Immunogenic composition for preventing and treating chlamydia psittaci infection and application

Abstract

The invention relates to a novel fusion protein, an immunogenic composition, a recombinant vaccine and the like for preventing and treating chlamydia psittaci infection. The fusion protein provided by the invention contains fusion proteins coded by four genes of rplE, rpsC, rpsE and rpsG, and has good immunogenicity. Compared with a traditional antibiotic therapy, the nucleic acid vaccine constructed by the fusion protein can provide an effective immune protection effect, remarkably inhibit chlamydia psittaci infection, effectively eliminate chlamydia psittaci colonization and avoid repeated infection, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, particularly the field of immunopharmaceutical technology, and specifically relates to a fusion protein, immunogenic composition, and applications that can be used to prevent and treat Chlamydia psittaci infection. Background Technology

[0002] Chlamydia psittaci ( Chlamydia psittaci Chlamydia psittaci, also known as avian chlamydia, is a strictly intracellular parasite with zoonotic risks. It not only causes highly pathogenic outbreaks in birds but also poses a serious threat of zoonotic infectious diseases. In human medicine, Chlamydia psittaci infection primarily leads to psittacosis, clinically manifested as high fever, severe headache, and atypical pneumonia (commonly known as "bird plague"). Severe cases can rapidly progress to severe pneumonia and multiple organ failure, with a mortality rate as high as 10%-20%, especially serious in pregnant women and immunocompromised individuals. In livestock and poultry farming, this pathogen can cause a sharp decline in egg production in laying hens (up to 20% or more), fibrinous pericarditis and hepatitis in broilers (with a herd mortality rate of up to 30%), epidemic abortions in calves and pregnant ewes (abortion rate exceeding 40%), and pneumonia and polyarthritis in pigs, causing billions of dollars in economic losses to the global livestock industry annually.

[0003] Chlamydia psittaci rapidly invades the host's respiratory epithelial cells and macrophages through surface antigens such as the major outer membrane protein (MOMP), forming intracellular inclusion bodies to evade host immune clearance. This causes persistent immune stimulation and inflammatory damage, leading to immunosuppression and increased susceptibility to secondary infections with other pathogens. In poultry farms, mixed infections with Escherichia coli, Bordetella avianis, and other pathogens are common, complicating the disease and increasing mortality rates to over 50%. Notably, Chlamydia psittaci exhibits strong resistance to common disinfectants and environmental factors, surviving for months in dried feces. Its aerosol transmission efficiency is extremely high, posing a significant infection risk to occupational groups such as poultry farmers, veterinarians, and poultry processing plant workers.

[0004] Safe and effective vaccines are considered a key means to fundamentally control Chlamydia psittaci infection and transmission. Currently, internationally researched Chlamydia psittaci vaccines mainly include inactivated whole-cell vaccines, subunit vaccines (such as key antigens like MOMP, PmpD, and CPAF), and emerging viral vector vaccines. These candidate vaccines can induce a certain immune response in experimental animals and reduce the pathogen load after acute infection, but they generally fail to provide complete sterilizing immunity and cannot effectively clear intracellular parasites, block persistent infection, or vertical transmission. In recent years, domestic researchers have also been actively exploring novel vaccine strategies, such as a recombinant vaccine based on the Chlamydia psittaci outer membrane protein N-PmpD disclosed in patent CN102643336A (publication date 2012-08-22), and a live vector vaccine based on the MOMP protein disclosed in patent CN1864747A (publication date 2006-11-22).

[0005] The ribosomal proteins rplE / rpsC / rpsE / rpsG encode ribosomal proteins with the 50S large and 30S small subunits. These are highly conserved housekeeping genes, primarily involved in ribosome assembly and protein synthesis. These proteins are generally not found on the surface of bacteria, but mainly within the cell; therefore, current technologies do not disclose or teach their use in constructing vaccines for pathogen control.

