening and sequence optimization involved: Existing patents only vaguely describe the "nucleic acid sequence encoding type XVII collagen", without systematically screening and optimizing the specific antigenic regions with strong immunogenicity and high biological activity in type XVII collagen. Different antigenic regions have significantly different abilities to induce cellular immune responses and promote hair follicle stem cell function. The lack of targeted antigen screening may lead to unsatisfactory biological activity of mRNA-translated proteins.

[0004] (2) The stability and translation efficiency of the mRNA sequence have not been systematically optimized: Existing technologies do not involve systematic optimization of the 5'UTR, 3'UTR, codon bias, etc., of type XVII collagen mRNA to improve the stability and translation efficiency of mRNA in cells. Previous studies have shown that optimizing nucleic acids, such as codons, makes the optimized nucleic acids more stable and more conducive to efficient expression. mRNA is unstable in tissues and is easily degraded or diffused. The stability and translation efficiency of mRNA directly affect the expression level and duration of the target protein and are key factors determining the effectiveness of the technology.

[0005] (3) The existing technology has not designed the mRNA sequence specifically for the molecular characteristics of type XVII collagen: The full-length coding sequence of the type XVII collagen gene (COL17A1) is about 4.5kb and contains multiple functional domains. The existing technology has not designed the sequence for the specific functional regions of type XVII collagen in the microenvironment of hair follicle stem cells, making it difficult to maximize its biological functions in promoting the self-renewal of hair follicle stem cells and inhibiting hair follicle aging.

[0006] Therefore, existing mRNA techniques encoding type XVII collagen lack systematic antigen sequence analysis and screening, failing to identify the dominant antigenic region in type XVII collagen that exhibits the strongest immunogenicity and is most directly related to biological function regulation. This results in the translated protein potentially containing a large number of non-functional regions, wasting the encoding capacity of the mRNA and potentially affecting translation efficiency and proper protein folding due to excessively large protein molecules. There is an urgent need in this field for a method to obtain dominant antigenic sequences of human type XVII collagen with high immunogenicity and biological activity through systematic antigen sequence analysis and screening, and to construct sequence-optimized mRNA molecules based on these sequences. This would enable efficient and stable in vitro transcription preparation of type XVII collagen mRNA, providing a superior technical solution for subsequent protein function research and application development. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing human type XVII collagen mRNA based on antigen sequence screening, thereby addressing the problems existing in the prior art. This invention significantly shortens the length of the dominant antigen sequence (only 234 nucleotides) obtained through screening, improving the utilization rate of the mRNA's coding capacity, while avoiding interference from non-functional regions on translation efficiency and protein folding. This enables efficient and stable in vitro transcription preparation of type XVII collagen mRNA.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a human type XVII collagen mRNA, the mRNA containing a nucleotide sequence encoding the extracellular segment of human type XVII collagen NC16A, the amino acid sequence of the extracellular segment of NC16A is shown in SEQ ID NO:2; the mRNA contains, from the 5′ end to the 3′ end, a 5′ untranslated region, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2, and a 3′ untranslated region; the nucleotide sequence of the 5′ untranslated region is shown in SEQ ID NO:4, and the nucleotide sequence of the 3′ untranslated region is shown in SEQ ID NO:5.

[0009] Furthermore, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2 has been codon-optimized, and its nucleotide sequence is shown in SEQ ID NO:3.

[0010] Furthermore, the full-length nucleotide sequence of the mRNA is shown in SEQ ID NO:6.

[0011] The present invention also provides a recombinant plasmid containing the coding sequence of the above-mentioned human type XVII collagen mRNA.

[0012] Furthermore, the vector for the recombinant plasmid is pUC57; the coding sequence for human type XVII collagen mRNA is inserted between the NcoI and EcoRI sites of the pUC57 vector.

[0013] This invention also provides a method for preparing human type XVII collagen mRNA, comprising the following steps: (1) Provides a nucleotide sequence encoding the extracellular segment of human type XVII collagen NC16A, the amino acid sequence of which is shown in SEQ ID NO:2; (2) The above nucleotide sequence was cloned into a vector to construct a recombinant plasmid; (3) Transform the recombinant plasmid into Escherichia coli, amplify and culture, and then extract the recombinant plasmid; (4) Linearize the recombinant plasmid by single enzyme digestion with restriction endonucleases; (5) Using the linearized recombinant plasmid as a template, an in vitro transcription reaction was performed to obtain mRNA; (6) Purify mRNA.

