Fusion protein and application thereof

Abstract

The invention discloses a fusion protein and application thereof. Belongs to the technical field of biological medicine. The invention provides a novel fusion molecular architecture which comprises fusion proteins coded by wza and hphA genes and can be used for research and development of nucleic acid vaccines or subunit vaccines. The invention finds that the new fusion molecule has good expression and secretion ability and immunogenicity, can provide effective immune protection effect, and significantly inhibits acinetobacter baumannii infection and colonization. The invention also provides corresponding recombinant nucleic acid, a gene expression cassette, a vector, a host cell, a pharmaceutical composition, a vaccine, application and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a fusion protein and its applications. Background Technology

[0002] Acinetobacter baumannii ( Acinetobacter baumannii Acinetobacter baumannii is an opportunistic Gram-negative bacterium widely found in hospital environments and is one of the important multidrug-resistant (MDR) pathogens causing nosocomial infections in recent years. Acinetobacter baumannii infection mainly leads to ventilator-associated pneumonia, bloodstream infections, meningitis, wound infections, and urinary tract infections. The mortality rate of severely infected patients can be as high as 30% to 50%.

[0003] Acinetobacter baumannii rapidly colonizes and invades the host lungs and surfaces of medical devices through various virulence factors, including its capsular polysaccharide, outer membrane proteins, lipopolysaccharide, and biofilm. It possesses extremely strong environmental survival capabilities, able to survive on dry surfaces for weeks or even months, and spreads through biofilms formed by ventilators, catheters, and medical devices. This pathogen carries multidrug-resistant (MDR) and even extensively drug-resistant (XDR) genes, and its resistance rate to last-line antibiotics such as carbapenems and polymyxins continues to rise, leading to high clinical treatment failure rates, significantly prolonged hospital stays, and a sharp increase in medical costs.

[0004] Safe and effective vaccines are considered a strategic means to fundamentally prevent Acinetobacter baumannii infection, reduce antibiotic use, and curb the spread of drug resistance. Current vaccine development for Acinetobacter baumannii mainly focuses on inactivated vaccines, recombinant protein vaccines, polysaccharide-protein conjugate vaccines, and outer membrane vesicle vaccines. However, these vaccines often suffer from problems such as single antigen, weak immune response, and limited protection. Furthermore, traditional inactivated vaccines have drawbacks such as complex production processes and high immunopathological risks, limiting their clinical application.

[0005] Therefore, there is an urgent need to find new antigen targets, new antigen combinations, or new vaccine preparation technologies to develop preventive or therapeutic vaccines against Acinetobacter baumannii. Summary of the Invention

[0006] In view of this, the present invention provides a fusion protein and its application.

[0007] To address the aforementioned problems, this invention proposes a novel fusion protein design scheme, aiming to develop a highly effective vaccine against Acinetobacter baumannii infection.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A fusion protein comprising wza protein or an antigen-selected fragment thereof, and hphA protein or an antigen-selected fragment thereof.

[0010] Furthermore, the antigen is derived from Gram-negative bacteria; preferably, from Acinetobacter baumannii (…). Acinetobacter baumannii ).

[0011] Furthermore, the fusion protein includes a wza antigen-selected fragment and an hphA antigen-selected fragment; The selected fragments of the wza antigen are N-terminal truncated, C-terminal truncated, transmembrane truncated, or extracellular fragments of the wza protein, used to stimulate a specific immune response against the wza antigen. The selected hphA antigen fragment is an N-terminal truncated, C-terminal truncated, or functional domain fragment of the hphA protein, used to stimulate a specific immune response against the hphA antigen.

[0012] Furthermore, the amino acid sequence of the wza antigen selected fragment is shown in SEQ ID No. 1; the amino acid sequence of the hphA antigen selected fragment is shown in SEQ ID No. 2.

[0013] Furthermore, the linker sequences are linked between the wza antigen-selected fragment and the hphA antigen-selected fragment.

[0014] Furthermore, the amino acid sequence of the linker sequence is shown in SEQ ID No. 3.

[0015] Furthermore, its amino acid sequence is shown in SEQ ID No. 4.

[0016] A recombinant nucleic acid molecule encoding the aforementioned fusion protein.