[0006] However, existing strategies for preventing and treating Chlamydia psittaci still face significant bottlenecks: inactivated whole-cell vaccines have weak immunogenicity and pose an immunopathological risk due to Th2 bias; while live vector vaccines can induce cellular immunity, they carry the potential safety hazard of virulence reversion; and while MOMP-based subunit vaccines are highly safe, they primarily induce antibody production and cannot eliminate intracellular Chlamydia psittaci. With a deeper understanding of Chlamydia invasion mechanisms and host immune responses, and the rapid development of reverse vaccinology, structural biology, and multi-antigen co-delivery technologies, developing novel genetically engineered vaccines that can serve both preventative and therapeutic purposes and have high safety has become a major direction for breakthroughs in the field of Chlamydia psittaci vaccines. There is an urgent need in this field for a vaccine that can effectively eliminate Chlamydia psittaci colonization and prevent reinfection. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a novel immunogenic composition, fusion protein, recombinant vaccine, and molecular architecture design and application for the prevention and treatment of Chlamydia psittaci infection. This invention provides a novel fusion molecular architecture comprising fusion proteins encoded by four genes: rplE, rpsC, rpsE, and rpsG, which can be used in the development of nucleic acid vaccines. This invention reveals that the novel fusion molecule exhibits good immunogenicity and provides effective immune protection, significantly inhibiting Chlamydia psittaci infection, effectively clearing Chlamydia psittaci colonization, and preventing reinfection. This invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, and applications.

[0008] One aspect of the present invention provides a fusion protein, characterized in that the fusion protein comprises a 50S ribosomal protein L5 (rplE) antigen or an antigenic fragment thereof, a 30S ribosomal protein S3 (rpsC) antigen or an antigenic fragment thereof, a 30S ribosomal protein S5 (rpsE) antigen or an antigenic fragment thereof, and a 30S ribosomal protein S7 (rpsG) antigen or an antigenic fragment thereof.

[0009] Furthermore, the antigen or its antigenic fragment is derived from Chlamydia; preferably, from Chlamydia psittaci (…). Chlamydia psittaci ).

[0010] Further, the amino acid sequence of the rplE antigen has at least 95% sequence identity with SEQ ID NO: 1, preferably 96%, 97%, 98%, 99%, or 100%; the amino acid sequence of the rpsC antigen has at least 95% sequence identity with SEQ ID NO: 2, preferably 96%, 97%, 98%, 99%, or 100%; the amino acid sequence of the rpsE antigen has at least 95% sequence identity with SEQ ID NO: 3, preferably 96%, 97%, 98%, 99%, or 100%; the amino acid sequence of the rpsG antigen has at least 95% sequence identity with SEQ ID NO: 4, preferably 96%, 97%, 98%, 99%, or 100%; more preferably, the amino acid sequence of the rplE antigen is as shown in SEQ ID NO: 1, the amino acid sequence of the rpsC antigen is as shown in SEQ ID NO: 2, the amino acid sequence of the rpsE antigen is as shown in SEQ ID NO: 3, and the amino acid sequence of the rpsG antigen is as shown in SEQ ID NO: 4. NO:4 shown.

[0011] Furthermore, antigens or their antigenic fragments may optionally be linked by a linker sequence; preferably, the amino acid sequence of the linker sequence is shown in SEQ ID NO: 5.

[0012] Furthermore, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 6.

[0013] Furthermore, the N-terminus of the fusion protein also includes a signal peptide and / or any secretion-promoting element, and / or the C-terminus also includes a protein stabilization motif element.

[0014] Another aspect of the present invention provides a recombinant nucleic acid molecule characterized in that it encodes the fusion protein described in any one of the present invention.

[0015] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention.

[0016] Furthermore, the recombinant gene expression cassette also includes one or more of a promoter, a terminator, and a regulatory sequence.

[0017] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention or the recombinant gene expression cassette described in the present invention.

[0018] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0019] Furthermore, the prokaryotic vector includes, but is not limited to, Escherichia coli vectors.

[0020] Furthermore, the Escherichia coli vector includes, but is not limited to, pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0021] Furthermore, the eukaryotic vector includes, but is not limited to, yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0022] Furthermore, the yeast expression vector includes, but is not limited to, pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0023] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention, or the recombinant gene expression cassette described in the present invention, or the recombinant vector described in the present invention.

[0024] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0025] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0026] Furthermore, the yeast cells include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0027] Furthermore, the prokaryotic cells include, but are not limited to, Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0028] Furthermore, the *E. coli* cells include, but are not limited to, BL21(DE3), DH5α, TOP10, and Rosetta.

