MRNA encoding anti-staphylococcus aureus single-chain antibody and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to mRNA (messenger Ribonucleic Acid) for coding a single-chain antibody resisting staphylococcus aureus and application of the mRNA. On the basis of a specific scFv with high affinity to staphylococcus aureus lysate, cell wall protein and toxin, the mRNA is optimized by a gene sequence and is matched with a functional element suitable for in-vivo expression of the mRNA, so that stable and efficient expression of the mRNA in a target tissue is ensured. The mRNA is further prepared into an mRNA-LNP preparation, and the preparation is beneficial to delivery of the mRNA to local tissues of the mammary gland of the dairy cow, expresses scFv protein with a specific antibacterial function in epithelial cells of the mammary gland, can be used for replacing traditional antibiotics to treat the mastitis of the dairy cow caused by staphylococcus aureus, and has an outstanding treatment effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an mRNA encoding a single-chain antibody against Staphylococcus aureus and its applications. Background Technology

[0002] Staphylococcus aureus is one of the main pathogens causing mastitis in dairy cows. It has a strong ability to colonize and form biofilms. Traditional antibiotic treatment often has problems such as unstable efficacy, easy emergence of drug-resistant strains, and high risk of drug residues.

[0003] To address this issue, previous studies have attempted to utilize single-chain antibodies (scFv) to target and neutralize the virulence factors or surface antigens of Staphylococcus aureus, thereby blocking its infection process. For example, the research group of Zhu Jianguo at Shanghai Jiao Tong University (Wang Man. Preparation and protective effect of bovine Staphylococcus aureus single-chain antibody [D]. Shanghai Jiao Tong University, 2016. DOI:10.27307 / d.cnki.gsjtu.2016.003446.) constructed a bovine anti-Staphylococcus aureus scFv antibody library and screened high-affinity scFv candidate strains. After expression and purification in a prokaryotic expression system, these strains were used for in vitro antibacterial experiments and therapeutic experiments in a mouse mastitis model. The results showed that the scFv antibody could inhibit the inflammatory response and reduce bacterial load in animals. However, the above studies used DNA plasmids for antibody expression, which carries risks such as transcription efficiency depending on nuclear entry, delayed expression initiation, and potential gene integration. Other reports indicate that extending the half-life of scFv by fusing it with the Fc fragment can enhance its in vivo antibacterial effect (Hay, CE, et al., The Development and Characterization of an scFv-Fc Fusion-Based Gene Therapy to Reduce the Psychostimulant Effects of Methamphetamine Abuse. J Pharmacol Exp Ther, 2020. 374(1): p. 16-23). ​​However, this method still mainly uses recombinant proteins, which require in vitro expression and purification processes, resulting in problems such as long production cycles and poor formulation stability.

[0004] Currently, mRNA-based therapeutic strategies have achieved breakthroughs in vaccines (such as mRNA-1273 developed by Moderna) and antibody drugs. Studies have shown that mRNA can be used to encode therapeutic antibodies and express them in situ in vivo, thus avoiding complex protein preparation processes (Rybakova, Y., et al., mRNA Delivery for Therapeutic Anti-HER2 Antibody Expression In Vivo. Mol Ther, 2019, 27(8): p. 1415-1423.). For example, Li et al. (Li, Y., et al., An anti-FAP-scFv-functionalized exosome-carrying hydrogel delivers SKI mRNA to fibrotic nucleus pulposus cells to alleviate intervertebral disc degeneration by regulating FOXO3. Theranostics, 2025, 15(9): p. 3877-3899.) constructed an anti-tumor system combining anti-FAP-scFv mRNA expression with a hydrogel exosome system, successfully expressing functional scFv in mice and achieving good therapeutic effects. However, there are currently no reports of applying the strategy of mRNA-encoded scFv to the treatment of local bacterial infections in large animals such as dairy cows. Therefore, there is still a lack of biological agents that target Staphylococcus aureus infection and are suitable for local expression in the mammary tissue of large animals, especially in the treatment of bovine mastitis, where rapid expression, high tissue targeting, and low residual risk are still to be achieved.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an mRNA encoding a single-chain antibody against Staphylococcus aureus and its application.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides an mRNA encoding a single-chain antibody against Staphylococcus aureus, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] This invention utilizes phage display technology to isolate antibody variable region genes (VH and VL) from peripheral blood lymphocytes in cases of bovine mastitis. Overlap extension PCR is used to construct scFv sequences, and specific scFvs exhibiting high affinity for Staphylococcus aureus lysates, cell wall proteins, and toxins are screened. Then, based on the mRNA coding system, the screened scFv gene sequences are optimized and cloned into a suitable mRNA template construction system for in vivo translation and expression. This system includes functional elements such as a 5' cap structure, optimized UTR, open reading frame (ORF), and a 3' polyadenylated tail, ensuring stable and efficient expression in target tissues.

