Circular RNA and its use in trichuris vaccine

Abstract

The application discloses circular RNA and application thereof in pigeon trichomonad vaccine. The circular RNA comprises a coding element capable of encoding recombinant pigeon trichomonad AP33 or AP65 protein. The coding element has a sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or a conservatively variant sequence thereof. The circular RNA can induce protein expression and generate high-level antibodies in an animal body. When the circular RNA is applied to the pigeon trichomonad vaccine, the circular RNA does not contain infectious worm component, can be degraded into nucleotides in the body, cannot be integrated into a host genome, has no risk for pigeons, can stimulate pigeons to generate a stronger immune response, including humoral immunity and cellular immunity, and can effectively resist parasitic infection. Meanwhile, the vaccine has high stability, the vaccine of the application can be prepared on a large scale through a molecular technology, and production cost is low.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a circular RNA and its application in a pigeon trichomoniasis vaccine. Background Technology

[0002] Pigeon trichomoniasis is a common parasitic infectious disease caused by Trichomonas gallinae, a protozoan disease that is prevalent and seriously harmful in the pigeon industry. Its typical symptoms include lesions in the digestive tract, such as necrotic ulcers in the mouth, crop, esophagus, and other parts of the body. Infected pigeons often die due to obstruction of the digestive and respiratory tracts, which hinders their eating and normal breathing, and seriously damages the health and production performance of the flock.

[0003] As a parasite, Trichomonas vaginalis lives inside cells and has a complete life cycle, making it difficult for the immune system to completely eliminate it. This makes the development of its vaccine quite challenging. Currently, the development of Trichomonas vaginalis vaccines mainly focuses on live vaccines and subunit vaccines. However, there are no mature technologies to refer to for either the issue of complete inactivation of the parasite and the balance between immunogenicity in inactivated vaccines or the issue of screening effective antigen targets for subunit vaccines.

[0004] On the other hand, circular RNA (circRNA) vaccines possess significant advantages in animal vaccine development due to their high stability, high biocompatibility, and sustained translation efficiency. Circular RNA is a single-stranded RNA with a covalently closed structure. Compared to traditional linear mRNA, circular RNA has a more stable molecular structure. The covalently closed structure endows it with resistance to nuclease degradation, and its half-life is 3-5 times longer than that of linear RNA. It can achieve sustained antigen expression in vivo, thereby stimulating a stronger and more durable immune response. Furthermore, it can induce Th1 / CTL polarization without exogenous adjuvants, achieving synergistic humoral and cellular immunity. The neutralizing antibody titers induced by this technology are higher, and it has a broader response to pathogen mutations, while reducing the risk of antibody-dependent enhancement (ADE). Furthermore, circular RNA vaccines tend to elicit a protective Th1-type immune response dominated by IgG2, which helps mitigate adverse reactions such as vaccine-associated respiratory disease (VAERD). Crucially, circular RNA vaccines exhibit excellent stability at ambient temperatures, allowing for storage and transportation without the need for a cold chain system, thus offering significant convenience. The in vitro transcription-circularization process enables mass production within 7 days, and a single vector can tandemly support multiple antigenic epitopes. Therefore, circular RNA vaccines are more suitable for emergency prevention and control of emerging animal diseases. However, current research and development of circular RNA vaccines primarily focuses on mammalian vaccines, with very few avian vaccines. This is because the avian immune system differs significantly from that of mammals. The RNA recognition mechanisms of pattern recognition receptors such as TLR3 and TLR7 in birds differ from those in mammals, requiring targeted optimization of circular RNA, increasing manufacturing costs, and thus hindering the large-scale, industrial-scale application of circular RNA vaccines.

[0005] Given the aforementioned problems with pigeon trichomoniasis vaccines, and the limitations of chemical drug control methods, the prominent issue of drug resistance, and the lack of a mature pigeon trichomoniasis vaccine, developing a safe and effective vaccine has become a key strategy for the prevention and control of pigeon trichomoniasis. Summary of the Invention

[0006] The main objective of this invention is to provide a circular RNA vaccine for Trichomonas vaginalis, its preparation method, and its application. By using molecular cloning technology, the invention achieves efficient construction and large-scale preparation of circular RNA, significantly reducing vaccine production costs and accelerating the industrial application of Trichomonas vaginalis vaccines, thereby overcoming the shortcomings of existing technologies.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution.

