Fusion protein and immunogenic composition for preventing plasmodium infection and application

Abstract

The invention relates to a fusion protein, an immunogenic composition, a recombinant vaccine and the like for preventing plasmodium infection. The invention provides a novel fusion molecular architecture which comprises fusion proteins coded by four genes of ADF1, Pfj4, 14-3-3 protein and SUI1, or fusion proteins coded by two genes of an N-terminal structural domain of a TRAP antigen and a C-terminal structural domain of a CSP antigen, and further provides a composition of the two fusion proteins, and the composition can be used for research and development of nucleic acid vaccines or subunit vaccines. The fusion protein provided by the invention has good immunogenicity, can provide an effective immune protection effect against plasmodium infection, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, especially the technical field of immunopharmaceuticals, and specifically relates to a fusion protein for preventing Plasmodium infection, an immunogenic composition, and applications thereof, etc. Background Art

[0002] Malaria is an infectious disease caused by Plasmodium ( Plasmodium ), and is mainly transmitted by Anopheles mosquitoes ( Anopheles mosquito ). Plasmodium is a single-celled parasite that widely exists in tropical and subtropical regions, especially in sub-Saharan Africa, South Asia, and South America. Currently, the main pathogenic Plasmodium species include: Plasmodium falciparum ( Plasmodium falciparum ), Plasmodium vivax ( Plasmodium vivax ), Plasmodium ovale ( Plasmodium ovale ), and Plasmodium malariae ( Plasmodium malariae ). Among them, Plasmodium falciparum is the most lethal Plasmodium species, which can cause severe malaria symptoms such as high fever, anemia, cerebral malaria, and multiple organ failure, resulting in hundreds of thousands of deaths every year, especially among children and pregnant women.

[0003] The pathogenicity of Plasmodium mainly stems from its complex life cycle and various virulence factors in the host. After Plasmodium enters the human body through mosquito bites, it first multiplies in the liver and then invades red blood cells, triggering red blood cell rupture and immune responses, leading to typical malaria symptoms. The virulence factors of Plasmodium include surface proteins (such as merozoite surface protein MSP1), antigenic variation proteins (such as PfEMP1), and immune escape mechanisms, which enable Plasmodium to evade the host immune system and survive in the host for a long time.

[0004] For the treatment of malaria, traditional methods mainly rely on antimalarial drugs such as chloroquine, artemisinin and its derivatives. However, Plasmodium has developed widespread resistance to antimalarial drugs. In particular, the resistance of Plasmodium falciparum to artemisinin has emerged in Southeast Asia and is gradually spreading to other regions, posing a severe challenge to global malaria prevention and control. Vaccination is a more effective solution for malaria prevention. Compared with drug treatment, vaccines can activate the host's immune system to produce specific antibodies and immune cells against Plasmodium, thus providing long-term protection and preventing infection. In addition, effective vaccines can reduce the dependence on antimalarial drugs, inhibit the emergence and spread of drug-resistant Plasmodium, and relieve the pressure of global malaria prevention and control. However, due to the complex life cycle of Plasmodium and its powerful immune escape mechanism, developing effective malaria vaccines faces huge challenges. Currently, the only approved malaria vaccine is RTS,S / AS01 (trade name Mosquirix), but its protective effect is limited and the duration is short. RTS,S / AS01 is not available in China, and there is currently a lack of commercially available malaria vaccines in China.

[0005] Currently, several vaccines based on the major antigens of Plasmodium have entered the clinical trial stage. For example, the RTS,S / AS01 vaccine is based on the circumsporozoite protein (CSP) of Plasmodium falciparum and has been promoted in many African countries, but its protective effect is limited and the duration is short. The PfSPZ vaccine is a vaccine based on attenuated live Plasmodium and is currently in clinical trials. Preliminary results show that it has a high protective effect, but the production cost of live vaccines is high, the risk is great, and it is difficult to promote. CIS43LS and L9LS are two monoclonal antibodies targeting the circumsporozoite protein (CSP) of Plasmodium falciparum, which can block the infection of Plasmodium in the liver. CIS43LS showed a high protective effect in phase I clinical trials, and the protective effect could last for several months after a single injection; L9LS is an improved version of CIS43LS, with stronger neutralizing ability and a longer half-life, and is currently in phase II clinical trials. Preliminary results show that the protective effect of L9LS against Plasmodium falciparum infection in malaria-endemic areas exceeds 80%. However, the protective effect of both CIS43LS and L9LS lasts for a relatively short time, and the production cost is high, also facing the problem of being difficult to promote on a large scale.

[0006] Although multiple malaria vaccines have entered clinical trials, most of them still face problems such as the inability to provide long-term protection and cross-immune protection. Research has shown that the long-term protective effect after Plasmodium infection depends on T cell-mediated immune responses. However, traditional vaccines (such as the subunit vaccine RTS,S / AS01 and the antibody vaccines CIS43LS, L9LS) mainly rely on inducing humoral immune responses and are difficult to effectively activate T cell immunity, thus unable to solve the problem of long-term protection. In addition, since most current research is based on the CSP antigen of Plasmodium falciparum for vaccine design, and there are significant differences in the CSP protein among different Plasmodium species, although these vaccines can provide specific protection against Plasmodium falciparum, they cannot prevent or treat infections caused by other Plasmodium species such as Plasmodium vivax and Plasmodium malariae.