[0010] In a second aspect, the present invention provides a pharmaceutical composition comprising the mRNA described in the first aspect above, and further comprising a delivery vector for delivering the mRNA to local tissue of a bovine mammary gland.

[0011] Preferably, the delivery carrier is an LNP.

[0012] Preferably, the LNP has an average particle size of 80-100 nm and a PDI < 0.2.

[0013] Preferably, the LNP is prepared using the SM102 formulation.

[0014] In a more specific embodiment, this invention provides an mRNA-LNP formulation (M73), which uses the SM102 formulation to prepare lipid nanoparticles (LNPs) and employs a Myanna Nano L+ microfluidic device to encapsulate the mRNA. Key preparation parameters are as follows: mRNA dosage: 6.0 mg; preparation volume: 32.00 mL; final yield: 53.69%. This mRNA-LNP formulation can be used to encode and express a single-chain antibody (scFv) against Staphylococcus aureus, demonstrating significant advantages in functional validation, formulation characteristics, and application effects, mainly in the following aspects:

[0015] (1) High in vivo expression efficiency and strong protein targeting: This invention ensures high-efficiency expression of mRNA in mammary epithelial cells through optimized mRNA sequence design and LNP delivery system, successfully achieving local and sustained expression of scFv protein. Animal experimental results show that, under the same dosage conditions, this formulation can induce high-level antibody expression in the mammary gland within 24-48 hours after injection, effectively covering the early peak period of bacterial infection. Compared with traditional recombinant proteins, it avoids degradation loss during protein preparation and modification, and significantly improves targeted delivery efficiency.

[0016] (2) It has significant antibacterial and anti-inflammatory effects: The mouse mastitis model induced by Staphylococcus aureus was used for verification. After injection of medium and high doses of M73 (20 μg and 40 μg), the bacterial load in the mammary tissue decreased to below the detection limit within 3 days, the infiltration of inflammatory cells was significantly reduced, the number of somatic cells in milk decreased by more than 50%, and the inflammatory pathological score was comparable to that of the positive drug penicillin group and significantly better than that of the PBS control group, showing its strong inhibitory and anti-inflammatory ability against Staphylococcus aureus.

[0017] (3) It has the advantage of non-antibiotic treatment: Compared with traditional antibiotic treatment, the mRNA-LNP delivery system used in this invention has no drug residues, does not induce resistance, and can be used to control infections of cryptic and drug-resistant strains. It is in line with the current trend of food safety of animal-derived food and reduction of antibiotic use, and has obvious substitution potential.

[0018] (4) The formulation process is stable and easy to industrialize: The mRNA sequence of this invention has a Cap1 structure, 5'UTR / 3'UTR stabilizing elements and a polyA tail, which is conducive to stable translation in vivo. The LNP delivery system used has the characteristics of concentrated nanoparticle size distribution, high encapsulation rate and good freeze-drying stability. The preparation process is easy to standardize, batch and scale up production, and has a clear foundation for the industrial application of the technology.

[0019] (5) It has broad adaptability and scalability: In addition to being used for the treatment of mastitis in dairy cows, this technical solution can also be used to express specific proteins or antibodies against other pathogens (such as Haemophilus parasuis and Mycoplasma sheep), and has broad-spectrum versatility and cross-species application potential in the fields of local infectious diseases in animals and mucosal immune regulation.

[0020] Thirdly, the present invention provides the use of the mRNA encoding the anti-Staphylococcus aureus single-chain antibody or the pharmaceutical composition in the preparation of a medicament for treating bovine mastitis.