[0008] As a first aspect of the invention, the present invention provides a circular RNA that encodes proteins associated with Trichomonas vaginalis invasion.

[0009] The circular RNA provided by the present invention comprises at least a coding element that encodes a polypeptide containing Trichomonas vaginalis protein.

[0010] Furthermore, the Trichomonas vaginalis protein is at least one of AP33 protein and AP65 protein, which enables the circular RNA to provide at least 80% immune protection when used as a vaccine to prevent Trichomonas vaginalis infection.

[0011] In one embodiment, the Trichomonas vaginalis protein is the AP33 protein, and the corresponding coding element has the sequence shown in SEQ ID NO:1 or an extension or truncated sequence thereof, for example, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:1. More preferably, the sequence of the coding element is as shown in SEQ ID NO:1.

[0012] In one embodiment, the Trichomonas vaginalis protein is the AP65 protein, and the corresponding coding element has the sequence shown in SEQ ID NO:2 or an extension or truncated sequence thereof, for example, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:2. More preferably, the sequence of the coding element is as shown in SEQ ID NO:2.

[0013] Preferably, the coding element encodes a polypeptide containing the Trichomonas pigeonis AP65 protein, which enables the circular RNA to provide at least 90% immune protection when used as a vaccine to prevent Trichomonas pigeonis infection.

[0014] This invention optimizes the nucleotide sequence of Trichomonas vaginalis proteins, including codon optimization of the AP33 and / or AP65 protein coding sequences. The optimized nucleotide sequence is better suited to the host cell's translation system, thereby achieving and significantly improving the expression efficiency and stability of the target gene in the host cell. The optimized antigen can induce a strong protective immune response after a single immunization, helping to reduce the dependence on and abuse of clinical drugs against Trichomonas vaginalis.

[0015] As a second aspect of the invention, the present invention also provides a method for preparing circular RNA, comprising linearizing a recombinant DNA plasmid expressing Trichomonas vaginalis protein, transcribing it in vitro to synthesize RNA, and then circularizing it to obtain circular RNA.

[0016] For example, the present invention uses T7 in vitro transcription-self-circularization technology to realize the in vitro transcription and circularization of circular RNA. The precursor RNA is generated by in vitro transcription mediated by T7 RNA polymerase, and then a covalently closed circular structure is formed by self-circularization mechanism. It has the characteristics of simple process and scalability.

[0017] In one embodiment, the preparation method may include the following steps:

[0018] (1) Design, optimize and synthesize nucleotide sequences encoding AP33 and AP65 proteins of Trichomonas pigeonis;

[0019] (2) The target nucleotide sequence is ligated into a plasmid to obtain a recombinant vector, and linearized using restriction endonucleases;

[0020] (3) Using T7 RNA polymerase as a template, T7 RNA is transcribed and self-circularized, and then purified to obtain the target circular RNA. The purification methods used include, but are not limited to, lithium chloride precipitation and high performance liquid chromatography (HPLC) purification.

[0021] As a third aspect of the invention, the present invention also provides a host cell comprising HEK293 cells transfected with the said circular RNA.

[0022] As a fourth aspect of the invention, the present invention also provides a complex (LNP-circular RNA) comprising lipid nanoparticles encapsulating said circular RNA.

[0023] Furthermore, the lipid nanoparticles exhibit an encapsulation efficiency of greater than 90% for circular RNA.

[0024] Furthermore, the surface potential of the composite is either electroneutrally neutral or electronegative.

[0025] In one embodiment, the LNP-circular RNA is composed of the circular RNA and lipid nanoparticles (LNPs), wherein the lipid nanoparticles encapsulate the circular RNA with an encapsulation efficiency greater than 90%.

[0026] For example, this invention uses a commercially available LNP transfection kit (HanzBio) to improve the transfection efficiency and stability of circular RNA, thereby further enhancing immunogenicity and protective efficacy.

[0027] Preferably, the LNP-circular RNA composition has an electroneutrally neutral or electronegative surface potential to improve its biosafety.