[0029] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

[0030] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins as described in any one of the present invention, and / or one or more recombinant nucleic acid molecules as described in the present invention, and / or one or more recombinant gene expression cassettes as described in the present invention, and / or one or more recombinant vectors as described in the present invention, and / or one or more recombinant host cells as described in the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions as described in the present invention. Preferably, the recombinant vaccine is a nucleic acid vaccine; more preferably, the nucleic acid vaccine is a DNA vaccine or an RNA vaccine.

[0031] Another aspect of the present invention provides the use of one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present invention, and / or one or more recombinant vaccines according to the present invention in the preparation of vaccines or pharmaceuticals for prevention, treatment and / or inoculation.

[0032] Another aspect of the present invention provides a method for preventing and / or treating infection or disease in humans, mammals, or birds caused by Chlamydia psittaci, characterized in that it comprises administering to a subject one or more fusion proteins according to any one of the present inventions, and / or one or more recombinant nucleic acid molecules according to the present inventions, and / or one or more recombinant gene expression cassettes according to the present inventions, and / or one or more recombinant vectors according to the present inventions, and / or one or more recombinant host cells according to the present inventions, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present inventions, and / or one or more recombinant vaccines according to the present inventions.

[0033] Furthermore, the drug is used to prevent and / or treat infections or diseases in humans, mammals, or birds caused by Chlamydia psittaci; preferably, the infection or disease in humans or mammals caused by Chlamydia psittaci is psittaci; preferably, the infection or disease in birds caused by Chlamydia psittaci is avian plague; more preferably, the birds are selected from poultry.

[0034] The fusion proteins, pharmaceutical compositions, recombinant vaccines, etc. of the present invention have the following beneficial technical effects: (1) The present invention creatively screens out four antigens: rplE, rpsC, rpsE and rpsG. The prior art has not disclosed the use of the above antigens to construct vaccines for the prevention and treatment of pathogens. However, the present invention uses the above four antigens to construct fusion protein and nucleic acid vaccines for the prevention and treatment of Chlamydia psittaci infection, and has achieved unexpected technical effects.

[0035] (2) The rplE-rpsC-rpsE-rpsG vaccine of the present invention can be significantly expressed in cells and significant expression can be detected in cell lysates, proving that the vaccine designed based on the present invention can be correctly expressed in eukaryotic cells and the expressed protein structure is correct and stable, which is conducive to the presentation of immune epitopes.

[0036] (3) The results of Example 4 of the present invention show that 73% of the individuals in vaccine treatment group 1 (five immunizations) turned negative on Day 28, and all turned negative by Day 42. The retest on Day 70 was still negative, indicating that Chlamydia psittaci colonization was effectively eliminated and no reinfection occurred. In vaccine treatment group 2 (four immunizations), 59% of the individuals turned negative on Day 21, and 99% reached negative on Day 35. The retest on Day 70 was negative for all of them, which also confirms that the vaccine can completely eliminate the pathogen and prevent reinfection. In contrast, although all of the individuals in the antibiotic treatment group turned negative on Day 28, some individuals tested positive again on Day 42, and all tested positive again on Day 70. This indicates that antibiotic treatment cannot completely eliminate pathogen colonization, and the animals still face the risk of reinfection.

[0037] (4) For Chlamydia psittaci infection, traditional antibiotic therapy is long-term, complicated and the protective effect is not sustainable. However, the antigen and fusion protein architecture provided by this invention can induce an effective immune response in experimental parrots, showing good therapeutic and preventive efficacy against Chlamydia psittaci infection. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the molecular structure of the fusion protein expressed in the vaccine of the present invention.

[0039] Figure 2 This is a schematic diagram of a template plasmid containing the present invention.

[0040] Figure 3 This is a quality control peak diagram and purity test results for a nucleic acid vaccine containing the antigen sequence of this invention.

[0041] Figure 4 This is the expression effect of the vaccine of the present invention after in vitro transfection into HEK293T cells.

[0042] Figure 5 This invention describes the immunization, treatment, and sampling procedures for the vaccine and antibiotic treatment group in a parrot population infected with Chlamydia psittaci.