[0017] Furthermore, the nucleotide sequence of the recombinant nucleic acid molecule is shown in SEQ ID No. 5.

[0018] A recombinant gene expression cassette comprising the aforementioned recombinant nucleic acid molecules.

[0019] A recombinant vector comprising the aforementioned recombinant gene expression cassette.

[0020] A recombinant host cell comprising the aforementioned recombinant vector.

[0021] An immunogenic composition or pharmaceutical composition comprising the above-mentioned fusion protein, the above-mentioned recombinant nucleic acid molecule, the above-mentioned recombinant gene expression cassette, the above-mentioned recombinant vector, and the above-mentioned recombinant host cell; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

[0022] A recombinant vaccine comprising the aforementioned fusion protein, the aforementioned recombinant nucleic acid molecule, the aforementioned recombinant gene expression cassette, the aforementioned recombinant vector, the aforementioned recombinant host cell, and the aforementioned immunogenic composition or pharmaceutical composition.

[0023] The above-mentioned fusion protein, recombinant nucleic acid molecule, recombinant gene expression cassette, recombinant vector, and recombinant host cell are used in the preparation of drugs for the treatment or prevention of diseases caused by Acinetobacter baumannii.

[0024] Furthermore, the diseases caused by Acinetobacter baumannii include pneumonia, bloodstream infections, meningitis, wound infections, and urinary tract infections.

[0025] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: The wza antigen and hphA antigen fusion protein provided by this invention can induce effective protective immunity in mouse model animals, demonstrating good preventive effects against Acinetobacter baumannii infection. Therefore, this invention not only provides a novel antigen design option but also offers a multi-antigen fusion expression strategy, which can effectively improve the protective potency of vaccines, possessing extremely high commercial value and broad application prospects. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the molecular structure of the fusion protein expressed by the recombinant nucleic acid vaccine in Example 1 of the present invention.

[0028] Figure 2 This is a schematic diagram of an expression cassette containing the antigen sequence of the present invention in Embodiment 1 of the present invention.

[0029] Figure 3 This is a quality control peak diagram and purity test results of the recombinant nucleic acid in Example 3 of the present invention.

[0030] Figure 4 This is the expression effect of recombinant nucleic acid in HEK293T cells after in vitro transfection in Example 3 of the present invention.

[0031] Figure 5 This describes the immunization, challenge, and detection process in Embodiment 5 of the present invention.

[0032] Figure 6 The image shows the changes in bacterial load in the lungs of mice in Example 5 of this invention. In the image, A is a comparison photograph of a culture dish, and B is the statistical result of bacterial load. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Terms and Definitions The term "Acinetobacter baumannii" refers to Acinetobacter baumannii It is an aerobic, Gram-negative, opportunistic pathogen widely found in both natural and hospital environments. It exhibits strong environmental adaptability and drug resistance, commonly colonizing patient skin, respiratory tract, and the surfaces of medical equipment, and can cause hospital-acquired infections. This pathogen can lead to pneumonia, bloodstream infections, meningitis, urinary tract infections, and wound infections in humans. It is a significant nosocomial pathogen, particularly prevalent in intensive care unit (ICU) patients, mechanically ventilated individuals, immunocompromised populations, and trauma patients. It is a multidrug-resistant and extensively drug-resistant strain.

[0035] The term "wza protein" refers to a polysaccharide biosynthesis / export protein, a key outer membrane channel protein in the capsular polysaccharide export system of the Gram-negative bacterium Acinetobacter baumannii. As an octamer complex, it forms a transmembrane channel responsible for transporting intracellularly synthesized capsular polysaccharides and secreting them onto the cell surface, playing a crucial role in capsule formation, bacterial virulence, anti-phagocytic capacity, and environmental resistance. The wza antigen-selected fragments of this invention, such as N-terminal truncation, C-terminal truncation, transmembrane truncation, or extracellular fragments, can all stimulate a specific immune response against the wza antigen.