[0021] Preferably, in the application, the drug has the effect of blocking the adhesion and colonization of Staphylococcus aureus on the surface of breast tissue.

[0022] Preferably, in the application, the drug can effectively reduce the accumulation of bacteria on the surface of mammary ducts and alveoli.

[0023] Preferably, in the application, the drug can inhibit the formation and maturation of bacterial biofilms.

[0024] Preferably, in the application, the drug can relieve breast swelling, induration, and decreased milk production caused by bacterial infection.

[0025] In a more specific embodiment, the present invention delivers an mRNA-LNP preparation to breast tissue via ductal injection, using breast epithelial cells as an expression platform to enable scFv to be efficiently expressed locally and secreted into the breast cavity, thereby achieving targeted neutralization and biofilm intervention against Staphylococcus aureus.

[0026] This invention systematically validated the antibacterial and membrane-inhibiting effects of the scFv-mRNA-LNP formulation in an in vitro biofilm formation model and a mouse mastitis animal model, demonstrating that it can significantly reduce the infection burden, superior to the control group. The scFv-mRNA-LNP expression system constructed in this invention possesses high modularity and target customizability, and has the potential to be extended to the expression of other targeted proteins and other animal infectious diseases (such as pigs and sheep), constituting a novel veterinary precision treatment platform.

[0027] Beneficial effects:

[0028] This invention provides an mRNA encoding a single-chain antibody against Staphylococcus aureus and its application. The mRNA is based on a specific scFv protein with high affinity for Staphylococcus aureus lysates, cell wall proteins, and toxins. The gene sequence has been optimized and combined with functional elements suitable for its in vivo expression, thereby ensuring stable and efficient expression in target tissues. Furthermore, this invention formulates the mRNA into an mRNA-LNP preparation, which facilitates the delivery of the mRNA to local mammary gland tissue in dairy cows and expresses the scFv protein with specific antibacterial function in mammary epithelial cells. This preparation can be used as an alternative to traditional antibiotics for the treatment of bovine mastitis caused by Staphylococcus aureus, demonstrating outstanding therapeutic efficacy. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0030] Figure 1 This is the pVAX.1-M73 plasmid map of the present invention.

[0031] Figure 2 The results are the integrity detection results of the mRNA stock solution in Example 1 of this invention.

[0032] Figure 3 The results of the mRNA stock solution capping rate detection in Example 1 of this invention are shown. Figure A is the high-performance liquid chromatography (HPLC) analysis chromatogram; Figure B is the mass spectrometry (MS) analysis chromatogram.

[0033] Figure 4The results of M73 mRNA Poly-A tail length detection in Example 1 of this invention are shown. Figure A is the high-performance liquid chromatography (HPLC) analysis chromatogram; Figure B is the mass spectrometry (MS) analysis chromatogram.

[0034] Figure 5 The images show the Western blot (WB) results of transfection of bovine mammary epithelial cells with M73 mRNA stock solution in Example 1 of this invention. The top image shows the WB results of proteins in the cell lysate; the bottom image shows the WB results of proteins in the cell culture supernatant.

[0035] Figure 6 This is the particle size distribution of the mRNA-LNP sample in Example 2 of the present invention.

[0036] Figure 7 The figures show the changes in somatic cell count and live bacteria count in milk before and after ICR mouse modeling in Example 3 of this invention. Figure A shows the changes in somatic cell count; Figure B shows the changes in live bacteria count in milk.

[0037] Figure 8 The figures show the changes in somatic cell count and milk somatic cell count in ICR mice after drug administration in Example 3 of this invention. Figure A shows the changes in somatic cell count; Figure B shows the changes in live bacteria count in milk.