[0043] Figure 6A , Figure 6B , Figure 6C The dynamic changes in bacterial load and positivity rate were recorded in vaccine treatment group 1, vaccine treatment group 2, and antibiotic treatment group, respectively. Detailed Implementation

[0044] Terms and Definitions The term "Chlamydia psittaci" refers to Chlamydia psittaci Chlamydia avium is a strictly intracellular, Gram-negative pathogen, also known as avian chlamydia. It is primarily found in birds such as parrots, pigeons, and poultry, and can contaminate the environment through its secretions and excrement, causing zoonotic infectious diseases such as psittacosis. This pathogen can cause atypical pneumonia, high fever, and bacteremia in humans, and in poultry and mammals, it can cause abortion, pneumonia, and systemic infections. It is a significant occupational pathogen, especially among poultry farmers, veterinarians, and pet bird owners. Diseases caused by Chlamydia avium infection include psittacosis and chlamydial diseases.

[0045] The term "rplE" refers to 50S ribosomal protein L5, a core component of the 50S ribosomal large subunit in prokaryotes. It plays a crucial role not only in the structure and assembly of the ribosome but also specifically binds to 5S rRNA, jointly stabilizing the three-dimensional conformation of the large subunit, and is an essential protein for maintaining the normal function of the protein synthesis machinery. The rplE antigen of this invention comprises the full-length rplE antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length truncation, all of which can stimulate an immune response against the rplE antigen. Preferably, the amino acid sequence of the rplE antigen is shown in SEQ ID NO: 1.

[0046] The term "rpsC" refers to 30S ribosomal protein S3, a key protein of the 30S ribosomal subunit in prokaryotes. Located near the decoding center of the ribosome, this protein directly participates in the localization and transport of messenger RNA and maintains translational fidelity, playing a crucial role in ensuring correct codon-anticodon pairing. The rpsC antigen of this invention comprises the full-length rpsC antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length truncation, all of which can stimulate an immune response against the rpsC antigen. Preferably, the amino acid sequence of the rpsC antigen is shown in SEQ ID NO: 2.

[0047] The term "rpsE" refers to 30S ribosomal protein S5, a core structural and functional protein of the prokaryotic 30S ribosomal small subunit. This protein plays a crucial role in maintaining the overall structural stability of the small subunit and participates in regulating translation accuracy; mutations in this protein are often associated with alterations in translational fidelity. The rpsE antigen of this invention comprises the full-length rpsE antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length truncation, all of which can stimulate an immune response against the rpsE antigen. Preferably, the amino acid sequence of the rpsE antigen is shown in SEQ ID NO: 3.

[0048] The term "rpsG" refers to 30S ribosomal protein S7, a core component of the head domain of the 30S ribosomal small subunit in prokaryotes. This protein binds to 16S rRNA early in ribosome assembly, playing a crucial "molecular scaffolding" role in the correct folding of the 30S small subunit's three-dimensional conformation, and directly participates in the formation of messenger RNA binding channels, thereby stabilizing the mRNA-ribosome complex. The rpsG antigen of this invention comprises the full-length rpsG antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length truncation, all of which can stimulate an immune response against the rpsG antigen. Preferably, the amino acid sequence of the rpsG antigen is shown in SEQ ID NO: 4.

[0049] The term "immune response" refers to a humoral response, a cellular response, or both in an organism. Immunity should be measurable 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.

[0050] 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, rectal or vaginal administration, or by combination of routes. Intramuscular administration in the neck muscles of animals is preferred. Boosting regimens can be used to adjust the administration regimen to provide optimal immunization.

[0051] 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.

[0052] 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," a "recombinant protein," or a "fusion protein."

[0053] 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 a 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 disclosure 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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), rodents (e.g., rabbits, mice, and rats), and birds (e.g., parrots, pigeons, and poultry).

[0059] The terms “transformation,” “transfection,” and “transduction” have the meanings generally understood by those skilled in the art: the process of introducing exogenous DNA or RNA into a host.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art described herein.

[0064] This invention discloses a fusion molecular structure for effectively preventing and treating Chlamydia psittaci infection, a method for preparing a recombinant vaccine based on this structure, 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.

[0065] The fusion protein and encoding nucleic acid and their elements provided by this invention, as well as the preparation method and application, all utilize commercially available raw materials and reagents. Based on conventional knowledge in molecular cloning, expression construction, vaccine preparation, and immunization, those skilled in the art can implement the methods and embodiments of this invention.

[0066] The present invention will be further illustrated below with reference to the embodiments. Preferably, a nucleic acid vaccine architecture is selected for the preparation of the recombinant vaccine.