[0028] As a fifth aspect of the invention, the present invention also provides an immune composition comprising:

[0029] The circular RNA or the complex described herein;

[0030] And, pharmaceutically acceptable carriers.

[0031] As a sixth aspect of the invention, the present invention also provides the use of the said circular RNA, the said complex, or the said immune composition in the production of an agent for inducing an immune response against Trichomonas vaginalis infection in test animals.

[0032] As a seventh aspect of the invention, the present invention also provides the use of the said circular RNA, the said complex, or the said immune composition in the preparation of an agent for the prevention and / or treatment of animals infected with Trichomonas vaginalis.

[0033] As an eighth aspect of the invention, the present invention also provides the use of the said circular RNA or the said complex in the preparation of a pigeon trichomoniasis vaccine.

[0034] As a ninth aspect of the invention, the present invention also provides a circular RNA vaccine against Trichomonas vaginalis, comprising:

[0035] The circular RNA or the complex described herein;

[0036] And, pharmaceutically acceptable carriers.

[0037] Compared with the prior art, the beneficial effects of the present invention are at least as follows:

[0038] (1) This invention provides a novel form of circular RNA vaccine construction, which realizes efficient construction and large-scale preparation of circular RNA through molecular cloning technology, greatly reducing vaccine production costs and facilitating industrial application.

[0039] (2) This invention significantly improves the expression efficiency and stability of Trichomonas vaginalis AP33 and AP65 proteins in host cells by codon optimization, and further enhances the self-circulation efficiency of circular RNA. The optimized antigen can induce a strong protective immune response after a single immunization, which helps to reduce the dependence and abuse of clinical drugs against Trichomonas vaginalis.

[0040] (3) This invention is the first to apply circular RNA technology to the research and development of pigeon trichomoniasis vaccine, and at the same time uses type II intron self-splicing technology, which can rely on the hydroxyl groups inside the nucleic acid sequence to trigger splicing. This technology does not require additional in vitro circularization operations, and breaks through the bottleneck problems of complex production process, high cost and limited immune effect of existing protein vaccine, providing a new strategy and technical approach for the prevention and control of pigeon trichomoniasis.

[0041] (4) This invention uses a circular RNA vaccine, which does not contain infectious parasite components. It degrades into nucleotides in vivo and does not integrate into the host genome, posing no risk to pigeons. The circular RNA vaccine can stimulate pigeons to produce a strong immune response, including humoral immunity and cellular immunity.

[0042] (5) The circular RNA vaccine of the present invention has high stability and good safety. It can provide at least 80% immune protection when preventing Trichomonas cubitus infection, especially the Trichomonas cubitus AP65 circular RNA vaccine can provide at least 90% immune protection. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of this application 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 some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a schematic diagram illustrating the principle of constructing circular RNA in Example 1 of the present invention.

[0045] Figure 2 This is a schematic diagram of agarose gel electrophoresis of the circular RNAs encoding AP33 and AP65 proteins, respectively, in Example 1 of the present invention.

[0046] Figure 3 This is a comparison diagram of the expression of circular RNAs encoding AP33 and AP65 proteins in HEK293 cells, as shown in Example 1 of this invention.

[0047] Figure 4 This is a comparison of serum IgY antibody levels in pigeons immunized 21 days after primary immunization, using LNP-circular RNA encoding AP33 and AP65 proteins, respectively, in Example 2 of this invention.

[0048] Figure 5 This is a comparison diagram showing the effects of LNP-circular RNA encoding AP33 protein and AP65 protein, respectively, on the prevention and treatment of Trichomonas vaginalis in pigeons in Example 2 of the present invention. Detailed Implementation

[0049] The invention will be more fully understood through the following detailed description, which should be read in conjunction with the accompanying drawings. Detailed embodiments of the invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, the specific functional details disclosed herein should not be construed as limiting, but rather as the basis for the claims and as intended to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiment.

[0050] Because different species and cells have codon biases, in order to enable the vaccine-using species (avian birds) to obtain stable and highly expressed AP33 and AP65 proteins by taking advantage of their codon biases, this invention optimizes the CDS of AP33 and AP65 proteins.

[0051] To further improve the self-circulation efficiency of circular RNA, sequence-level adjustments were made to the codon optimization sequence.