[0014] Further, the vector in step (2) is pUC57; the restriction endonuclease in step (4) is BspQI; the in vitro transcription reaction in step (5) uses T7 RNA polymerase and introduces a 5′ cap structure by co-transcription and capping; the purification in step (6) uses Oligo dT affinity chromatography.

[0015] Furthermore, the nucleotide sequence encoding the extracellular segment of human type XVII collagen NC16A has been codon-optimized, and its nucleotide sequence is shown in SEQ ID NO:3.

[0016] Furthermore, the full-length nucleotide sequence of the mRNA is shown in SEQ ID NO:6.

[0017] The present invention also provides the application of the above-mentioned human type XVII collagen mRNA in the preparation of pharmaceuticals, medical devices, biomaterials, tissue-engineered products or cosmetics for treating hair loss and gray hair.

[0018] The present invention discloses the following technical effects: Compared with the prior art, the present invention has the following beneficial effects: 1. Antigen screening improves mRNA coding efficiency This invention is the first to systematically screen COL17A1 antigen sequences. Based on quantitative data from Kolaskar & Tongaonkar antigenicity analysis and Emini surface accessibility analysis, combined with UniProt functional domain annotation, a dominant antigenic region (SEQ ID NO:2) with strong immunogenicity, located in the NC16A immunodominant region, and relatively high surface accessibility was identified. Compared with existing techniques using the full-length COL17A1 sequence (approximately 4.5 kb), the dominant antigenic sequence screened in this invention (only 234 nucleotides) is significantly shorter, improving the utilization of mRNA coding capacity while avoiding interference from non-functional regions on translation efficiency and protein folding.

[0019] 2. Sequence optimization improves mRNA stability and translation efficiency. This invention systematically optimized the sequence of COL17A1 mRNA, including codon optimization, 5′UTR optimization, and 3′UTR optimization. Codon optimization adjusted the GC content from approximately 67% of the native sequence to 59.0%, better aligning with the codon usage preferences of human cells. 3′UTR optimization effectively reduced ARE-mediated mRNA degradation by decreasing the number of AU-enriched elements (AREs). Combined with these optimization measures, the COL17A1 mRNA prepared by this invention exhibits significantly better intracellular stability and translation efficiency than the unoptimized sequence.

[0020] 3. Optimized in vitro transcription template design improves mRNA product purity. This invention designs linearization sites downstream of the target gene and uses a BspQI single-enzyme digestion linearization strategy to prepare a linearized template, ensuring precise termination of transcription at the preset termination site and avoiding non-specific long RNA byproducts generated by transcription readthrough. In vitro transcription employs a co-transcriptional capping technique to synthesize 5′-capped mRNA in one step, combined with Oligo dT affinity chromatography purification, effectively improving the purity of the mRNA product.

[0021] 4. The preparation process is simple and the cost is controllable. This invention employs a technical route where gene synthesis is outsourced to a commercial company, and subsequent molecular cloning and in vitro transcription can be completed under standard laboratory conditions. The entire gene is chemically synthesized and delivered in glycerol-containing bacterial form. Compared to recombinant protein technologies, this invention avoids complex protein purification processes, resulting in a shorter preparation cycle and lower costs.

[0022] 5. Product quality is controllable This invention ensures the quality control of the product by detecting multiple quality indicators such as concentration, purity (A260 / A280 and A260 / A230 ratios) and integrity (CE detection of main peak content ≥80%). Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a cross-sectional view of antigenicity / surface accessibility; Figure 2 Map of COL17A1 mRNA recombinant plasmid; Figure 3 This is a graph showing the mRNA integrity detection results for Example 4. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1 Screening of dominant antigen sequences of human type XVII collagen The XVII type collagen sequence was found in NCBI (accession number: NCBI Reference Sequence: NP_000485.3), and its amino acid sequence is shown in SEQ ID NO:1:

[0031] Using the full-length amino acid sequence of the α1 chain of human type XVII collagen as shown in NCBI Reference Sequence: NP_000485.3 (UniProtKB: Q9UMD9), antigenicity analysis was performed using a combination of bioinformatics methods.