[0036] The term "hphA protein" refers to a heme carrier protein, a key protein in the highly efficient iron acquisition system expressed by *Acinetobacter baumannii* to adapt to iron-deficient host environments. It captures heme from host proteins such as hemoglobin with high affinity and transports it to receptors on the bacterial cell surface, ultimately delivering iron into the cell for bacterial growth. This protein plays a crucial role in bacterial overcoming the host's nutritional immune barrier and establishing and maintaining infection, and is an important virulence factor. The hphA antigen-selected fragments of this invention, such as N-terminal truncation, C-terminal truncation, or functional domain fragments, can all stimulate a specific immune response against the hphA antigen.

[0037] The term "immune response" refers to a humoral response, a cellular response, or both in an organism. Immune responses can be measured by assays, including but not limited to assays measuring the presence or amount of antibodies that specifically recognize proteins or cell surface proteins, assays measuring T cell activation or proliferation, and / or assays measuring the regulation of the activity or expression of one or more cytokines.

[0038] The terms "administration" or "inoculation" refer to the administration of the nucleic acid vaccine or vaccine composition based on the present invention, preferably via intramuscular or subcutaneous routes, although other routes of administration may also be used, such as oral, intranasal (e.g., aerosol or other non-injectable), intralymphatic, intradermal, intraperitoneal, groin, rectal, or vaginal administration, or by combination of routes. Inguinal administration in animals is preferred. Boosting regimens can be used to adjust the administration regimen to provide optimal immunization.

[0039] The term “expression” includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0040] The term "recombinant nucleic acid molecule" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can then be transformed into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," "recombinant protein," or "fusion protein."

[0041] The term "recombinant expression vector" refers to a DNA structure containing a polynucleotide encoding, for example, a desired polypeptide. A recombinant expression vector may include, for example, a set of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence transcribed into mRNA and translated into protein; and (3) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used. Possible vectors used in this invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, from viruses such as lentiviruses, retroviruses, vaccinia virus, adenovirus, fowlpox virus, baculovirus, SV40, and pseudorabies virus. Self-replicating vectors and non-self-replicating vectors are included.

[0042] The term "mRNA" refers to messenger RNA, which is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and can guide protein synthesis.

[0043] The term "5'-UTR" refers to the "5' untranslated region" or "5'UTR," which is a portion of a gene transcribed into a primary RNA transcript (precursor mRNA) and located upstream of the coding sequence. Primary transcripts are the initial RNA products, containing introns and exons, produced by DNA transcription. Many primary transcripts must undergo RNA processing to form physiologically active RNA. The processing to form mature mRNA includes end modification, intron removal, capping, and / or cleavage of individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of mRNA is a portion of mRNA that is not translated into protein and is located upstream of the coding sequence. In the genome sequence, the 5'UTR is generally defined as the region between the transcription start site and the start codon. The length of the 5' untranslated region (5'UTR) of vertebrate mRNA can range from tens to hundreds of bases.

[0044] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR," which refers to the region located at the 3' end of a gene, downstream of the stop codon in a protein-coding region, and which is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule. The 3'-UTR typically extends from the stop codon of the translation product to a poly(A) sequence that usually attaches after transcription. The 3'-UTR of mammalian mRNA typically has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-UTR may contain one or more inverted repeats that can fold to create stem-loop structures that act as barriers to ribonucleases or interact with proteins known to enhance RNA stability, such as RNA-binding proteins.

[0045] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells.

[0046] The terms “individual,” “patient,” or “subject” include mammals. Mammals include, but are not limited to, domesticated animals (e.g., pigs, cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats).

[0047] The terms "transformation," "transfection," and "transduction" refer to the process of introducing exogenous DNA or RNA into a host.

[0048] The term "pharmaceutical combination" or "pharmaceutical composition" refers to excipients widely used in the pharmaceutical manufacturing industry. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, and also to provide a method for its effective absorption in a subject. Pharmaceutically acceptable carriers can be inert fillers or active ingredients that provide a function to the pharmaceutical combination (e.g., stabilizing the overall pH of the composition or preventing degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, flow aids, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0049] The term "treatment" refers to exposing a subject to (e.g., administering medication) a recombinant vaccine, composition, etc., based on the present invention after contracting a disease, thereby reducing the symptoms of the disease compared to when not exposed, and does not imply the necessity of completely suppressing the symptoms of the disease. Contracting a disease means that the body has developed symptoms of a disease.