[0052] The optimized nucleotide sequence of AP33 is shown in SEQ ID NO.1; the optimized nucleotide sequence of AP65 is shown in SEQ ID NO.2.

[0053] Key considerations for codon optimization include: codon preference of the target expression species, adaptation to restriction endonuclease sites of the vector used, overall GC content of the sequence, and other sequence features that may affect expression or stability.

[0054] This invention constructs a recombinant vector containing the above-mentioned optimized nucleotide sequence. This vector can be any commonly used cloning vector or expression vector. This invention does not limit the type of vector, but is limited to achieving the technical effects of this invention.

[0055] Furthermore, the present invention provides a method for constructing circular RNA, specifically comprising:

[0056] (1) Synthesize the nucleotide sequence encoding Trichomonas vaginalis protein as the target nucleotide sequence;

[0057] (2) The target nucleotide sequence is ligated into a DNA recombinant plasmid to obtain a recombinant vector, and linearized DNA template is obtained by linearization using restriction endonucleases;

[0058] (3) Use T7 RNA polymerase to perform T7 RNA transcription and complete self-circularization using the linearized DNA template from the previous step;

[0059] (4) The target circular RNA was obtained by precipitation and purification using lithium chloride method and further purification by HPLC.

[0060] The technical solution of the present invention will be described in detail below through specific embodiments.

[0061] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0062] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0063] Example 1: Construction of circular RNA from Trichomonas vaginalis

[0064] 1. DNA template synthesis

[0065] The recombinant DNA template plasmid was synthesized by Genscript Biotech Co., Ltd. This recombinant plasmid contains a T7 promoter, a 5' intron fragment, IRES, the target antigen sequence, a 3' intron fragment, and a terminator. See [link to documentation]. Figure 1 This is a schematic diagram of the structure of the recombinant DNA template plasmid.

[0066] The recombinant plasmids were stored in Escherichia coli DH5α competent strains. Single clones were randomly selected and inoculated into 200 mL of LB liquid medium containing penicillin resistance. The clones were cultured in a shaker at 37 °C. The DNA recombinant plasmids expressing AP33 and AP65 proteins were extracted according to the instructions of the Omega endotoxin-free plasmid DNA extraction kit. The concentrations were measured and the plasmids were stored for use.

[0067] 2. Preparation of DNA linearization plasmid template

[0068] (1) The RNA synthesis plasmid constructed according to Example 1 was linearized by Xba I restriction enzyme digestion.

[0069] First, take 10 μg of recombinant DNA plasmid and digest it with Xba I enzyme at 37℃ for 30 min. The prepared enzyme digestion reaction system is shown in Table 1.

[0070] Table 1 Enzyme digestion system

[0071]

[0072] Then, the template DNA obtained by PCR amplification was extracted with phenol-chloroform at a 1:1 volume ratio, and then precipitated with 2.5 volumes of anhydrous ethanol for purification to obtain a linearized plasmid template.

[0073] 3. In vitro transcription and circularization of RNA

[0074] (1) Circular RNA of antigen protein

[0075] a) First, take 0.5–1 μg of linearized plasmid template and synthesize RNA in vitro using T7 RNA polymerase (Promega) at 37°C. The in vitro transcription reaction system is as follows:

[0076] Table 2 In vitro transcription system

[0077]

[0078] b) The in vitro transcription system was incubated at 37°C for 4 hours. During this process, the natural self-splicing ribonuclease activity of type II introns was utilized. By designing EBS-IBS complementary pairing, RNA folding into a catalytic conformation was induced, causing the 5' and 3' ends of the target sequence to approach each other and undergo a two-step transesterification reaction, achieving head-to-tail ligation. After splicing, the introns were removed, and the target sequence formed a closed loop.

[0079] c) Then add 1 μL of DNase and incubate at 37°C for 30 min to digest the DNA template. For further enrichment, purify the circular RNA by precipitation using lithium chloride.

[0080] d) Purify circular RNA by HPLC to remove small linear RNA.

[0081] HPLC conditions were as follows: gel size exclusion column: Waters B EH450A, column temperature: 40℃, flow rate: 1 min / mL, elution conditions: 0~30 min, 100% buffer A (10 mM Tris, 0.5 mM EDTA, DEPC water).