[0067] Example 1: Construction of the recombinant nucleic acid vaccine of the present invention Different antigens were screened to obtain four antigens—rplE, rpsC, rpsE, and rpsG—suitable for constructing fusion proteins and nucleic acid vaccines to prevent and treat Chlamydia psittaci infection.

[0068] To prepare a recombinant nucleic acid vaccine containing the antigen of the present invention, exemplarily, Figure 1 This is a schematic diagram of the molecular structure of the fusion protein expressed in the vaccine of the present invention. In order to prepare a vaccine capable of producing... Figure 1 The recombinant nucleic acid vaccine with the molecular structure shown first constructs a gene expression cassette for expressing the antigen sequence described in this invention. A schematic diagram of the template plasmid containing this invention is shown below. Figure 2 As shown, the expression cassette, from the 5' end to the 3' end, sequentially comprises: a 5' UTR, a CDS region, a 3' UTR, and PolyA, wherein the CDS region contains the fusion molecular structure described in this invention. Subsequently, the complete gene expression cassette sequence was optimized based on codon degeneracy, and the DNA sequence was directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence was inserted into an expression vector suitable for in vitro RNA transcription, yielding a vector plasmid for preparing recombinant nucleic acid vaccines.

[0069] According to the above method, a carrier for use in subsequent embodiments is prepared: Step a: Synthesize the “rplE antigen-rpsC antigen-rpsE antigen-rpsG antigen” gene fragments, linking them together using a linker sequence as shown in SEQ ID NO: 5. The amino acid sequence of the fusion protein encoded by the rplE antigen-rpsC antigen-rpsE antigen-rpsG antigen fusion gene is shown in SEQ ID NO: 6. Specifically, the amino acid sequence of the rplE antigen is shown in SEQ ID NO: 1, the amino acid sequence of the rpsC antigen is shown in SEQ ID NO: 2, the amino acid sequence of the rpsE antigen is shown in SEQ ID NO: 3, and the amino acid sequence of the rpsG antigen is shown in SEQ ID NO: 4.

[0070] Step b: Construct a nucleic acid vaccine architecture carrier.

[0071] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0072] Step c: Prepare recombinant plasmids.

[0073] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine preparation vector based on the present invention.

[0074] Table 1. Protein amino acid sequences of the architectural elements involved in this invention. amino acid sequence and sequence number rplE antigen MSRLKKLYTEEIRKTLQEKFGYSNTMQIPVLKKIVISMGLAEAAKDKNLFQAHLEELSMISGQKPLVTKARNSIAGFKLREGQGIGAKVTLRGQRMYDFMDRFCHIVSPRIRDFRGFSSKGDGRGCYSLGLDDQQIFPEVDLDRVKRTQGMNITWVTTAKTDVECTTLLELMGLRFK (SEQID NO: 1) rpsC antigen YGNKQEFGKFLIEDVKIREFLRKKPSCQGAAGFVVRRMSGKIEVTIQTARPGLVIGKKGAEVDLLKEELRKLTGKEVWVEIAEIKRPELNAKLVADNIAKQIERRVSFRRAMKKAMQSVMDAGAVGVKIQVSGRLAGAEIARSEWYKNGRVPLHTLRADIDYATASAATTYGIIGVKVWINLGEKVST (SEQ ID NO: 2) rpsE antigen KEDQLEEKVLVVNRCSKVVKGGRKFSFSALILVGDGKGRLGYGFAKANELTDAIRKGGEAARKNLITIESLEGDSIPHEVLVDQDGAQLLLKPAKPGTGIVAGSRIRLILEMAGVKNIVAKSLGSNNPMNQVKAAFKALLSLSS (SEQ ID NO: 3) rpsG antigen PIYGSVTLERFINKVMMHGKKSVARKIVYNALERFAKKIGAENVLEAFEEALENAKPLLEVRSRRVGGATYQVPVEVAAGRRDCLAMQWIIKFARAKPGKSMEVGLATELVDCFNKQGATIKKREDTHRMEANKAFAH (SEQ ID NO: 4) linker sequence GGSGGGGSGG (SEQ ID NO: 5) rplE antigen-rpsC antigen-rpsE antigen-rpsG antigen fusion protein MSRLKKLYTEEIRKTLQEKFGYSNTMQIPVLKKIVISMGLAEAAKDKNLFQAHLEELSMISGQKPLVTKARNSIAGFKLREGQGIGAKVTLRGQRMYDFMDRFCHIVSPRIRDFRGFSSKGDGRGCYSLGLDDQQIFPEVDLDRVKRTQGMNITWVTTAKTDVECTTLLELMGLRFKGGSGGGGSGGYGNKQEFGKFLIEDVKIREFLRKKPSCQGAAGFVVRRMSGKIEVTIQTARPGLVIGKKGAEVDLLKEELRKLTGKEVWVEIAEIKRPELNAKLVADNIAKQIERRVSFRRAMKKAMQSVMDAGAVGVKIQVSGRLAGAEIARSEWYKNGRVPLHTLRADIDYATASAATTYGIIGVKVWINLGEKVSTGGSGGGGSGGKEDQLEEKVLVVNRCSKVVKGGRKFSFSALILVGDGKGRLGYGFAKANELTDAIRKGGEAARKNLITIESLEGDSIPHEVLVDQDGAQLLLKPAKPGTGIVAGSRIRLILEMAGVKNIVAKSLGSNNPMNQVKAAFKALLSLSSGGSGGGGSGGPIYGSVTLERFINKVMMHGKKSVARKIVYNALERFAKKIGAENVLEAFEEALENAKPLLEVRSRRVGGATYQVPVEVAAGRRDCLAMQWIIKFARAKPGKSMEVGLATELVDCFNKQGATIKKREDTHRMAEANKAFAH (SEQ ID NO: 6) Example 2: Preparation of the recombinant nucleic acid vaccine of the present invention (1) Preparation of capped mRNA vaccines Step a: Linearize the vector plasmid used in Example 1 for producing capped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0075] Step b: The linearized plasmid was subjected to an in vitro co-transcriptional capping reaction to add a 7-methylguanylate cap structure to the 5' end of the transcribed mRNA and the template DNA was degraded.