[0050] The term "prevention" refers to the reduction of symptoms after contracting a disease by exposing (e.g., administering medication) a subject to a recombinant vaccine, composition, etc. based on the present invention before contracting the disease, compared to the absence of exposure, and does not imply the necessity of completely suppressing the disease.

[0051] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] This invention discloses a fusion molecular architecture that effectively improves the antigen presentation efficiency of cells, a method for preparing recombinant vaccines based on this architecture, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0053] The fusion protein and encoding nucleic acid and its elements provided by this invention, as well as the preparation method and application, all use commercially available raw materials and reagents.

[0054] Example 1 Construction of recombinant nucleic acid vaccines Figure 1 This is a schematic diagram of the molecular structure of the fusion protein expressed by the recombinant nucleic acid vaccine of the present invention.

[0055] In order to prepare a product capable of producing such Figure 1 The recombinant nucleic acid vaccine with the molecular structure shown first involves constructing a gene expression cassette to express the antigen sequence of this invention. The expression cassette is as follows: Figure 2 As shown, from the 5' end to the 3' end, it sequentially includes: 5'UTR, CDS region, 3'UTR, and PolyA, wherein the CDS region contains the molecular structure of the fusion protein of the present invention.

[0056] Subsequently, the complete gene expression cassette sequence was optimized based on codon degeneracy, and the DNA sequence was directly obtained through gene synthesis (commissioned to GenScript).

[0057] Finally, the synthesized gene expression cassette DNA sequence was inserted into an expression vector that can be used for in vitro RNA transcription to obtain a vector plasmid for preparing recombinant nucleic acid vaccines.

[0058] According to the above method, a carrier for use in subsequent embodiments is prepared: Step a: Synthesize the “wza antigen-selected fragment-hphA antigen-selected fragment” gene fragments, which are linked together by a linker sequence as shown in SEQ ID No. 3. The nucleic acid sequence of the “wza antigen-selected fragment-hphA antigen-selected fragment” fusion gene is shown in SEQ ID No. 5, and the amino acid sequence of the fusion protein encoded by this fusion gene is shown in SEQ ID No. 4. Specifically, the amino acid sequence of the wza antigen-selected fragment is shown in SEQ ID No. 1, and the amino acid sequence of the hphA antigen-selected fragment is shown in SEQ ID No. 2.

[0059] MCAVTSGLQTYDIPSEGVYKTDLGTTVNVVKISQETLPAIQPAQIDYQRDYASLFKTQQSIYRLSPGDILSIQLWAYPEITPPVNGISSEQSIQANGYPIDQSGYIQFPLVGRYKAAGKTLAQVNRELHNQLARFLKNPDVIVRVLSYEGQRFSVQGSVTKGQFYLSDQPVSIYTALGMAGGVTTTGDNTYIQLIRNGRTYNLNTIDLEKAGYSLHKLLVQPNDTIYVSTRENQKIYVMGESGKNQALPMRDQGMTLTDALGESLGINPNSASASRIYVVRTNPNDHVTEIYHLNLMSLGDFGLANQFRLRSNDIVYIDATGLTRWQRVVNQIIPFSNALYNIDRLGQ, SEQ ID No. 1.

[0060] GIDGISSNESNIKIGAAANASHPGGVAAVSVQAAGAPYNAFTGFSSLKGLAQAFAAQGTSNTNVAVGSKTFNISHIPVSAMPPSHSALGNFNFGQVGTQEVYFGEWWKAGDTPASASHTVYYAGDNTNTT, SEQ ID No. 2.

[0061] GGSGGGGSGG, SEQ ID No. 3.