[0038] Figure 9 This is an HE staining image of mammary gland tissue from ICR mice on day 14 after drug administration in Example 3 of the present invention. Detailed Implementation

[0039] While existing studies have reported strategies for expressing plasmid DNA-encoded single-chain antibodies (scFv) in animals for antibacterial therapy, these methods generally rely on plasmids entering the cell nucleus for transcription, leading to issues such as delayed expression, dependence on cell proliferation status, and unstable transcription efficiency. Furthermore, the long-term presence of plasmids in cells may pose safety risks such as gene integration or chronic inflammatory responses, hindering clinical promotion and large-scale use. On the other hand, while recombinant protein forms of scFv-Fc fusion antibodies can enhance in vivo efficacy by extending their half-life, their preparation relies on complex in vitro protein expression and purification processes, requiring stringent cold chain transportation and storage conditions, resulting in poor formulation stability and long production cycles, making it difficult to meet the rapid intervention needs for acute local infections such as bovine mastitis. Moreover, there are currently no reports, either domestically or internationally, of applying mRNA platform technology to the treatment of bovine mastitis. Although existing studies have explored the in vivo expression of mRNA-encoded antibodies in tumors or immune diseases, these studies have largely focused on small animal models or systemic delivery scenarios. Research on targeted delivery and expression in large animal tissues (such as bovine mammary glands) remains lacking, with a lack of mature product development pathways and stable expression systems. Therefore, existing technologies cannot effectively solve key problems such as rapid expression, tissue targeting, and controllable residues in local infections of the mammary glands of dairy cows.

[0040] The purpose of this invention is to provide an mRNA sequence encoding an anti-Staphylococcus aureus single-chain antibody (scFv), its mRNA-LNP formulation, and its application in the treatment of mastitis in dairy cows, in order to solve the problems of low scFv expression efficiency, unstable delivery mechanism, complex protein preparation, and lack of effective means for local application in large animals in the prior art.

[0041] This invention constructs an scFv sequence that specifically recognizes Staphylococcus aureus surface antigen and encapsulates it in the form of mRNA in lipid nanoparticles (LNPs) for delivery to bovine mammary epithelial cells for expression. This enables rapid, controllable, and localized expression of antimicrobial proteins, thereby effectively intervening in the bacterial infection process and achieving the goal of treating bovine mastitis. It has significant advantages such as high formulation safety, high bioavailability, and no risk of drug residues.

[0042] In this invention, the nucleotide sequence of the anti-Staphylococcus aureus single-chain antibody gene (named M73 in this invention) is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0043] The nucleotide sequence of M73:

[0044] AGGGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATCAAGCTTACCgccaccATGGACGCCATGAAGCGGGGCCTGTGTTGCGTGCTGCTGCTGTGCGGGGCCGTGTTCGTGAGCAACTCCCAGGCTGTGCTGACGCAGCCCAGCAGCGTCAGCGGAAGCCTGGGCCAGCGCGTGAGCATCACCTGCAGCGGCAGCAGCAGCAACATCGGCTCCGACAACGTGGGCTGGTACCGCCAGGTGCCTGGCAGCGGCCTGCGCACCATCATCTACGGCAGCAGTAGTCGGCCCTCCGGCGTGCCTGACAGGTTCTCCGGGTCCAAGAGTGGCAACACCGCCACTCTGACCATCAGCAGCCTGCAGGCCGAGGACGAGGCCGACTACTTCTGCGTGGCCTACGACAGCTCCATCAACACCGCTATCTTCGGCTCAGGCACCACCCTGACCGTGCTGGGCGGGGGCGGATCTGGAGGAGGAGGAAGCGGAGGAGGCGGCTCTCAGGTGCAGCTGCGCGAGAGCGGCCCCTCCCTGGTGAAGCCCTCCCAGACCCTGTCCCTGACCTGCACCGTGTCAGGGTTCAGCCTGAGCGACAATAGCGTGGACTGGGTGCGGCAGGCCCCTGGCAAGGCCCTGGAGTGGCTGGGGGAGCTGAACAGGGACGGGACCATCGACGACAACCCTGCTCTGAAATCCAGGCTGTCCATCACCAAGGACAGCACCAAGAACCAGGTGAGCCTGTCCCTGAGCTCCGCCACCACCGAGGACACCGCTACCTACTACTGCGGAAGGTCCACCGGACCTTACGGCGGCACCGCCCACGTGGACGCCTGGGGCCAGGGCCTGCTGGTGACGGTGTCCTCCACCAGCCACCACCACCACCACCACTGA GGACTAGTGCA TCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTT TTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCT GTGCTTCAATTAATAAAAATGGAAAGAACCTAGATCT aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa(SEQ ID NO.1)。

[0045] In the nucleotide sequence of M73 as mRNA, all "T"s in the sequence shown in SEQ ID NO.1 are replaced with "U".