[0076] (2) Preparation of uncapped mRNA vaccines Step a: Linearize the vector plasmid used in Example 1 for producing uncapped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0077] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0078] (3) DNA vaccine preparation Step a: Amplify the vector plasmid used in Example 1 for producing DNA vaccines to obtain a large number of target plasmids for purification.

[0079] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0080] Example 3: Quality control and expression efficacy verification of the recombinant nucleic acid in vitro transcription of the present invention. The recombinant nucleic acid vaccine based on the present invention was prepared using the method for preparing the capped mRNA vaccine in Example 2. The purity of the produced recombinant nucleic acid was tested. Figure 3 The results of the quality control peak diagram and purity test of the nucleic acid vaccine containing the antigen sequence of the present invention show that the purity of the recombinant nucleic acid used in the experiment is 90.3%, which meets the quality requirements for cell transfection experiments and vaccine production.

[0081] The above-mentioned vaccine was transfected into HEK293T cells using cell transfection reagents. After 24 hours of in vitro culture, the protein was collected and analyzed by Western blot.

[0082] Figure 4The results of in vitro expression analysis (WB) on HEK293 cells transfected with the vaccine were presented. The expressed antigen is a cellular immune antigen, which theoretically should be significantly expressed in cell lysates, with an estimated protein molecular weight of 104.2 kDa. The results show that the vaccine can be significantly expressed intracellularly and detected significantly in cell lysates, proving that the vaccine designed based on this invention can be correctly expressed in eukaryotic cells, and the expressed protein structure is correct and stable, which is beneficial for immune epitope presentation.

[0083] Example 4: Prevention and treatment effect of the recombinant nucleic acid vaccine of the present invention against Chlamydia psittaci infection. To verify whether the recombinant nucleic acid vaccine of this invention can elicit an effective immune protective response in target animals, this embodiment conducted vaccine immunization and systematic tracking monitoring in a parrot population naturally infected with Chlamydia psittaci. By continuously monitoring changes in the infection status of the population, the actual effectiveness of the vaccine in preventing infection can be indirectly assessed.