[0062] MCAVTSGLQTYDIPSEGVYKTDLGTTVNVVKISQETLPAIQPAQIDYQRDYASLFKTQQSIYRLSPGDILSIQLWAYPEITPPVNGISSEQSIQANGYPIDQSGYIQFPLVGRYKAAGKTLAQVNRELHNQLARFLKNPDVIVRVLSYEGQRFSVQGSVTKGQFYLSDQPVSIYTALGMAGGVTTTGDNTYIQLIRNGRTYNLNTIDLEKAGYSLHKLLVQPNDTIYVSTRENQKIYVMGESGKNQALPMRDQGMTLTDALGESLGINPNSASASRIYVVRTNPNDHVTEIYHLNLMSLGDFGLANQFRLRSNDIVYIDATGLTRWQRVVNQIIPFSNALYNIDRLGQGGSGGGSGGGIDGISSNESNIKIGAAANASHPGGVAAVSVQAAGAPYNAFTGFSSLKGLAQAFAAQGTSNTNVAVGSKTFNISHIPVSAMPPSHSALGNFNFGQVGTQEVYFGEWKAGDTPASASHTVYYAGDNTNTT, SEQ ID No. 4.

[0063]

[0064] Step b: Construct a nucleic acid vaccine architecture carrier.

[0065] The nucleic acid vaccine architecture vector is a DNA plasmid template for in vitro transcription of mRNA, namely pKC57IVT, which sequentially contains a T7 promoter, a 5'-UTR, a template CDS sequence, a 6×His tag sequence, a 3'-UTR, a polyA, a plasmid replication origin, an antibiotic resistance gene and its promoter.

[0066] Step c: Prepare recombinant plasmids.

[0067] By seamlessly cloning the nucleotide sequence synthesized in step a (as shown in SEQ ID No. 5) to replace the template CDS sequence in the structural vector of step b, a recombinant nucleic acid vaccine preparation vector based on the present invention is obtained. The specific operation steps are as follows: (1) The target gene was amplified by high-fidelity DNA polymerase PCR to ensure no mutation; the plasmid DNA other than the template CDS sequence was amplified by PCR to obtain a linearized vector with homologous sequences at both ends to the inserted fragment.

[0068] (2) Use gel recovery to recover PCR products with high purity and remove primers, enzymes and templates.

[0069] (3) Perform Gibson assembly reaction: Prepare the mixture on ice according to the instructions of NEB commercial kit Gibson Assembly® HiFi MasterMix, place the reaction tube in the PCR instrument, and incubate at 50 °C for 60 minutes.

[0070] (4) Transformation: The cooled assembly reaction product (usually 2~5 μL) is directly added to competent cells DH5α for heat shock transformation.

[0071] (5) Screening of positive bacterial clones: The transformed bacterial culture was plated on LB agar plates containing the corresponding antibiotic (based on the vector resistance marker) and incubated overnight at 37 °C. The molecular weight of the target gene was identified by colony PCR. Clones with preliminarily verified correct molecular weights were sent for sequencing to ensure that the sequences were completely correct and seamless.

[0072] (6) Sequencing identification and plasmid extraction: The correctly sequenced cloned bacteria were amplified by liquid LB shaking, and the plasmid was extracted using a plasmid extraction kit and the size and purity of the plasmid were detected by agarose gel electrophoresis. Finally, the vector plasmid for mRNA production was obtained.

[0073] Example 2 Preparation of recombinant nucleic acids (I) Preparation of Capped mRNA Step a: Linearize the recombinant plasmid obtained in step c of Example 1 by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0074] Enzymatic digestion was performed using the BspQI restriction endonuclease of nearshore proteins. The reaction system is as follows: 10×BspQI Reaction Buffer 5 μL, DNA 1 μL, BspQI 1 μL, RNase Free WaterUp to 50 μL.

[0075] Reaction conditions: React at 50 ℃ for 2 hours, and then incubate at 80 ℃ for 20 minutes after the reaction is completed to terminate the reaction.

[0076] Step b: The linearized plasmid was transcribed in vitro to degrade the template DNA, and a 7-methylguanylic acid cap structure was added to the 5' end of the transcribed mRNA. The specific steps are as follows: (1) The reaction system was prepared according to the T7 High Yield RNA Transcription Kit 2.0 for nearshore proteins and an in vitro transcription reaction was performed. The reaction system is as follows: 10×Transcription Buffer 2 μL, ATP / GTP / CTP / UTP (Each 200mM) 0.75~1 μL each, Enzyme Mix 2.0 1 μL, Template DNA 0.1~1 μg, RNase Free Water Up to 20 μL.

[0077] Reaction conditions: Gently mix the components, briefly centrifuge to collect, and incubate at 37 °C for 2 hours.