[0046] The amino acid sequence of the protein encoded by M73:

[0047] MDAMKRGLCCVLLLCGAVFVSNSQAVLTQPSSVSGSLGQRVSITCSGSSSNIGSDNVGWYRQVPGSGLRTIIYGSSSRPSGVPDRFSGSKSGNTATLTISSLQAEDEADYFCVAYDSSINTAIFGSGTTLTVLGGGGSGG GGSGGGGSQVQLRESGPSLVKPSQTLSLTCTVSGFSLSDNSVDWVRQAPGKALEWLGELNRDGTIDDNPALKSRLSITKDSTKNQVSLSLSSATTEDTATYYCGRSTGPYGGTAHVDAWGQGLLVTVSSTSHHHHHH(SEQ ID NO.2).

[0048] In a more specific embodiment of the present invention, the product is a messenger RNA (mRNA) formulation encoding a single-chain variable region antibody (scFv) against Staphylococcus aureus. Its preparation process mainly includes steps such as scFv sequence construction, screening, optimization, in vitro transcription, and lipid nanoparticle (LNP) encapsulation. Specifically:

[0049] (1) Construction and screening of scFv sequences:

[0050] First, based on the immune response characteristics of dairy cows infected with Staphylococcus aureus, peripheral blood lymphocytes (PBMCs) were collected from dairy cows with mastitis. Total RNA was extracted, and the gene fragments of the bovine antibody heavy chain variable region (VH) and light chain variable region (VL) were amplified using reverse transcription PCR (RT-PCR). The primers used for amplification were designed based on publicly available databases (such as IMGT and NCBI IgBLAST), and the specific sequences could be reasonably determined by those skilled in the art based on the conserved region sequences of the antibody genes.

[0051] Subsequently, overlap extension PCR (SOE-PCR) was used to splice the VH and VL fragments using a flexible linker (such as Gly4Ser) to form a single-chain variable region antibody (scFv) gene, and the resulting product was cloned into the phage display vector pCANTAB-5E. A bovine-derived scFv phage display library against Staphylococcus aureus was constructed by electrotransformation of Escherichia coli TG1 strain, and the library size, positive clone rate, and antibody diversity were evaluated.

[0052] Staphylococcus aureus whole-cell lysate was used as the coating antigen, and four rounds of biopanning were performed to enrich specifically binding phages. The enriched single clones were then screened using phage ELISA for high-throughput selection to obtain scFv antibody gene sequences with high affinity for Staphylococcus aureus antigens. Positive sequences were validated by sequencing, and structural modeling and affinity prediction were performed to select the optimal sequences for subsequent mRNA construction.

[0053] (2) Construction of mRNA expression vector and in vitro transcription:

[0054] The selected scFv gene sequence was cloned into an mRNA synthesis template vector containing a T7 promoter, 5'UTR, 3'UTR, and a poly(A) tail. The linearized template was transcribed into mRNA using an in vitro transcription system (e.g., using T7 RNA polymerase) to generate mature mRNA with a 5' cap (Cap1) and a 120 nt poly(A) tail. After phosphatase treatment, purification, and removal of double-stranded RNA contamination, the mRNA underwent quality testing (including concentration, integrity, sterility, and endotoxin levels) to meet delivery requirements.

[0055] (3) Preparation of LNP delivery system and mRNA encapsulation:

[0056] A microfluidic system was used to rapidly mix mRNA with lipid components (including cationic lipids, structural lipids, cholesterol, and PEG-modified lipids) in an ethanol and buffer system to form lipid nanoparticles (LNPs). By adjusting the component ratios and mixing parameters, efficient mRNA encapsulation and particle size control (average particle size approximately 80–100 nm, PDI < 0.2) were achieved. Finally, the LNP-mRNA formulation was dialyzed to remove organic solvents, filtered for sterilization, and then cryopreserved.

[0057] The mRNA-LNP preparation obtained in this invention can be locally delivered to the mammary glands of dairy cows via injection through the nipple duct. After delivery, the mRNA enters the mammary epithelial cells, where it is translated and expressed in the cytoplasm as scFv antibodies and secreted extracellularly. These antibodies specifically bind to Staphylococcus aureus surface antigen, thereby blocking its adhesion and biofilm formation, achieving the purpose of intervening in infection and controlling inflammation.