[0007] In summary, although multiple malaria vaccines have entered the research and development and clinical trial stages, there is still an urgent need in this field for a malaria vaccine with cross-immunogenicity and the ability to provide long-term protection. Through precise molecular design, nucleic acid vaccines can fuse and express multiple effective antigens, breaking through the difficulties of cross-immune protection. At the same time, by expressing antigen proteins intracellularly, nucleic acid vaccines can activate both humoral immune and T cell immune responses, providing a new direction for the development of malaria vaccines. Therefore, as an emerging technology, nucleic acid vaccines are expected to overcome the limitations of traditional vaccines and provide new solutions for malaria prevention and control. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a new fusion protein for preventing Plasmodium infection, an immunogenic composition, a recombinant vaccine, as well as a molecular architecture design and application. The present invention provides a new fusion molecular architecture, which includes a fusion protein encoded by four genes, namely ADF1, Pfj4, 14-3-3 protein, and SUI1, or a fusion protein encoded by two genes, namely the N-terminal domain of the TRAP antigen and the C-terminal domain of the CSP antigen, and a composition of the above two fusion proteins, which can be used for the research and development of nucleic acid vaccines or subunit vaccines. The present invention discovers that the new fusion molecule has good immunogenicity and can provide effective immune protection against Plasmodium infection, and almost completely inhibits Plasmodium amplification in some individuals. The present invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, uses, etc.

[0009] One aspect of the present invention provides a fusion protein, which is characterized in that it is a fusion protein selected from any one of the following: (1)Fusion protein A, which comprises an actin-depolymerizing factor 1 (ADF1) antigen, a heat shock protein DNAJ-like Pfj4 (Pfj4) antigen, a 14-3-3 protein antigen, and a translation initiation factor SUI1 (SUI1) antigen; (2)Fusion protein B, which comprises a thrombospondin related anonymous protein (TRAP) antigen and a circumsporozoite protein (CSP) antigen.

[0010] Furthermore, the fusion protein further comprises a combination of the fusion protein A and the fusion protein B.

[0011] Furthermore, the antigen is from Plasmodium.

[0012] Furthermore, the antigen is from Plasmodium falciparum ( Plasmodium falciparum ), Plasmodium vivax ( Plasmodium vivax ), Plasmodium ovale ( Plasmodium ovale ), Plasmodium malariae ( Plasmodium malariae ), or Plasmodium knowlesi ( Plasmodium knowlesi ), or one or more thereof.

[0013] Furthermore, the antigen is from Plasmodium falciparum ( Plasmodium falciparum ).

[0014] Furthermore, a functional element is added to the N-terminus or C-terminus of the fusion protein A or fusion protein B to promote the expression of the fusion protein or epitope presentation.

[0015] Furthermore, the fusion protein A sequentially comprises the actin-depolymerizing factor 1 antigen, the heat shock protein DNAJ-like Pfj4 antigen, the 14-3-3 protein antigen, and the translation initiation factor SUI1 antigen from the N-terminus to the C-terminus; optionally, the antigens are linked by a linker.

[0016] Further, for the fusion protein A, the amino acid sequence of the actin depolymerizing factor 1 antigen is as shown in SEQ ID NO: 1, the amino acid sequence of the DNAJ-like heat shock protein Pfj4 antigen is as shown in SEQ ID NO: 2, the amino acid sequence of the 14-3-3 protein antigen is as shown in SEQ ID NO: 3, the amino acid sequence of the translation initiation factor SUI1 antigen is as shown in SEQ ID NO: 4, and the amino acid sequence of the linker is as shown in SEQ ID NO: 12.

[0017] Further, for the fusion protein B, the TRAP antigen is the full-length sequence or the N-terminal domain of TRAP, and the CSP antigen is the full-length sequence or the C-terminal domain of CSP.

[0018] Further, the N-terminus of the fusion protein B further comprises a signal peptide and / or an Fc domain; more preferably, the N-terminus of the fusion protein B sequentially comprises a signal peptide and an Fc domain.

[0019] Further, the signal peptide is the signal peptide of human blucetin protein, and the Fc domain is the Fc domain of human IGHG1 protein; optionally, the TRAP antigen and the Fc domain are linked by a spacer sequence.

[0020] Further, for the fusion protein B, the TRAP antigen is the N-terminal domain of TRAP, and the amino acid sequence of the N-terminal domain of TRAP is as shown in SEQ ID NO: 5; the CSP antigen is the C-terminal domain of CSP, and the amino acid sequence of the C-terminal domain of CSP is as shown in SEQ ID NO: 6; the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 9; the amino acid sequence of the Fc domain is as shown in SEQ ID NO: 10; the amino acid sequence of the spacer sequence is as shown in SEQ ID NO: 11.

[0021] Further, the amino acid sequence of the fusion protein A is as shown in SEQ ID NO: 7, and the amino acid sequence of the fusion protein B is as shown in SEQ ID NO: 8.

[0022] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it comprises a nucleic acid encoding the fusion protein according to any one of the present invention.

[0023] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule according to the present invention.

[0024] Further, the recombinant gene expression cassette further comprises one or more of a promoter, a terminator, and a regulatory sequence.

[0025] Another aspect of the present invention provides a recombinant vector, characterized by comprising the recombinant nucleic acid molecule of the present invention, or the recombinant gene expression cassette of the present invention.

[0026] Further, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0027] Further, the prokaryotic vector comprises an Escherichia coli vector.