[0078] (2) Degradation of template DNA: Add 2-4 U of DNase I to the reaction system and incubate at 37°C for 15 minutes to digest the transcribed DNA template.

[0079] (3) Product purification: For every 20 μL of transcription product, add 30 μL of lithium chloride precipitation solution (7.5 M Lithium Chloride, 50 mM EDTA) and 30 μL of RNase-free water, mix well, and incubate at -20 ℃ for 30 minutes. Centrifuge at 12000 rpm for 15 minutes, discard the supernatant, and collect the precipitate. Wash three times with pre-cooled 75% ethanol. After reconstitution with RNase-free water, measure OD260 / 280.

[0080] (4) Capping reaction: The following steps were performed using the Cap 1 Capping System for nearshore proteins: ① Dilute an appropriate amount of RNA to 67 μL with RNase-free water; ② Heat the RNA at 65 ℃ for 5 minutes, then place it on ice for 5 minutes after heating. ③ Add the following components in sequence: Denatured RNA 67 μL, 10x Capping Reaction Buffer 10 μL, GTP (10 mM) 10 μL, SAM (20 mM) 2.5 μL, Recombinant RNase Inhibitor (40 U / μL) 2.5 μL, mRNA Cap 2´-O-Methyltransferase (100 U / μL) 4 μL, Vaccinia Capping Enzyme (10 U / μL) 4 μL; ④ After mixing, react at 37 ℃ for 30 minutes. RNA capping is complete, and subsequent experiments can be carried out.

[0081] (II) Preparation of Uncapped mRNA Vaccines Step a: Linearize the recombinant plasmid obtained in step c of Example 1 by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0082] Enzymatic digestion was performed using the BspQI restriction endonuclease of nearshore proteins. The reaction system is as follows: 10×BspQI Reaction Buffer 5 μL, DNA 1 μL, BspQI 1 μL, RNase Free WaterUp to 50 μL.

[0083] Reaction conditions: React at 50 ℃ for 2 hours, and then incubate at 80 ℃ for 20 minutes after the reaction is completed to terminate the reaction.

[0084] Step b: Perform in vitro transcription of the linearized plasmid, the specific steps of which are as follows: (1) The reaction system was prepared according to the T7 High Yield RNA Transcription Kit 2.0 for nearshore proteins and an in vitro transcription reaction was performed. The reaction system is as follows: 10×Transcription Buffer 2 μL, ATP / GTP / CTP / UTP (Each 200mM) 0.75~1 μL each, Enzyme Mix 2.0 1 μL, Template DNA 0.1~1 μg, RNase Free Water Up to 20 μL.

[0085] Reaction conditions: Gently mix the components, briefly centrifuge to collect, and incubate at 37 °C for 2 hours.

[0086] (2) Degradation of template DNA: Add 2-4 U of DNase I to the reaction system and incubate at 37 °C for 15 minutes to digest the transcribed DNA template.

[0087] (3) Product purification: For every 20 μL of transcription product, add 30 μL of lithium chloride precipitation solution (7.5 M Lithium Chloride, 50 mM EDTA) and 30 μL of RNase-free water, mix well, and incubate at -20 ℃ for 30 minutes. Centrifuge at 12000 rpm for 15 minutes, discard the supernatant, and collect the precipitate. Wash three times with pre-cooled 75% ethanol. After reconstitution with RNase-free water, measure OD260 / 280.

[0088] (III) DNA vaccine preparation Step a: The Escherichia coli DH5α containing the recombinant plasmid obtained in step c of Example 1 was amplified and cultured in shake flasks (37 ℃, LB medium) until OD600≈4~6, and the bacterial cells were collected by centrifugation.

[0089] Step b: Plasmid purification: The plasmid was purified using an endotoxin-free plasmid extraction kit (EndoFree® Plasmid Kit) via alkaline lysis combined with column chromatography. After purification, the plasmid concentration, purity (A260 / A280≈1.8~2.0) and endotoxin level (<0.1 EU / μg DNA) were measured, and the plasmid was stored at -20℃ for later use.

[0090] Example 3 Quality control and expression efficacy verification of recombinant nucleic acids Subsequent experiments were conducted using the capped mRNA prepared in step (i) of Example 2.