[0058] Unless otherwise specified, the above mRNA construction and LNP packaging steps can be optimized and scaled up by referring to the publicly available technical conditions of existing technology platforms such as Moderna and Acuitas.

[0059] The mRNA-LNP preparation provided by this invention is mainly used to prevent and / or treat infectious inflammatory responses caused by Staphylococcus aureus in bovine mastitis, and is particularly suitable for controlling refractory Staphylococcus aureus colonization and biofilm-related infections. The mRNA-LNP preparation is delivered to the mammary gland of the cow by local injection through the teat duct. Lipid nanoparticles mediate the entry of mRNA into mammary epithelial cells. After intracytoplasmic translation and expression, scFv antibodies can be released into the mammary cavity and local interstitial fluid via the secretory pathway, specifically binding to Staphylococcus aureus surface antigen, thereby achieving the following biological functions and clinical effects: (1) blocking the adhesion and colonization of Staphylococcus aureus on the surface of mammary tissue, effectively reducing the accumulation of bacteria on the surface of mammary ducts and alveoli; (2) inhibiting the formation and maturation of bacterial biofilms, overcoming the problem that antibiotics have difficulty penetrating biofilms; (3) reducing bacterial load and the degree of inflammation in mammary tissue, alleviating mammary swelling, induration and decreased milk production caused by bacterial infection; (4) enhancing the local immune barrier function of the body, shortening the recovery period and reducing the risk of recurrence. The function of scFv in this invention has been verified by the following experiments: (1) Binding activity verification: Through phage ELISA and co-immunoprecipitation (Co-IP) experiments, it was confirmed that the scFv obtained by screening can bind to Staphylococcus aureus whole bacterial lysate, cell wall-related proteins and secreted toxins with high affinity, verifying its ability to target and recognize key Staphylococcus aureus antigens; (2) Biofilm inhibition verification: In the in vitro biofilm model established by 96-well plates, the scFv treatment group significantly inhibited the formation of Staphylococcus aureus biofilm, and the formation area was significantly reduced compared with the control group, suggesting that it has the ability to interfere with bacterial adhesion and initial aggregation, which helps to overcome the treatment difficulties of anti-biofilm infection; (3) Antibacterial and anti-inflammatory effects: In the mouse mastitis model, the injection of mRNA lipid nanoparticles expressing scFv can significantly reduce the bacterial load, somatic cell number and pathological score in breast tissue, and the inflammation relief effect is better than that of the control group. Therefore, the mRNA-LNP formulation of this invention not only provides a novel, non-antibiotic, residue-free biological treatment method, but also has advantages such as standardized production process, high tissue specificity, and low local immune risk. It offers an industrially viable technical solution for the precise prevention and treatment of bovine mastitis, and is particularly suitable for refractory or subclinical mammary gland infections caused by drug-resistant strains. Furthermore, the technical solution provided by this invention can be extended to the treatment of other animal infectious diseases (such as Haemophilus parasuis pneumonia in pigs and Mycoplasma mastitis in sheep) or to therapeutic strategies targeting the expression of other functional proteins, demonstrating promising cross-species application prospects and industrial promotion potential.

[0060] The mRNA sequence and LNP delivery system provided by this invention can solve various problems existing in the background art, and has significant technical innovation and application prospects.

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0062] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0063] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0064] 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.

[0065] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0066] Example 1

[0067] This embodiment describes the process of obtaining scFv-encoded mRNA.

[0068] (I) Construction and screening of anti-Staphylococcus aureus scFv

[0069] Total RNA was extracted from peripheral blood lymphocytes of dairy cows suffering from Staphylococcus aureus mastitis. RT-PCR amplification of the heavy chain variable region (VH) and light chain variable region (VL) of bovine antibodies was performed using specific primers. After verification by agarose gel electrophoresis, the resulting fragments were spliced ​​together using overlap extension PCR (SOE-PCR) to form the complete scFv gene. The gene product was cloned into the phage display vector pCANTAB-5E and electroporated into Escherichia coli TG1 strain to construct a bovine anti-Staphylococcus aureus phage antibody library.