[0028] Further, the Escherichia coli vector includes but is not limited to pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, pBR vector.

[0029] Further, the eukaryotic vector includes but is not limited to yeast expression vectors, insect expression vectors, mammalian cell expression vectors.

[0030] Further, the yeast expression vectors include but are not limited to pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, pPIC9 vector.

[0031] Another aspect of the present invention provides a recombinant host cell, characterized by comprising the recombinant nucleic acid molecule of the present invention, or the recombinant gene expression cassette of the present invention, or the recombinant vector of the present invention.

[0032] Further, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0033] Further, the eukaryotic cell comprises mammalian cells, insect cells, yeast cells.

[0034] Further, the yeast cells include but are not limited to Saccharomyces cerevisiae, Pichia pastoris, Hansenula anomala.

[0035] Further, the prokaryotic cells include but are not limited to Escherichia coli cells, Bacillus subtilis cells, Pseudomonas cells.

[0036] Further, the Escherichia coli cells include but are not limited to BL21(DE3), DH5α, TOP10, Rosetta.

[0037] Another aspect of the present invention provides an immunogenic composition or a pharmaceutical composition, characterized by comprising one or more of the fusion proteins of any one of the present invention, and / or one or more of the recombinant nucleic acid molecules of the present invention, and / or one or more of the recombinant gene expression cassettes of the present invention, and / or one or more of the recombinant vectors of the present invention, and / or one or more of the recombinant host cells of the present invention.

[0038] Furthermore, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0039] Furthermore, the immunogenic composition or pharmaceutical composition comprises the fusion protein A and the fusion protein B.

[0040] Another aspect of the present invention provides a recombinant vaccine, characterized by comprising one or more of the fusion proteins according to any one of the present invention, and / or one or more of the recombinant nucleic acid molecules according to the present invention, and / or one or more of the recombinant gene expression cassettes according to the present invention, and / or one or more of the recombinant vectors according to the present invention, and / or one or more of the recombinant host cells according to the present invention, and / or one or more of the immunogenic compositions or pharmaceutical compositions according to the present invention.

[0041] Furthermore, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine.

[0042] Furthermore, the recombinant vaccine is a nucleic acid vaccine.

[0043] Another aspect of the present invention provides the use of one or more of the fusion proteins according to any one of the present invention, and / or one or more of the recombinant nucleic acid molecules according to the present invention, and / or one or more of the recombinant gene expression cassettes according to the present invention, and / or one or more of the recombinant vectors according to the present invention, and / or one or more of the recombinant host cells according to the present invention, and / or one or more of the immunogenic compositions or pharmaceutical compositions according to the present invention, and / or one or more of the recombinant vaccines according to the present invention in the preparation of a vaccine or a drug for biologic immunization for prevention, treatment and / or vaccination.

[0044] Furthermore, the drug is used for preventing and / or treating Plasmodium infection.

[0045] Furthermore, the Plasmodium is Plasmodium falciparum ( Plasmodium falciparum ), Plasmodium vivax ( Plasmodium vivax ), Plasmodium ovale ( Plasmodium ovale ), Plasmodium malariae ( Plasmodium malariae ), or Plasmodium knowlesi ( Plasmodium knowlesi ), or one or more thereof.

[0046] Furthermore, the Plasmodium is Plasmodium falciparum ( Plasmodium falciparum ).

[0047] Another aspect of the present invention provides a method for preventing and / or treating a disease, characterized by comprising administering to a subject one or more of the fusion proteins described in any one of the present invention, and / or one or more of the recombinant nucleic acid molecules described in the present invention, and / or one or more of the recombinant gene expression cassettes described in the present invention, and / or one or more of the recombinant vectors described in the present invention, and / or one or more of the recombinant host cells described in the present invention, and / or one or more of the immunogenic compositions or pharmaceutical compositions described in the present invention, and / or one or more of the recombinant vaccines described in the present invention.

[0048] Further, the disease is malaria parasite infection.

[0049] Further, the malaria parasite is Plasmodium falciparum ( Plasmodium falciparum ), Plasmodium vivax ( Plasmodium vivax ), Plasmodium ovale ( Plasmodium ovale ), Plasmodium malariae ( Plasmodium malariae ), or Plasmodium knowlesi ( Plasmodium knowlesi ), or one or more thereof.

[0050] Further, the malaria parasite is Plasmodium falciparum ( Plasmodium falciparum ).

[0051] The fusion proteins, immunogenic compositions, recombinant vaccines, etc. of the present invention have the following beneficial technical effects: 1. Starting from the perspective of reverse vaccinology, the present invention gradually screens and excludes, and finally preferably obtains four antigens, namely ADF1, Pfj4, 14-3-3 protein, and SUI1, for constructing a new fusion protein.

[0052] 2. Existing technologies show that simply selecting the RTS domain of CSP cannot provide sufficient protective efficacy. Based on the design strategy for the CSP antigen in the RTS,S / AS01 vaccine, the present invention further optimizes the design, reselects the C-terminal domain of CSP, and adds the N-terminal domain of the TRAP antigen to form another new fusion protein, providing more effective epitopes while ensuring the stability of the molecular conformation.

[0053] 3. The present invention discovers that the new fusion molecule has good immunogenicity and can provide an immune protection effect, and can be used for the research and development of nucleic acid vaccines or subunit vaccines. The immunogenic composition of the present invention can provide effective immune protection against malaria parasite infection.