[0032] Specifically, the following analyses were performed using online tools provided by the IEDB Analysis Resource platform: antigenicity was analyzed using the Kolaskar & Tongaonkar Antigenicity method; surface accessibility was analyzed using the Emini Surface Accessibility method; and the annotation information of the protein's functional domains was comprehensively evaluated with reference to the UniProt database.

[0033] Based on UniProt topological annotations (TOPO_DOM / TRANSMEM) and domain annotations, the domain distribution of COL17A1 is as follows: the intracellular region (Cytoplasmic) is located at amino acids 1–467; the transmembrane region is located at amino acids 468–488; and the extracellular region is located at amino acids 489–1497. Important functional domains include: the NC16 non-helical region (positions 1–566, with the extracellular region at positions 489–566 being the immunodominant NC16A region), the triple-helical region (positions 567–1482), and the NC1 region (positions 1483–1497).

[0034] Kolaskar & Tongaonkar antigenicity analysis used a sliding window of 7 residues, taking the mean of the antigenicity scale within the window, with a threshold of 1.0 (>1.0 indicates a potential antigenic determinant). The analysis showed 23 regions with antigenicity >1.0 detected in the extracellular region, with the highest peak located at positions 468–500 (peak value 1.196, mean 1.091), and its extracellular portion at positions 489–500 overlapping the N-terminus of NC16A; the remaining antigenicity peaks were mostly located in the triple-helix GXY repeat region (e.g., positions 1057–1070, 1120–1138, etc.).

[0035] Emini surface accessibility analysis used a sliding window of 6 residues, taking the product of the surface probability scales within the window, with a threshold of 1.0 (>1.0 indicates surface accessibility). The results showed that no extracellular region reached the absolute threshold of 1.0, consistent with the structural characteristics of low surface accessibility in collagen. A relative threshold of 0.293 (mean + 1 SD) was used for screening, detecting 5 regions, 4 of which were located within the NC16A domain.

[0036] Based on the above analysis results: (1) The Kolaskar & Tongaonkar antigenic peak region (positions 468–500) overlaps with the N-terminus of NC16A; (2) 4 / 5 of the Emini relatively high accessibility region falls within NC16A; (3) NC16A is located in the extracellular region and is a non-triple-helix flexible fragment—finally, one dominant antigen region was obtained, namely the extracellular segment of NC16A (positions 489–566, 78 amino acids), and its amino acid sequence is shown in SEQ ID NO:2: AEEVRKLKARVDELERIRRSILPYGDSMDRIEKDRLQGMAPAAGADLDKIGLHSDSQEELWMFVRKKLMMEQENGNLR.

[0037] See antigenicity / surface accessibility profile. Figure 1 .

[0038] Example 2 Design and optimization of COL17A1 mRNA sequence 1. Codon optimization for E. coli expression systems Based on the amino acid sequence of the dominant antigen region identified in Example 1, its encoding nucleotide sequence was designed. Codon optimization was performed using the following methods: Reverse translation and codon optimization of SEQ ID NO:2 were performed using an online tool (Yunzhou Biotechnology Online Website): The optimized sequence is SEQ ID NO:3: ATGGCCGAGGAGGTGAGAAAGCTGAAGGCCAGAGTGGACGAGCTGGAGAGAATCAGAAGAAGCATCCTGCCTTACGGCGACAGCATGGACAGAATCGAGAAGGACAGACTGCAGGGCATGGC TCCTGCCGCCGGCGCCGACCTGGACAAGATCGGCCTGCACAGCGACAGCCAGGAGGAGCTGTGGATGTTCGTGAGAAAGAAGCTGATGATGGAGCAGGAGAACGGCAACCTGAGATAATGA.

[0039] (1) The optimization principle is based on ensuring that the amino acid sequence is completely consistent with the natural sequence.