[0084] Prior to the experiment, veterinarians assessed the clinical symptoms of sick parrots, selecting individuals exhibiting typical psittacosis symptoms. Mixed swab samples were collected from their mouths, noses, and conjunctivae. After positive qPCR testing for Chlamydia psittaci, these parrots were included in the experiment and randomly assigned to groups (see Table 2). Each parrot was numbered for tracking throughout the process. Details of the immunization, treatment, and sampling procedures can be found in [link to table]. Figure 5 The immunization and sampling procedures for vaccine treatment group 1 are as follows: Figure 5 As shown in A, the immunization and sampling procedures for vaccine treatment group 2 are as follows: Figure 5 As shown in B, the treatment and sampling procedures for the antibiotic treatment group are as follows: Figure 5 As shown in C.

[0085] Table 2. Grouping and Treatment Procedures of Experimental Animals in Example 4

[0086] The specific medical orders listed in Table 2 are as follows: 1. 10% Enrofloxacin Injection (Bayer) ® ): 0.1 mL / animal / day.

[0087] 2. 1 mL of dexamethasone sodium phosphate injection + 2 mL of amikacin sulfate injection + 2 mL of normal saline were used for nebulization of 10 mice. The antibiotic treatment group of this invention consisted of 5 mice, with the dosage halved, once a day.

[0088] 3. Doxycycline hydrochloride tablets: 10 tablets / day. After crushing in a high-speed blender, place in water and stir. The antibiotic treatment group of this invention consists of 5 tablets, with the dosage halved.

[0089] Establish an independent case file for each sick parrot, and systematically observe and record its clinical symptoms every 3–5 days, including respiratory symptoms (sneezing, runny nose, difficulty breathing, respiratory noises, etc.), gastrointestinal symptoms (watery diarrhea, undigested food residue, etc.), eye lesions (redness, swelling, discharge), and general condition (depression, loss of appetite, weight loss, etc.). Record any abnormalities such as worsening of the condition or death promptly.

[0090] Sample collection: ① Use a sampling swab to collect conjunctival secretions; ② Insert the swab into the parrot's mouth, rotate the swab, and collect oral secretions; ③ Use a syringe to assist in aspirating nasal secretions and collect them with a swab; In the above sample collection, ensure that at least two different sampling locations of obvious secretions are collected from each parrot, and place the swabs into a sampling tube for subsequent qPCR pathogen detection.

[0091] In qPCR (quantitative real-time PCR) detection, the CT value refers to the number of cycles required for the fluorescence signal intensity to reach a pre-set detection threshold during qPCR amplification. A lower CT value indicates a higher pathogen DNA content in the sample; conversely, a higher CT value indicates a lower pathogen content, or even no pathogen. In this field, the detection thresholds for Chlamydia psittaci are: CT value < 40 is positive, values ​​between 40 and 45 (excluding 45) are suspected positive, and values ​​≥ 45 are negative.

[0092] Tracking test results such as Figures 6A - 6C As shown. Figure 6A and Figure 6B The dynamic changes in bacterial load and positivity rate in the two vaccine treatment groups (vaccine treatment group 1 and vaccine treatment group 2) are shown separately. Figure 6C The data corresponds to the antibiotic treatment group. Results showed that in vaccine treatment group 1, 73% of individuals (8 animals) tested negative on Day 28, and all tested negative by Day 42. Retesting on Day 70 also showed negative results, indicating that *Chlamydia psittaci* colonization was effectively eliminated and no reinfection occurred. In vaccine treatment group 2, 59% of individuals (7 animals) tested negative on Day 21, and this figure reached 99% (11 animals) on Day 35. Retesting on Day 70 showed all animals were negative, similarly confirming that the vaccine could completely eliminate the pathogen and prevent reinfection. In contrast, although all individuals in the antibiotic treatment group tested negative on Day 28, some tested positive again on Day 42, and all tested positive again on Day 70. This indicates that antibiotic treatment cannot completely eliminate pathogen colonization, and the animals still face the risk of reinfection.

[0093] In summary, traditional antibiotic therapies are characterized by long treatment courses, complex procedures, and unsustainable protective effects. In contrast, the antigen and fusion protein architecture provided by this invention can induce an effective immune response in parrots, demonstrating excellent therapeutic and preventative efficacy against Chlamydia psittaci infection. This groundbreaking research fills a key gap in the current field of Chlamydia psittaci vaccine development, providing an innovative solution for the development and production of animal immunotherapies, and possesses significant commercial value and application prospects.