[0054] 4. The present invention also discovers that the new fusion molecule can be highly expressed in eukaryotic cells, providing correct and sufficient immune epitopes and good immune protection effects, providing new technologies for the field of immune drug design of malaria parasite multivalent vaccines.

[0055] 5. Example 6 of the present invention shows that both Vaccine A and Vaccine B designed based on the present invention can be correctly expressed in eukaryotic cells, and the expressed proteins have correct and stable structures, which is beneficial to the presentation of immune epitopes.

[0056] 6. Example 7 of the present invention shows that in a mouse infection model, Vaccine A, Vaccine B, and the combination of Vaccine A and Vaccine B based on the present invention can all provide good immune protection effects. Whether it is cellular immunity or humoral immunity, they can significantly inhibit the amplification of Plasmodium in the liver stage.

[0057] 7. Example 7 of the present invention also shows that the combined use of Vaccine A and Vaccine B can provide a superimposed immune protection effect, achieving unexpected technical effects. This result also indicates that the development of malaria vaccines needs to take into account both humoral immunity and cellular immunity.

[0058] 8. Examples 6 and 7 of the present invention together show that the vaccines based on the present invention can induce effective immunity in the model animal mice and have good preventive effects against Plasmodium infection. The present invention can be applied to the production and research and development of human and animal immune drugs, filling the gap in the current field of malaria vaccine research and development, and having extremely high commercial value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figures 1A - 1D Homology analysis of four cellular immune targets screened for the present invention in different Plasmodium; Figure 1A Homology analysis of the ADF1 cellular immune target in different Plasmodium; Figure 1B Homology analysis of the Pfj4 cellular immune target in different Plasmodium; Figure 1C Homology analysis of the 14-3-3 cellular immune target in different Plasmodium; Figure 1D Homology analysis of the SUI1 cellular immune target in different Plasmodium.

[0060] Figure 2 Schematic diagram of the truncation strategy for the N-terminal domain of the TRAP antigen and the C-terminal domain of the CSP antigen of the present invention.

[0061] Figure 3 Non-limiting molecular schematic diagrams of Vaccine A and Vaccine B of the present invention.

[0062] Figures 4A - 4B Quality control result diagrams of Vaccine A and Vaccine B of the present invention respectively.

[0063] Figures 5A - 5B Expression conditions of Vaccine A and Vaccine B of the present invention after in vitro transfection of HEK293 cells respectively.

[0064] Figure 6Schematic diagram of the vaccination schedule for mice with the vaccine of the present invention.

[0065] Figure 7 Results of qPCR relative quantification of parasite load in liver tissues of mice vaccinated with the vaccine of the present invention and challenged with the virus, 42 h after challenge.

[0066] Figure 8 Results of ELISA detection of CSP-specific IgG antibodies in sera of mice vaccinated with the vaccine of the present invention and challenged with the virus, 42 h after challenge. Detailed implementation manners

[0067] Terms and definitions The term "Plasmodium" refers to single-celled parasites belonging to the genus Plasmodium ( Plasmodium ), mainly including Plasmodium falciparum ( Plasmodium falciparum ), Plasmodium vivax ( Plasmodium vivax ), Plasmodium ovale ( Plasmodium ovale ), Plasmodium malariae ( Plasmodium malariae ), and Plasmodium knowlesi ( Plasmodium knowlesi ), etc. Plasmodium is transmitted by mosquito vectors and infects the red blood cells and liver cells of mammals (including humans), causing malaria.

[0068] The term "malaria infection" refers to an infection caused by parasites of the genus Plasmodium, including diseases caused by the infection of mammals (preferably human hosts) by species such as Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Malaria infection is usually transmitted through the bite of infected Anopheles mosquitoes, and its characteristics include periodic fever, anemia, hepatosplenomegaly, and in severe cases, it can lead to organ failure or death.

[0069] The term "ADF1" refers to the actin-depolymerizing factor 1 (ADF1) antigen from Plasmodium. Proteins containing the isolated wild-type ADF1 polypeptide and its segments of Plasmodium and variants that can stimulate an immune response against the Plasmodium ADF1 protein. Preferably, the amino acid sequence of ADF1 is as shown in SEQ ID NO: 1.

[0070] The term "Pfj4" refers to the heat shock protein DNAJ-like Pfj4 (Pfj4) antigen from Plasmodium. Proteins containing the isolated wild-type Pfj4 polypeptide and its segments of Plasmodium and variants that can stimulate an immune response against the Plasmodium Pfj4 protein. Preferably, the amino acid sequence of Pfj4 is as shown in SEQ ID NO: 2.

[0071] The term "14-3-3 protein" refers to the 14-3-3 protein antigen from Plasmodium, also known as "14-3-3". Proteins containing the isolated wild-type 14-3-3 polypeptide of Plasmodium and its segments, and variants that can stimulate an immune response against the Plasmodium 14-3-3 protein. Preferably, the amino acid sequence of the 14-3-3 protein is as shown in SEQ ID NO: 3.

[0072] The term "SUI1" refers to the translation initiation factor SUI1 antigen from Plasmodium. Proteins containing the isolated wild-type SUI1 polypeptide of Plasmodium and its segments, and variants that can stimulate an immune response against the Plasmodium SUI1 protein. Preferably, the amino acid sequence of SUI1 is as shown in SEQ ID NO: 4.