[0040] (2) Codon preference: based on high-frequency human codons (R→AGA, P→CCT, etc.) (3) Optimize the GC content of the sequence to maintain it in the range of 40% to 60%, and the optimized GC content is 59.0% (the natural sequence is about 67%, which is higher and is beneficial to mRNA translation). (4) Repeated sequences of four or more identical nucleotide residues consecutively (to avoid transcription slip). (5) Common restriction enzyme sites: No conflicts with EcoRI / XbaI / BamHI / HindIII / NotI / XhoI / NheI / BsaI, etc.

[0041] 2. Design of the non-translated region (5′UTR) A 5′UTR sequence was designed upstream of the coding sequence. The nucleotide sequence of the 5′UTR is shown in SEQ ID NO:4: SEQ ID NO: 4: TAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC.

[0042] The 5′UTR design must meet the following requirements: a. Length between 20 and 150 nucleotides; b. Does not contain upstream open reading frames (uORF); c. Does not contain the Internal Ribosome Entry Site (IRES) sequence; d. GC content is between 30% and 60%; e. Failure to form stable secondary structures hinders the assembly of the translation initiation complex.

[0043] 3. Design of the Untranslated Region (3'UTR) A 3′UTR sequence was designed downstream of the coding sequence. The nucleotide sequence of the 3′UTR is shown in SEQ ID NO:5.

[0044] SEQ ID NO:5: CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGC AGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC.

[0045] The 3'UTR design meets the following requirements: a. Length between 50 and 1000 nucleotides; b. Contains at least one element that enhances mRNA stability. c. The number of AU enriched elements (AREs) is reduced or eliminated; d. Does not contain a hidden poly(A) signal sequence; e. The inability to form a stable secondary structure interferes with translation termination.

[0046] 4. Determination of the full-length mRNA sequence The 5′UTR sequence, coding sequence, and 3′UTR sequence were sequentially linked in the 5′→3′ direction to obtain the target full-length COL17A1 sequence, the nucleotide sequence of which is shown in SEQ ID NO:6.

[0047] SEQ ID NO:6: .

[0048] 5. Sequence design and whole-genome chemical synthesis Design of restriction enzyme cleavage sites To facilitate subsequent cloning into the expression vector, restriction endonuclease recognition sites were added to both ends of the synthesized gene: NcoI (C↓CATGG) and EcoRI (G↓AATTC) sites were added at the 5′ and 3′ ends of the sequence. The designed sequence was entrusted to Nanjing GenScript Biotech Co., Ltd. for whole-genome chemical synthesis. Nanjing GenScript Biotech delivered the gene in glycerol-containing bacterial form. The COL17A1 mRNA recombinant plasmid map is shown below. Figure 2 .

[0049] Example 3 Amplification and extraction of recombinant plasmids 1. Preparation and preservation of glycerol bacteria The obtained glycerol bacteria were inoculated into LB liquid medium containing kanamycin (100 μg / mL) and cultured at 37°C with shaking until the logarithmic growth phase.

[0050] Mix 500 μL of bacterial culture with 500 μL of sterile 50% glycerol (final glycerol concentration 25%), dispense into sterile cryovials, and store at -80°C.

[0051] 2. Plasmid expansion culture and extraction Glycerol bacteria were removed from -80°C, streaked onto LB plates containing kanamycin (100 μg / mL), and incubated overnight at 37°C.

[0052] Pick a single colony and inoculate it into 5 mL of LB liquid medium containing kanamycin (100 μg / mL), and incubate overnight at 37°C with shaking.

[0053] The overnight culture was transferred at a ratio of 1:100 to 200 mL of LB liquid medium containing kanamycin (100 μg / mL) and cultured at 37°C with shaking for 12–16 hours.

[0054] The recombinant plasmid pUC57-COL17A1 was extracted using the Thermo Fisher Scientific plasmid large-scale extraction kit. The following steps were added during extraction to ensure plasmid purity: a. Add an RNase A digestion step after the lysis step to remove RNA contamination; b. Increase the number of washes (3 times) with high-salt washing solution during the washing process to remove protein contamination.

[0055] The concentration and purity of the extracted plasmids were determined by ultraviolet spectrophotometry, with an A260 / A280 ratio of ≥1.8.