[0070] Four rounds of biological panning were performed using Staphylococcus aureus whole-cell lysate as the coating antigen to enrich phage clones that specifically bind to Staphylococcus aureus. Candidate scFv sequences with high binding capacity to Staphylococcus aureus were then screened using phage ELISA. The obtained sequences were validated by sequencing; some His-tagged expression products showed distinct specific bands in the prokaryotic system, confirming expression.

[0071] (II) Construction and expression verification of scFv-encoded mRNA.

[0072] Using the high-affinity scFv73 sequence obtained through screening as a template, and combining optimization algorithms (CAI optimization, MFE stability, GC content, restriction site exclusion, etc.), a linear DNA template containing a T7 promoter, 5'UTR, Kozak sequence, scFv ORF, 3'UTR, and polyA tail was designed and synthesized. Based on this template, in vitro transcription was performed using a T7 RNA polymerase system with ATP, GTP, CTP, and N1-Me-pUTP as substrates. Simultaneously, a one-step chemical capping process was performed using CAP5011 reagent (Shenji).

[0073] The mRNA transcript SJ453-73 was obtained. The detection results are as follows:

[0074] SJ453-73: Concentration 1054.2 ng / μL, A260 / 280 1.97, integrity 97.8% (see [link to product]). Figure 2 The capping rate was 95.31% (see...). Figure 3 The tail length of polyA is 96–111 Å (see [reference]). Figure 4 ).

[0075] The above mRNA was transfected into MAC-T cells (bovine mammary epithelial cell line). Cells were collected 24 hours later for Western blotting analysis of His-tagged proteins. Results showed successful expression of scFv, with high protein expression levels and clear bands in SJ453-73 cells (see [link to relevant documentation]). Figure 5 The molecular weight is as expected.

[0076] Example 2

[0077] This embodiment describes the preparation and physicochemical property verification process of the mRNA-LNP formulation.

[0078] Lipid nanoparticles (LNPs) were prepared using the SM102 formulation. The SJ453-73-mRNA formulation was encapsulated using a Myanna Nano L+ microfluidic device. Key preparation parameters were as follows: mRNA dosage: 6.0 mg; preparation volume: 32.00 mL; final yield: SJ453-S-73-LNP 3.22 mg (yield 53.69%).

[0079] The LNP quality test results are shown in Table 1 below.

[0080] Table 1

[0081]

[0082] mRNA-LNP formulations all exhibited uniform physicochemical properties and moderate particle size (see [reference]). Figure 6 It features low PDI and high encapsulation efficiency, meeting the physicochemical standards for local delivery in animals.

[0083] Example 3

[0084] This embodiment uses an animal model to verify the effect of the mRNA-LNP formulation.

[0085] To verify the effectiveness of the mRNA-LNP formulation expressing anti-Staphylococcus aureus single-chain antibody (scFv) (hereinafter referred to as "M73") described in this invention in treating a bovine mastitis animal model, this embodiment uses ICR lactating female mice to establish a Staphylococcus aureus mastitis model and evaluates the antibacterial and anti-inflammatory effects of M73 under different dosage conditions.

[0086] (I) Establishment of animal models:

[0087] SPF-grade, 8-10 week old, 30-40g, postpartum lactating female ICR mice were selected. Staphylococcus aureus (CVCC3702 strain) bacterial suspension (1×10⁻⁶) was injected via the mammary duct. 7 Mice were infected with CFU / mL (100 μL) in their fourth pair of mammary glands, and milk was collected 48 hours later for somatic cell counting and viability testing. The average somatic cell count increased to 15.3 × 10⁻⁶ after modeling. 5 / mL, with an average viable count of 1.53×10⁹ / mL. 5 CFU / mL, with a modeling success rate of 100% (15 / 15) (see...) Figure 7 ).

[0088] (II) Experimental Design and Dosing Protocol:

[0089] Mice were divided into five groups: experimental groups (A1, A2, A3) and control groups (C1, C2). The experimental groups were given M73 preparations at doses of 10 μg, 20 μg, and 40 μg, respectively; the control groups were given the same volume of PBS (negative control) and penicillin (positive control, 15 mg / milk area), respectively. Each group was given a repeat injection 72 hours after the first injection, and the observation period was 14 days (Table 2).