[0073] The term "N-terminal domain of TRAP" refers to the N-terminal domain of the thrombospondin-related anonymous protein (TRAP) antigen from Plasmodium. Proteins containing the isolated wild-type N-terminal polypeptide of TRAP of Plasmodium and its segments, and variants that can stimulate an immune response against the Plasmodium TRAP protein. Preferably, the amino acid sequence of the N-terminal domain of TRAP is as shown in SEQ ID NO: 5.

[0074] The term "C-terminal domain of CSP" refers to the C-terminal domain of the Circumsporozoite protein (CSP) antigen from Plasmodium. Proteins containing the isolated wild-type C-terminal polypeptide of CSP of Plasmodium and its segments, and variants that can stimulate an immune response against the Plasmodium CSP protein. Preferably, the amino acid sequence of the C-terminal domain of CSP is as shown in SEQ ID NO: 6.

[0075] The term "immune response" refers to a humoral response, a cellular response, or both a humoral and cellular response in an organism. The immune response can be measured by assays including, but not limited to, assays measuring the presence or amount of antibodies that specifically recognize a protein or cell surface protein, assays measuring T cell activation or proliferation, and / or assays measuring the regulation of the activity or expression of one or more cytokines.

[0076] The term "administration" or "inoculation" refers to the administration of the nucleic acid vaccine or vaccine composition of the present invention preferably via the intramuscular or subcutaneous route, although other administration routes can also be used, for example, oral, intranasal (such as aerosol or other non-injection administrations), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or via combined routes. Intramuscular administration in the neck of the animal is preferred. Boosting regimens can be employed to adjust the administration regimen to provide optimal immunity.

[0077] 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.

[0078] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used for transformation into a suitable host cell. Then the polynucleotide is expressed in the recombinant host cell to produce, for example, "recombinant polypeptide", "recombinant protein", "fusion protein", etc.

[0079] The term "recombinant expression vector" refers to a DNA construct used for expressing, for example, a polynucleotide encoding a desired polypeptide. The recombinant expression vector can include, for example, a transcriptional subunit containing (1) a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structure or coding sequence that is transcribed into mRNA and translated into a protein; and (3) appropriate transcriptional and translational start and stop sequences. The recombinant expression vector is constructed in any suitable manner and can use any vector, including plasmids, viruses, phages and transposons. Possible vectors for the present disclosure include but are not limited to chromosomal, non-chromosomal and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmid and phage DNA, DNA from viruses such as lentivirus, retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40 and pseudorabies. Include self-replicating vectors and non-self-replicating vectors.

[0080] The term "mRNA" refers to messenger RNA, which is a single-stranded ribonucleic acid transcribed from one strand of DNA and carrying genetic information to direct protein synthesis.

[0081] The term "5'-UTR" refers to the "5'-untranslated region" or "5'UTR", which is a part of a gene that is transcribed into a primary RNA transcript (precursor mRNA) and is located upstream of the coding sequence. The primary transcript is the initial RNA product that contains introns and exons and is produced by transcription of DNA. Many primary transcripts must undergo RNA processing to form a physiologically active RNA. The processing steps to form mature mRNA include modifying the termini, excising introns, capping, and / or cleaving out individual rRNA molecules from the precursor RNA. Thus, the 5'UTR of mRNA is the part of the mRNA that is not translated into protein and is located upstream of the coding sequence. In genomic sequences, the 5'UTR is typically defined as the region between the transcription start site and the start codon. The length of the 5'-untranslated region (5'UTR) of vertebrate mRNAs can range from a few dozen to several hundred bases.

[0082] 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 the protein-coding region, and which is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule. The 3'-untranslated region typically extends from the stop codon of the translation product to the poly(A) sequence that is usually appended after transcription. The 3'-untranslated region of mammalian mRNAs typically has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence may be the poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-untranslated region can contain one or more inverted repeats that can fold to produce a stem-loop structure that serves as a barrier to exonucleases or interacts with proteins (e.g., RNA-binding proteins) known to enhance RNA stability.

[0083] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include both primary transformed cells and progeny derived therefrom. A host cell is any type of cell system that can be used to produce a recombinant vaccine based on the present invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells.

[0084] The terms "individual", "patient", or "subject" include mammals. Mammals include, but are not limited to, domestic animals (e.g., pigs, cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats).

[0085] The terms "transformation, transfection, transduction" have the meanings generally understood by those skilled in the art, that is, the process of introducing exogenous DNA or RNA into a host.

[0086] The term "drug combination" or "pharmaceutical composition" refers to an auxiliary material widely used in the field of drug production. The main purpose of using a carrier is to provide a pharmaceutically safe, stable in nature and / or functionally specific pharmaceutical composition, and also to provide a method for effective absorption in the body of a subject. A pharmaceutically acceptable carrier can be an inert filler or an active ingredient that provides a certain function for the pharmaceutical combination (such as stabilizing the overall pH value of the composition or preventing the 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, glidants, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweetening agents, etc.

[0087] The term "treatment" means that after a subject suffers from a disease, the subject is exposed to (such as administered) a recombinant vaccine, composition, etc. based on the present invention, so that the symptoms of the disease are alleviated compared with when not exposed, and it does not necessarily mean completely suppressing the symptoms of the disease. Suffering from a disease means that the body shows symptoms of the disease.