[0091] The purity of recombinant nucleic acids was determined using capillary electrophoresis. Figure 3 The results showed that the purity of the recombinant nucleic acid used in the experiment was 90.6%, which meets the quality requirements for cell transfection experiments and vaccine production.

[0092] The recombinant nucleic acid was transfected into HEK293T cells using Lipofectamine 3000 (Thermo Fisher Scientific) transfection reagent according to the instructions. After culturing in vitro for 24 hours, the protein and cell culture supernatant were collected. Each protein sample (40 μg) was mixed with 5× loading buffer, vortexed, heated at 100 °C for 5 minutes, and then centrifuged at 12,000 rpm for 5 minutes at 4 °C.

[0093] Subsequently, Western blot (WB) detection was performed. The detection process included sample heating and denaturation, polyacrylamide gel electrophoresis (SDS-PAGE), wet transfer to a PVDF membrane, blocking with 5% skim milk (TBST), incubation and washing with Yamei His-Tag rabbit polyclonal antibody primary antibody, incubation and washing with Yamei HRP-labeled goat anti-rabbit IgG secondary antibody, and exposure with a fully automated chemiluminescence analyzer.

[0094] Figure 4 The results of in vitro Western blotting (WB) analysis of recombinant nucleic acid transfected into HEK293T cells were presented. The expressed antigen is a humoral immune antigen, which theoretically should be significantly expressed in the cell secretion supernatant, with an estimated protein molecular weight of 52.5 kDa. The results show that the recombinant nucleic acid can be expressed intracellularly and secreted into the supernatant, demonstrating that the recombinant nucleic acid designed based on this invention can be correctly expressed and secreted in eukaryotic cells, and the expressed protein structure is correct and stable, which is beneficial for immunoepitaxy presentation.

[0095] Example 4 Preparation of recombinant nucleic acid vaccines To further verify the immunomodulatory effects of recombinant nucleic acids in animal models, this embodiment encapsulates the capped mRNA prepared in Example 2 into lipid nanoparticles (LNPs). The specific steps are as follows: (1) Capped mRNA was encapsulated in SM102 LNPs via a microfluidic system: SM102, 1,2-distearate phosphatidylcholine (DSPC), cholesterol, and polyethylene glycol 2000 derivative (DMG-PEG 2000) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. The mRNA sample was diluted to the target final concentration with 20 mM sodium acetate buffer (pH 5.5). The aqueous phase and organic phase were mixed in a microfluidic mixing chip at a flow rate ratio of 95:5. The weight ratio of lipids to mRNA was approximately 25:1, allowing the positively charged lipids and negatively charged mRNA to spontaneously self-assemble to obtain mRNA-LNPs.

[0096] (2) Dilution and filtration: The obtained mRNA-LNP was adjusted to pH 7.0~7.4 with 1 M Tris-HCl (pH 8.0), filtered through a 0.22 μm filter membrane and stored at 2~8 °C.

[0097] Particle size, polydispersity index (PDI), and zeta potential (ZP) were determined using dynamic light scattering. mRNA-LNP encapsulation efficiency was determined using the Quant-iTRiboGreen RNA Quantification Kit (Thermo Fisher Scientific).

[0098] The test results showed that the encapsulation efficiency of the recombinant nucleic acid vaccine prepared by encapsulation was 85.5%, the particle size was 120.8 ± 33.6 nm, the PDI was 0.21, and the zeta potential was 2.5 mV.

[0099] Example 5 The preventive effect of recombinant nucleic acid vaccines against Acinetobacter baumannii infection. To verify whether the nucleic acid vaccine based on the present invention has an immune protective effect, this embodiment compares the immunization and challenge of vaccine-immunized mice (vaccine group, using the recombinant nucleic acid vaccine prepared in Example 4) with unimmunized mice (challenge control group). One mouse was neither immunized nor challenged and served as the negative control group.