[0094] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises the antigen of the large ribosomal protein L5 (50S ribosomal protein L5, rplE) or an antigenic fragment thereof, the antigen of the small ribosomal protein S3 (30S ribosomal protein S3, rpsC) or an antigenic fragment thereof, the antigen of the small ribosomal protein S5 (30S ribosomal protein S5, rpsE) or an antigenic fragment thereof, and the antigen of the small ribosomal protein S7 (30S ribosomal protein S7, rpsG) or an antigenic fragment thereof.

2. The fusion protein according to claim 1, characterized in that, The antigen or its antigenic fragment is derived from Chlamydia; preferably, from Chlamydia psittaci (…). Chlamydia psittaci ).

3. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of the rplE antigen has at least 95% identity with the sequence of SEQ ID NO: 1, preferably 96%, 97%, 98%, 99%, or 100%; the amino acid sequence of the rpsC antigen has at least 95% identity with the sequence of SEQ ID NO: 2, preferably 96%, 97%, 98%, 99%, or 100%; the amino acid sequence of the rpsE antigen has at least 95% identity with the sequence of SEQ ID NO: 3, preferably 96%, 97%, 98%, 99%, or 100%; the amino acid sequence of the rpsG antigen has at least 95% identity with the sequence of SEQ ID NO: 4, preferably 96%, 97%, 98%, 99%, or 100%; more preferably, the amino acid sequence of the rplE antigen is as shown in SEQ ID NO: 1, the amino acid sequence of the rpsC antigen is as shown in SEQ ID NO: 2, the amino acid sequence of the rpsE antigen is as shown in SEQ ID NO: 3, and the amino acid sequence of the rpsG antigen is as shown in SEQ ID NO: 4; NO:4 shown.

4. The fusion protein according to any one of claims 1-3, characterized in that, Antigens or their antigenic fragments may optionally be linked by a linker sequence; preferably, the amino acid sequence of the linker sequence is shown in SEQ ID NO:

5.

5. The fusion protein according to any one of claims 1-4, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

6.

6. The fusion protein according to any one of claims 1-5, characterized in that, The N-terminus of the fusion protein further includes a signal peptide and / or any secretion-promoting element, and / or the C-terminus further includes a protein stabilization motif element.

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

8. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule of claim 7.

9. A recombinant vector, characterized in that, It comprises the recombinant nucleic acid molecule of claim 7 or the recombinant gene expression cassette of claim 8.

10. A recombinant host cell, characterized in that, It comprises the recombinant nucleic acid molecule of claim 7, or the recombinant gene expression cassette of claim 8, or the recombinant vector of claim 9.

11. An immunogenic composition or pharmaceutical composition, characterized in that, The composition comprises one or more of the fusion proteins of any one of claims 1-6, and / or one or more of the recombinant nucleic acid molecules of claim 7, and / or one or more of the recombinant gene expression cassettes of claim 8, and / or one or more of the recombinant vectors of claim 9, and / or one or more of the recombinant host cells of claim 10; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

12. A recombinant vaccine, characterized in that, The recombinant vaccine comprises one or more fusion proteins according to any one of claims 1-6, and / or one or more recombinant nucleic acid molecules according to claim 7, and / or one or more recombinant gene expression cassettes according to claim 8, and / or one or more recombinant vectors according to claim 9, and / or one or more recombinant host cells according to claim 10, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 11; preferably, the recombinant vaccine is a nucleic acid vaccine; more preferably, the nucleic acid vaccine is a DNA vaccine or an RNA vaccine.

13. Use of one or more fusion proteins according to any one of claims 1-6, and / or one or more recombinant nucleic acid molecules according to claim 7, and / or one or more recombinant gene expression cassettes according to claim 8, and / or one or more recombinant vectors according to claim 9, and / or one or more recombinant host cells according to claim 10, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 11, and / or one or more recombinant vaccines according to claim 12 in the preparation of vaccines or pharmaceuticals for prevention, treatment and / or inoculation.

14. The use according to claim 13, characterized in that, The drug is used to prevent and / or treat infections or diseases in humans, mammals or birds caused by Chlamydia psittaci; preferably, the infection or disease in humans or mammals caused by Chlamydia psittaci is psittaci; preferably, the infection or disease in birds caused by Chlamydia psittaci is avian plague; more preferably, the birds are selected from poultry.

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