[0090] Table 2

[0091] Grouping preparation Dosage Administration method Animal number A1 M73 10μg 400 μL injected via mammary duct 3 A2 M73 20μg 400 μL injected via mammary duct 3 A3 M73 40μg 400 μL injected via mammary duct 3 C1 PBS 400μL 400 μL injected via mammary duct 3 C2 penicillin 15mg / breast area 400 μL injected via mammary duct 3

[0092] (III) Methods for evaluating drug efficacy:

[0093] Somatic cell count: Somatic cell count in breast milk was performed using a milkCELL100 instrument.

[0094] Viable bacteria count: Staphylococcus aureus colony forming units (CFU / mL) were measured in milk samples.

[0095] Pathological analysis: HE staining was used to observe the structure and inflammatory infiltration of breast tissue.

[0096] Observation of systemic and local clinical manifestations: including mental state, appetite, redness and swelling of the breast area, and induration.

[0097] (iv) Test Results:

[0098] (1) Decreased somatic cell count (see also) Figure 8 ):

[0099] M73 showed a dose-dependent anti-inflammatory effect on day 7: A1 group (10 μg): somatic cell count decreased by 44%; A2 group (20 μg): decrease by 53%; A3 group (40 μg): decrease by 61%; C2 group (penicillin): decrease by 63%; no significant change in somatic cells was observed in the PBS group.

[0100] (2) Sterilization effect (see Figure 8 ):

[0101] M73 completely eliminated Staphylococcus aureus in the mammary glands by day 3 in the medium- and high-dose groups (20 μg and 40 μg); approximately 1.2 × 10⁻⁶ μg was still detectable in the low-dose group (10 μg) by day 7. 4 CFU / mL residual bacteria; the bactericidal effect of the penicillin group and the high-dose M73 group was comparable; the bacterial load in the PBS group continued to increase.

[0102] (3) Pathological improvement (see Figure 9 ):

[0103] HE staining showed that the mammary alveoli in the PBS group were severely damaged and inflammatory cells were widely infiltrated; the mammary tissue structure in the high-dose M73 group was well restored, with only mild lymphocyte infiltration, which was comparable to that in the penicillin group; and the low-dose group still had moderate inflammation.

[0104] (4) Safety observation:

[0105] No significant toxic side effects were observed in any of the mice treated with M73 during the experiment, and there were no abnormal changes in indicators such as weight, activity, and appetite.

[0106] The above animal model validation results show that the mRNA-LNP preparation M73 of the present invention has good biocompatibility and therapeutic effect, can significantly inhibit mastitis caused by Staphylococcus aureus infection, reduce inflammatory markers, restore tissue structure, and has good safety, and has the potential to become a new strategy for the treatment of mastitis in dairy cows.

[0107] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention, used to illustrate the technical solutions of the present invention, and not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such 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. Therefore, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An mRNA encoding a single-chain antibody against Staphylococcus aureus, characterized in that, The nucleotide sequence of the mRNA is shown in SEQ ID NO.

1.

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mRNA of claim 1, and further comprises a delivery vector for delivering the mRNA to local tissue of the bovine mammary gland.

3. The pharmaceutical composition according to claim 2, characterized in that, The delivery carrier is an LNP.

4. The pharmaceutical composition according to claim 3, characterized in that, The LNP has an average particle size of 80-100 nm and a PDI < 0.

2.

5. The pharmaceutical composition according to claim 3 or 4, characterized in that, The LNP was prepared using the SM102 formulation.

6. The use of the mRNA encoding an anti-Staphylococcus aureus single-chain antibody as described in claim 1 or the pharmaceutical composition as described in any one of claims 2-5 in the preparation of a medicament for treating bovine mastitis.

7. The application according to claim 6, characterized in that, The drug has the effect of blocking the adhesion and colonization of Staphylococcus aureus on the surface of breast tissue.

8. The application according to claim 6 or 7, characterized in that, The drug can effectively reduce the accumulation of bacteria on the surface of mammary ducts and alveoli.

9. The application according to any one of claims 6-8, characterized in that, The drug can inhibit the formation and maturation of bacterial biofilms.

10. The application according to any one of claims 6-9, characterized in that, The drug can relieve breast swelling, induration, and decreased milk production caused by bacterial infection.

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