[0082] e) Take 1 μL, dilute it 10 times, and measure the RNA concentration using NanoDrop. Store the remaining RNA at -80℃.

[0083] f) After quantification, 500 ng was taken for gel electrophoresis to detect the synthesized circular RNA;

[0084] The results are as follows Figure 2 As shown, the AP33 gene circular RNA and AP65 gene circular RNA have been successfully synthesized using the above method.

[0085] 4. Detecting the effect of circular RNA on the production of target proteins

[0086] HEK293 cells in the logarithmic growth phase were harvested, digested with trypsin, and then diluted with cell culture medium to a cell density of 2.5 × 10⁻⁶. 5Cells were cultured at a concentration of 1 ml / well to form a cell suspension. The cell suspension was then added to 12-well cell culture plates and incubated at 37°C with 5% CO2. After 24 hours of culture, the supernatant was discarded, and the cells were washed once with sterile PBS. Cell culture medium was then added at 1 ml / well. The synthesized circular RNA carrying the target gene was then transfected into HEK293 cells using Lipofectamine MessengerMAX transfection reagent (Thermo Fisher Scientific). After 48 hours of transfection, the cell supernatant was removed, and the cells were lysed using RIPA lysis buffer. The cells were centrifuged at 12,000 rpm for 10 minutes at 4°C to separate the supernatant. The supernatant was subjected to SDS-PAGE with 5× Loading Buffer, transferred to a membrane, blocked with 5% milk, and incubated overnight at 4°C with the appropriate primary antibody. After adding secondary antibody and incubating at room temperature for 30 minutes, the cells were washed 2-3 times, and chromogenic buffer was added. Western blotting was then performed.

[0087] Figure 3 The results showed that HEK293 cells transfected with circular RNA carrying the Trichomonas vaginalis AP33 protein gene efficiently expressed AP33 protein, and HEK293 cells transfected with circular RNA carrying the Trichomonas vaginalis AP65 protein gene efficiently expressed AP65 protein. The control group, HEK293 cells not transfected with Trichomonas vaginalis protein genes, did not express the target proteins. Furthermore, unoptimized raw sequence circular RNA was simultaneously constructed and transfected in the experiment, and no expression of the target proteins was detected under the same experimental conditions. This indicates that the circular RNA synthesized using the protocol of this embodiment can effectively produce the target antigens AP33 and AP65 proteins, respectively.

[0088] Example 2: Immunization experiment of Trichomonas vaginalis circular RNA vaccine

[0089] 1. LNP-circular RNA preparation

[0090] Because circular RNA molecules are still susceptible to degradation by nucleases in vivo, and naked RNA has difficulty effectively crossing the cell membrane into the cytoplasm, direct administration results in low stability and delivery efficiency. Therefore, a delivery system is needed to coat them to improve their in vivo stability and bioavailability. Lipid nanoparticles (LNPs) can effectively encapsulate nucleic acid molecules, protecting them from degradation, and improve the in vivo delivery efficiency and expression level of RNA by promoting cellular uptake and endocytosis escape, while also exhibiting good biocompatibility and safety.

[0091] The circular RNA prepared in Example 1 was used to prepare lipid nanoparticle / RNA compositions using a commercial LNP transfection kit (HanzBio). The results showed that the LNPs carrying the circular RNA had small and uniform particle size, good encapsulation effect, and encapsulation efficiency greater than 90%. The surface potential was electroneutrally neutral or electronegative, avoiding cytotoxicity caused by positive charges on the nanoparticle surface, and the biocompatibility was good.

[0092] The above steps were used to obtain LNP-AP33 circular RNA and LNP-AP65 circular RNA, respectively.

[0093] 2. Immunological test

[0094] (1) Animal immunization

[0095] One-month-old pigeons were used as experimental animals and randomly divided into three groups of 10 pigeons each. The two LNP-encapsulated circular RNAs (LNP-AP33 and LNP-AP65) prepared in step 1 of this embodiment were used to immunize the pigeons by intramuscular injection at a dose of 0.1 mL / 10 μg. Pigeons inoculated with LNP served as negative controls (control group).