[0100] Eleven 6-8 week old BALB / c mice, weighing 18-25g, were selected for the experiment and housed in individual cages with constant temperature and humidity. These mice were acclimatized to the environment for 7 days prior to the experiment. The cage temperature was 20-26℃, and the humidity was 40-70%. A day-night cycle was implemented, with light from 8:00 AM to 8:00 PM and darkness from 8:00 PM to 8:00 AM the following day. Sufficient feed was continuously provided, with unlimited access to sterile water via a continuous water bottle. After acclimatization, the mice were randomly assigned to groups, and each mouse was ear-tagged. Details are shown in Table 1. In this table and below, dosages refer to the amount of active ingredient.

[0101] Table 1. Mouse immunization experiment grouping and immunization procedure

[0102] Each group of mice was immunized twice according to the immunization schedule in Table 1. On Day 35, 20 μL of Acinetobacter baumannii (product number: BNCC337173) bacterial suspension (1×10⁻⁶) was administered. 9 Mice were infected nasally with CFU / mL vaccine. On Day 37, mice were sacrificed, and lung tissue was collected to detect bacterial load and assess the immunoprotective effect of the vaccine. The immunization, challenge, and sampling schedule is as follows: Figure 5 As shown.

[0103] 10 mg of lung tissue was aseptically excised from each mouse and homogenized on ice with sterile PBS buffer until no significant precipitation occurred. The lung tissue homogenate was diluted 1:10, 1:100000, or undiluted using sterile PBS buffer. After thorough mixing, 100 μL of the diluted solution was evenly spread onto a solid culture medium and incubated for 24 h. The culture dish was then removed to count and photograph bacterial clones, and the protective effect of the vaccine on mice was observed. Results are as follows: Figure 6 As shown.

[0104] Figure 6Photo A shows a comparison of bacterial load (number of colonies on a plate) in the lung tissue of mice after immune challenge. The results show that the bacterial load in the negative control group was 0, confirming the validity of the comparison experiment. Figure 6 B is a statistical comparison of the bacterial load (number of colonies on agar plates) in the lung tissues of mice in the vaccine group, the challenge control group, and the negative control group after immunization and challenge. The results show that, compared with the challenge control group, the bacterial load of Acinetobacter baumannii in the vaccine group mice decreased by an average of about 1000 times.

[0105] Analysis of the above results shows that mice immunized with the vaccine of the present invention exhibited resistance to Acinetobacter baumannii infection, demonstrating that the vaccine of the present invention can induce immune protection against Acinetobacter baumannii in mice and prevent Acinetobacter baumannii infection.

[0106] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0107] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fusion protein, characterized in that, The fusion proteins include wza protein or its antigen-selected fragment, and hphA protein or its antigen-selected fragment.

2. The fusion protein as described in claim 1, characterized in that, The fusion protein includes a wza antigen-selected fragment and an hphA antigen-selected fragment; The selected fragments of the wza antigen are N-terminal truncated, C-terminal truncated, transmembrane truncated, or extracellular fragments of the wza protein, used to stimulate a specific immune response against the wza antigen. The selected hphA antigen fragment is an N-terminal truncated, C-terminal truncated, or functional domain fragment of the hphA protein, used to stimulate a specific immune response against the hphA antigen.

3. The fusion protein as described in claim 2, characterized in that, The amino acid sequence of the selected fragment of the wza antigen is shown in SEQ ID No. 1; the amino acid sequence of the selected fragment of the hphA antigen is shown in SEQ ID No.

2.

4. The fusion protein as described in claim 2, characterized in that, The linker sequence is linked between the wza antigen truncation fragment and the hphA antigen truncation fragment.

5. The fusion protein as described in claim 2, characterized in that, Its amino acid sequence is shown in SEQ ID No.

4.

6. A recombinant nucleic acid molecule, characterized in that, Encodes the fusion protein according to any one of claims 1 to 5.

7. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule as described in claim 6.

8. A recombinant vector, characterized in that, It includes the recombinant gene expression cassette as described in claim 7.

9. A recombinant host cell, characterized in that, It includes the recombinant vector as described in claim 8.

10. The use of the fusion protein according to any one of claims 1 to 5, the recombinant nucleic acid molecule according to claim 6, the recombinant gene expression cassette according to claim 7, the recombinant vector according to claim 8, and the recombinant host cell according to claim 9 in the preparation of a medicament for treating or preventing diseases caused by Acinetobacter baumannii.

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