[0088] The term "prevention" means that before a subject suffers from a disease, by exposing the subject to (such as administered) a recombinant vaccine, composition, etc. based on the present invention, so that the symptoms after suffering from the disease are alleviated compared with when not exposed, and it does not necessarily mean completely suppressing the onset of the disease.

[0089] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meanings as commonly understood by those of ordinary skill in the art to which this disclosure pertains.

[0090] The present invention discloses a fusion molecular architecture capable of preventing Plasmodium infection, a preparation method and application of a recombinant vaccine based on this architecture. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. 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 in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0091] In the fusion protein, encoding nucleic acid and its elements, preparation method and application provided by the present invention, the raw materials and reagents used can all be obtained commercially. Based on the basic knowledge of conventional molecular cloning, expression construction, vaccine preparation, immunology, etc. in the art, those skilled in the art can all implement the methods of the examples of the present invention.

[0092] The present invention will be further illustrated below in conjunction with examples. Among them, as a preference, a nucleic acid vaccine framework is selected for the preparation of recombinant vaccines.

[0093] Example 1 Screening of Cellular Immune Antigens and Construction of Fusion Proteins In order to screen antigens that can be used for cellular immunity, the present invention starts from the perspective of reverse vaccinology, downloads the transcriptome data of Plasmodium falciparum, Plasmodium berghei, Plasmodium malariae, Plasmodium vivax, and Plasmodium ovale from the NCBI database, and through technical analysis, obtains a series of candidate antigens with high transcriptional levels. These candidate antigens have the following characteristics in different Plasmodium species: high transcriptional level, and the sequence homology from different Plasmodium species is more than 80%. Since malaria vaccines are ultimately applied to human immunity, antigens with high homology to human proteins need to be excluded from the candidate antigens. At the same time, considering that when designing vaccines, proteins with too small molecular weights provide limited immune epitopes, while proteins with too large molecular weights are difficult to express and have certain difficulties in the production and preparation process, so screening and exclusion need to be carried out step by step. Finally, preferably, four antigens, ADF1 (actin-depolymerizing factor 1), Pfj4 (heat shock protein DNAJ-like Pfj4), 14-3-3 protein, and SUI1 (translation initiation factor SUI1), are obtained.

[0094] As Figure 1A shown, the homology of the ADF1 antigen in the five Plasmodium species is > 86%; as Figure 1B shown, the homology of the Pfj4 antigen in the five Plasmodium species is > 88%; as Figure 1C shown, the homology of the 14-3-3 protein (also known as 14-3-3) antigen in the five Plasmodium species is > 98%; as Figure 1D shown, the homology of the SUI1 antigen in the five Plasmodium species is > 93%. In addition, the amino acid lengths of the four preferred antigens are in the range of 100 - 300, which is the preferred length for constructing fusion proteins.

[0095] Example 2 Truncation of TRAP Antigen and CSP Antigen and Construction of Fusion Proteins According to existing research, antibodies that specifically bind to the CSP antigen of Plasmodium falciparum have been proven to be useful for blocking malaria parasite infection, with a protection rate of approximately 80%. However, due to the short protection time and the inability to induce long-term immune protection, the scope of use is limited and it is difficult to promote on a large scale. From the perspective of preventive vaccines, humoral immune vaccines are still indispensable. Currently, the RTS,S / AS01 vaccine uses the CSP antigen as an immunogen. Clinical results show that after four immunizations, the protection rate is around 30%. This proves that simply selecting the RTS domain of CSP cannot provide sufficient protective efficacy, and other humoral immune antigens need to be added to improve the protection effect.

[0096] Based on the design strategy for the CSP antigen in the RTS,S / AS01 vaccine, the present invention further optimizes the design. As Figure 2 shown, the present invention reselects the C-terminal domain of CSP and adds the N-terminal domain of the TRAP antigen to form a fusion protein, which provides more effective epitopes while ensuring the stability of the molecular conformation.

[0097] Example 3 Construction of the recombinant nucleic acid vaccine of the present invention In order to prepare a recombinant nucleic acid vaccine containing the antigen of the present invention, a non-limiting schematic diagram of the nucleic acid vaccine architecture of the present invention is as Figure 3 shown. Exemplarily, Figure 3 is a schematic diagram of the molecular structure of the fusion proteins expressed by Vaccine A and Vaccine B of the present invention. In order to prepare a recombinant nucleic acid vaccine capable of generating the molecular structure as Figure 3 shown, first, a gene expression cassette is constructed to express the antigen sequence of the present invention. The expression cassette sequentially includes from the 5' end to the 3' end: 5'UTR, CDS region, 3'UTR, PolyA, wherein the CDS region includes the fusion molecular architecture of the present invention. Subsequently, based on codon degeneracy, the complete gene expression cassette sequence is optimized, and the DNA sequence is directly obtained by gene synthesis (commissioned by GenScript Corporation for synthesis). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector available for in vitro RNA transcription to obtain a vector plasmid for preparing the recombinant nucleic acid vaccine.

[0098] According to the above method, vectors for subsequent examples are prepared: (1) Preparation of the vector based on the recombinant nucleic acid vaccine A of the present invention Step a: Synthesize the fusion fragment of the four genes "ADF1-Pfj4-[14-3-3]-SUI1", and the genes are connected by a linker sequence with the amino acid sequence as shown in SEQ ID NO: 12. The amino acid sequence of the fusion protein encoded by the ADF1-Pfj4-[14-3-3]-SUI1 fusion gene is as shown in SEQ ID NO: 7. Among them, the amino acid sequence of the ADF1 antigen is as shown in SEQ ID NO: 1, the amino acid of the Pfj4 antigen is as shown in SEQ ID NO: 2, the amino acid of the 14-3-3 antigen is as shown in SEQ ID NO: 3, and the amino acid of the SUI1 antigen is as shown in SEQ ID NO: 4.