[0096] (2) Serum antibody level detection

[0097] Twenty-one days after immunization, blood was collected from the subwing veins of all immunized pigeons and control pigeons, and serum samples were collected. The levels of specific antibodies against LNP-AP33 and LNP-AP65 in the immunized circular RNA vaccine were determined by indirect enzyme-linked immunosorbent assay (ELISA). The whole-worm antigen of Trichomonas vaginalis was diluted to 20 μg / ml with coating buffer CBS, and 100 μL was added to each well of the ELISA plate and incubated overnight at 4°C. The plate was then removed, the liquid was discarded, and the plate was washed three times for 3 minutes each with washing buffer PBST (containing 0.05% Tween-20, pH 7.4). Then, 100 μL / well of blocking buffer was added and the plate was incubated at 37°C for 2 hours. After washing the plate as before, add the sample to be tested (1:100 dilution) to the coated reaction wells (100 μL / well), incubate at 37℃ for 60 min, then add enzyme-labeled rabbit anti-pigeon IgY-HRP (1:7000 dilution), incubate at 37℃ for 30-60 min, wash the plate as before, finally add 100 μL of substrate / well, react for 10-15 min, add 50 μL of 2 mol / L H2SO4 to stop the reaction, and read the OD at 450 nm.

[0098] like Figure 4 As shown, both LNP-AP33 and LNP-AP65 immunization groups can effectively activate humoral immunity to produce specific antibodies against the target protein. Among them, the LNP-AP33 circular RNA vaccine can stimulate the body to produce higher levels of specific antibodies.

[0099] (3) Immunization challenge test

[0100] 21 days after immunization, each pigeon was given 1×10 mg orally. 7 One group of pigeons was infected with Trichomonas vaginalis (with a survival rate greater than 90%). The same amount and route of infection were used to infect the pigeons for 5 consecutive days. After the infection was completed, the pigeons were observed for 14 consecutive days, and the survival rate of each group was recorded.

[0101] like Figure 5 As shown, the survival rate of pigeons in both the LNP-AP33 and LNP-AP65 immunization groups was above 70% during the 14-day observation period after the inoculation. The LNP-AP65 immunization group had the highest survival rate, reaching 90%. In contrast, pigeons in the LNP control group began dying from the 5th day after the inoculation, with a mortality rate reaching 100% by the end of the observation period.

[0102] The above results indicate that a single immunization with the pigeon trichomoniasis circular RNA vaccine can significantly improve the pigeons' resistance to trichomoniasis infection, with a protection rate of over 70%, especially the LNP-AP65 immunization group, which showed the best immune protection effect.

[0103] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this document is not intended to limit the invention to the specific embodiments disclosed for carrying out the invention, but rather to include all embodiments falling within the scope of the appended claims.

Claims

1. A circular RNA, characterized in that, It includes a coding element and an internal ribosome entry site IRES, wherein the coding element is capable of encoding the Trichomonas pigeonis AP33 protein or AP65 protein, and the nucleotide sequence of the coding element is codon-optimized to adapt to the host cell's translation system; The nucleic acid sequence encoding the AP33 protein in the coding element is shown in SEQ ID NO.1; The nucleic acid sequence used to encode the AP65 protein in the coding element is shown in SEQ ID NO.

2.

2. A host cell, characterized in that, For transfecting HEK293 cells with the circular RNA of claim 1.

3. A complex, characterized in that, Including lipid nanoparticles carrying the circular RNA of claim 1.

4. The complex according to claim 3, characterized in that: The lipid nanoparticles encapsulate the circular RNA at a rate greater than 90%; and / or the surface potential of the complex is electroneutrally neutral or electronegative.

5. An immune composition, characterized in that, include: The circular RNA of claim 1 or the complex of any one of claims 3-4; And, pharmaceutically acceptable carriers.

6. Use of the circular RNA of claim 1, the complex of any one of claims 3-4, or the immune composition of claim 5 in the production of an agent for the prevention of infection of animals with Trichomonas vaginalis; wherein the animal is a pigeon.

7. The use of the circular RNA of claim 1, the complex of any one of claims 3-4, or the immune composition of claim 5 in the preparation of a pigeon trichomoniasis vaccine.

8. A circular RNA vaccine against Trichomonas vaginalis, characterized in that, include: The circular RNA of claim 1 or the complex of any one of claims 3-4; And, pharmaceutically acceptable carriers.

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