[0056] Example 4 Plasmid extraction, linearization, and mRNA synthesis 1. Plasmid amplification and extraction A small amount of bacterial culture was picked from the glycerol bacteria obtained in Example 3 and inoculated into LB liquid medium containing 100 μg / mL kanamycin (10 g / L soybean peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar; the antibiotic is kanamycin, added at 0.1%). The medium was cultured at 37°C and 200 rpm for 12 hours with shaking. Recombinant plasmids were extracted using an endotoxin-free plasmid extraction kit (Novizan). The plasmid concentration and A260 / A280 ratio (should be 1.8–2.0) were determined, and the plasmid conformation was confirmed by agarose gel electrophoresis.

[0057] 2. Linearization (single enzyme digestion of BspQ1, a necessary step) Linearization was performed using BspQ1 single enzyme digestion, and the specific steps are as follows: Reaction system (100 μL): 10 μg plasmid DNA, 10 μL 10×Cut buffer, 2 μL BspQ1 restriction endonuclease, and nuclease-free water to a final volume of 100 μL. (All reagents were purchased from Shanghai Zhaowei.) After gently mixing, incubate at 50°C for 3-4 hours.

[0058] The obtained reaction solution was concentrated and replaced with a 100KD ultrafiltration tube (purchased from Hangzhou Kebaite Filter Material Co., Ltd.) to remove ions and other impurities from the reaction system. The linear template was stored in nuclease-free water.

[0059] 3. In vitro transcription Preparation and purification of co-transcribed capped and tailed mRNA.

[0060] S1. In vitro transcription (IVT) reaction: For linearized DNA template, each reaction system (20 μL) contains: 4 μL of 5× transcription buffer, 1.5 μL of NTPs (1.5 μL each of ATP, CTP, GTP, and N1-methylpseudouridine triphosphate), 1.2 μL of cap analogue, 1 μg of linearized DNA template, 0.8 μL of T7 RNA polymerase, 0.5 μL of ribonuclease inhibitor, and 1 μL of inorganic pyrophosphatase (all purchased from Wuhan Tangzhi Pharmaceutical Co., Ltd.). After gentle mixing, react in a 37°C water bath for 2-3 hours.

[0061] S2, DNase digestion: After the in vitro transcription reaction is completed, add 2 μL of DNase I (RNase-free) to each system and incubate at 37°C for 15 minutes to degrade the DNA template. Then, add 2 μL of 0.5M EDTA to each reaction to terminate the reaction.

[0062] 4. mRNA purification and sample processing (1) Column preparation: Use an OligodT affinity chromatography column. Pre-equilibrate with high-salt binding buffer (0.5M NaCl, 10mM Tris-HCl, 1mM EDTA, pH 7.5) to ensure column stability and easy binding of Poly(A) tails to OligodT. Loading: Load the sample onto the chromatography column at a relatively low flow rate (5mL / min). Complete mRNA containing Poly(A) tails will specifically bind to the OligodT ligand on the column through base complementarity pairing, while impurities such as RNA fragments lacking Poly(A) tails, DNA, proteins, and unbound nucleotides will flow through directly.

[0063] (2) Washing: After loading the sample, use 3 column volumes (CV) of high-salt binding buffer to wash at a flow rate of 10 mL / min to further thoroughly wash away non-specifically bound impurities (unbound RNA fragments, proteins and free nucleotides).

[0064] (3) Elution: Elution was performed using enzyme-free water at a flow rate of 6 mL / min. The low ionic strength environment disrupted the hydrogen bond between the Poly(A) tail and OligodT, thereby specifically eluting high-purity mRNA. Prepare a 50 mL centrifuge tube, observe the computer chromatogram of the chromatography equipment, and when the peak is about to emerge, insert the collector into the 50 mL centrifuge tube to collect the elution peak.

[0065] (4) Column regeneration and storage: After elution, the column is washed with high-salt binding buffer to regenerate it, and then stored at room temperature.

[0066] Essential features: It is essential to ensure complete linearization and that the linearization site is located downstream of the target gene, without disrupting the target sequence.

[0067] In vitro transcription must involve co-transcriptional capping to ensure mRNA stability and translation efficiency.