[0099] Step b: Construct a nucleic acid vaccine framework vector.

[0100] The nucleic acid vaccine framework vector contains 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0101] Step c: Prepare a recombinant plasmid.

[0102] Insert the gene synthesized in step a into the vector framework of step b to obtain a preparation vector for the recombinant nucleic acid vaccine A based on the present invention.

[0103] (2) Preparation vector for the recombinant nucleic acid vaccine B based on the present invention Step a: Synthesize the gene fragment of "human signal peptide-Fc domain of human IGHG1-N-terminal domain of TRAP antigen-C-terminal domain of CSP antigen". Among them, the human signal peptide is the signal peptide of human Azurocidin protein, and its amino acid sequence is as shown in SEQ ID NO: 9. The amino acid of the Fc domain of human IGHG1 is as shown in SEQ ID NO: 10. The amino acid of the N-terminal domain of the TRAP antigen is as shown in SEQ ID NO: 5. The amino acid of the C-terminal domain of the CSP antigen is as shown in SEQ ID NO: 6. The Fc domain of human IGHG1 and the N-terminal domain of the TRAP antigen are connected by a spacer sequence with the amino acid sequence as shown in SEQ ID NO: 11. The amino acid sequence of the fusion protein encoded by the "N-terminal domain of the TRAP antigen-C-terminal domain of the CSP antigen" fusion gene is as shown in SEQ ID NO: 8.

[0104] Step b: Construct a nucleic acid vaccine framework vector.

[0105] The nucleic acid vaccine framework vector contains 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0106] Step c: Prepare a recombinant plasmid.

[0107] Insert the gene synthesized in step a into the vector framework of step b to obtain a vector for preparing the recombinant nucleic acid vaccine B based on the present invention.

[0108] Table 1 Protein amino acid sequences of the architecture elements involved in the present invention Amino acid sequences and sequence numbers ADF1 antigen GIRVNDNCVTEFNNMKIRKTCGWIIFVIQNCEIIIHSKGASTTLTELVQSIDKNNEIQCAYVVFDAVSKIHFFMYARESSNSRDRMTYASSKQAILKKIEGVNVLTSVIESAQDVADLK (SEQ ID NO: 1) Pfj4 antigen SRRVNYYEVLGVPQDADLTVIKKSYRTLAMKWHPDKNPNNKAEATERFKQISEAYEVLSDPKRRRKYDLYGTDENYMADENDEFSNFHKNFGFNDAQRIFEMFFGDSSPFGNDSFFSDVMGSSFVDKRRGRVPRSNDPFDNFFGSSFNVSFGSSFDNFMDGGS (SEQ ID NO: 2) 14 - 3 - 3 protein antigen KDELTVEERNLLSVAYKNAVGARRASWRIISSVEQKEMSKANVHNKNVAATYRKKVEEELNNICQDILNLLTKKLIPNTSESESKVFYYKMKGDYYRYISEFSCDEGKKEASNCAQEAYQKATDIAENELPSTHPIRLGLALNYSVFFYEILNQPHQACEMAKRAFDDAITEFDNVSEDSYKDSTLIMQLLRDNLTLWTSDLQGDQTEEKSKDEGLE (SEQ ID NO: 3) SUI1 antigen NATNLIHIRNQQRNGRKSVTTVQGLGKTFDLKKMVRALKKEFNCNGTIIEDIEHGSIIQLQGDKRNNVKEFLIREGICALEHIRIHGA (SEQ ID NO:4) N-terminal domain of the TRAP antigen RDVQNNIVDEIKYREEVCNDEVDLYLLMDCSGSIRRHNWVNHAVPLAMKLIQQLNLNENAIHLYANVFSNNAREIIRLHSDASKNKEKALIIIKSLLSTNLPYGRTNLTDALLQVRKHLNDRINRENANQLVVILTDGIPDSIQDSLKESRKLNDRGVKIAVFGIGQGINVAFNRFLVGCHPSDGKCNLYADSAWENVKNVIGPFMKAVCVEVEKTASCGVWDEWSPCSVTCGKGTRSRKREILHEGCTSELQEQCEEERCPPKREPLDVPD (SEQ ID NO: 5) C-terminal domain of the CSP antigen DPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCSSV (SEQ ID NO: 6) Fusion protein A GIRVNDNCVTEFNNMKIRKTCGWIIFVIQNCEIIIHSKGASTTLTELVQSIDKNNEIQCAYVVFDAVSKIHFFMYARESSNSRDRMTYASSKQAILKKIEGVNVLTSVIESAQDVADLKGGSGGGGSGGSRRVNYYEVLGVPQDADLTVIKKSYRTLAMKWHPDKNPNNKAEATERFKQISEAYEVLSDPKRRRKYDLYGTDENYMADENDEFSNFHKNFGFNDAQRIFEMFFGDSSPFGNDSFFSDVMGSSFVDKRRGRVPRSNDPFDNFFGSSFNVSFGSSFDNFMDGGSGGSGGGGSGGKDELTVEERNLLSVAYKNAVGARRASWRIISSVEQKEMSKANVHNKNVAATYRKKVEEELNNICQDILNLLTKKLIPNTSESESKVFYYKMKGDYYRYISEFSCDEGKKEASNCAQEAYQKATDIAENELPSTHPIRLGLALNYSVFFYEILNQPHQACEMAKRAFDDAITEFDNVSEDSYKDSTLIMQLLRDNLTLWTSDLQGDQTEEKSKDEGLEGGSGGGGSGGNATNLIHIRNQQRNGRKSVTTVQGLGKTFDLKKMVRALKKEFNCNGTIIEDIEHGSIIQLQGDKRNNVKEFLIREGICALEHIRIHGA (SEQ ID NO:7) Fusion protein B MTRLTVLALLAGLLASSRAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSGGSGGSGRDVQNNIVDEIKYREEVCNDEVDLYLLMDCSGSIRRHNWVNHAVPLAMKLIQQLNLNENAIHLYANVFSNNAREIIRLHSDASKNKEKALIIIKSLLSTNLPYGRTNLTDALLQVRKHLNDRINRENANQLVVILTDGIPDSIQDSLKESRKLNDRGVKIAVFGIGQGINVAFNRFLVGCHPSDGKCNLYADSAWENVKNVIGPFMKAVCVEVEKTASCGVWDEWSPCSVTCGKGTRSRKREILHEGCTSELQEQCEEERCPPKREPLDVPDDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCSSV (SEQ ID NO: 8) Signal peptide of human lentin protein MTRLTVLALLAGLLASSRA (SEQ ID NO: 9) Fc domain of human IGHG1 protein EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10) Spacer sequence GGSGGSGGSG (SEQ ID NO: 11) Linker sequence GGSGGGGSGG (SEQ ID NO: 12) Example 4 Preparation of the recombinant nucleic acid vaccine of the present invention (1) Preparation of capped mRNA vaccine Step a: Digest the vector plasmid used for producing the capped mRNA vaccine in Example 1 to linearize it, and obtain a linearized plasmid for in vitro transcription.