[0068] Optional features: Plasmid extraction can be performed using kits from different brands, or by preparing it in-house using the alkaline lysis method.

[0069] In vitro transcription can use other RNA polymerases (such as SP6 and T3), as long as they match the template promoter.

[0070] Purification methods can include LiCl precipitation, ethanol precipitation, or other affinity chromatography columns.

[0071] 5. Quality testing of mRNA The purified mRNA underwent the following quality tests: (1) Concentration detection: The mRNA concentration was detected by ultraviolet spectrophotometry, and the A260 value was recorded. The mRNA concentration C (ng / μL) in this embodiment was 1417 ng / μL.

[0072] (2) Purity detection: Record the A260 / A280 ratio and the A260 / A230 ratio, requiring the A260 / A280 to be between 1.8 and 2.2. In this example, the A260 / A280 of the mRNA was measured to be 1.9.

[0073] (3) Integrity detection: mRNA integrity was detected using capillary electrophoresis (CE). The main peak content should be ≥80%. For details of the mRNA integrity detection results in this example, please refer to [link to relevant documentation]. Figure 3 .

[0074] Example 5 mRNA preservation The purified mRNA was dissolved in nuclease-free water or TE buffer, aliquoted, and stored at -80°C. Repeated freeze-thaw cycles should be avoided.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A human type XVII collagen mRNA, characterized in that, The mRNA contains a nucleotide sequence encoding the extracellular segment of human type XVII collagen NC16A, the amino acid sequence of which is shown in SEQ ID NO:

2.

2. The human type XVII collagen mRNA according to claim 1, characterized in that, The mRNA comprises, from the 5′ end to the 3′ end, a 5′ untranslated region, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2, and a 3′ untranslated region; the nucleotide sequence of the 5′ untranslated region is shown in SEQ ID NO:4, and the nucleotide sequence of the 3′ untranslated region is shown in SEQ ID NO:5; the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2 has been codon-optimized, and its nucleotide sequence is shown in SEQ ID NO:

3.

3. The human type XVII collagen mRNA according to claim 1, characterized in that, The full-length nucleotide sequence of the mRNA is shown in SEQ ID NO:

6.

4. A recombinant plasmid, characterized in that, The recombinant plasmid contains the coding sequence of human type XVII collagen mRNA as described in any one of claims 1-3.

5. The recombinant plasmid according to claim 4, characterized in that, The vector for the recombinant plasmid is pUC57; the coding sequence of the human type XVII collagen mRNA is inserted between the NcoI and EcoRI sites of the pUC57 vector.

6. A method for preparing human type XVII collagen mRNA, characterized in that, Includes the following steps: (1) Provides a nucleotide sequence encoding the extracellular segment of human type XVII collagen NC16A, the amino acid sequence of which is shown in SEQ ID NO:2; (2) The nucleotide sequence is cloned into a vector to construct a recombinant plasmid; (3) Transform the recombinant plasmid into Escherichia coli, amplify and culture, and then extract the recombinant plasmid; (4) Linearize the recombinant plasmid by single enzyme digestion with restriction endonucleases; (5) Using the linearized recombinant plasmid as a template, an in vitro transcription reaction was performed to obtain mRNA; (6) Purify the mRNA.

7. The preparation method according to claim 6, characterized in that, The vector in step (2) is pUC57; the restriction endonuclease in step (4) is BspQI; the in vitro transcription reaction in step (5) uses T7 RNA polymerase and introduces a 5′ cap structure by co-transcription and capping; the purification in step (6) uses Oligo dT affinity chromatography.

8. The preparation method according to claim 6, characterized in that, The nucleotide sequence encoding the extracellular segment of human type XVII collagen NC16A has been codon-optimized, and its nucleotide sequence is shown in SEQ ID NO:

3.

9. The preparation method according to claim 6, characterized in that, The full-length nucleotide sequence of the mRNA is shown in SEQ ID NO:

6.

10. The use of human type XVII collagen mRNA according to any one of claims 1-3 in the preparation of pharmaceuticals, medical devices, biomaterials, tissue-engineered products or cosmetics for treating hair loss and gray hair.

Images