[0109] Step b: Perform an in vitro co-transcription capping reaction on the linearized plasmid, add a 7-methylguanylate cap structure to the 5' end of the transcribed mRNA, and degrade the template DNA.

[0110] (2) Preparation of uncapped mRNA vaccine Step a: Digest the vector plasmid used for producing the uncapped mRNA vaccine in Example 1 to linearize it, and obtain a linearized plasmid for in vitro transcription.

[0111] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid, and degrade the template DNA.

[0112] (3) Preparation of DNA vaccine Step a: Amplify the vector plasmid used for producing the DNA vaccine in Example 1 to obtain a large amount of target plasmid for purification.

[0113] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0114] Example 5 Quality control of in vitro transcription of the recombinant nucleic acid of the present invention and vaccine preparation Prepare vaccine A (recombinant nucleic acid vaccine A based on the present invention) and vaccine B (recombinant nucleic acid vaccine B based on the present invention) by the method for preparing the capped mRNA vaccine in Example 4. Detect the purity of the produced recombinant nucleic acid. The purity of the recombinant nucleic acid used in the experiment is greater than 80%. The quality control peak pattern of the recombinant nucleic acid based on the present invention is as shown in Figure 4A 、 Figure 4B Shown. Specifically described as: (1) Recombinant nucleic acid vaccine A based on the present invention, with a purity of 83.9%; (2) Recombinant nucleic acid vaccine B based on the present invention, with a purity of 86.8%. The above purities all meet the quality requirements for cell transfection experiments and vaccine production.

[0115] Example 6 In vitro expression effect of the recombinant nucleic acid of the present invention Using a cell transfection reagent, vaccines A and B in Example 5 were transfected into HEK293T cells, and the proteins were collected for Western blot detection. Figure 5A , Figure 5B showed the in vitro expression WB (Western blot) detection results of vaccines A and B transfected into HEK293 cells. Among them, the antigen expressed by vaccine A was a cellular immune antigen, and theoretically, significant expression could be detected in the cell lysate; the antigen expressed by vaccine B was a humoral immune antigen, and theoretically, significant expression could be detected in the supernatant. Vaccines containing multiple cellular immune antigens could detect significant protein expression in the cell lysate; vaccines containing multiple humoral immune antigens could detect significant protein expression in the supernatant.

[0116] The protein molecular weights of vaccines A and B are shown in Table 2. Among them, vaccine A could be significantly expressed intracellularly, proving that the molecular architecture provided by the present invention for promoting antigen fusion expression could enable multiple antigens to be successfully translated, correctly folded in eukaryotic cells, and had a stable structure and a long half-life. Vaccine B could be significantly expressed in the supernatant, proving that the molecular architecture provided by the present invention for promoting the secretory expression of fusion proteins could enable multiple antigens to be successfully translated, correctly folded, and secreted extracellularly in eukaryotic cells. Therefore, based on the vaccines designed by the present invention, whether it was a cellular immune antigen fusion protein or a humoral immune antigen fusion protein, it could be correctly expressed in eukaryotic cells, and the expressed proteins had a correct and stable structure, which was beneficial to immune epitope presentation.

[0117] Table 2 Protein molecular weights of vaccines A and B Name Protein molecular weight (kDa) Vaccine A 81.9 Vaccine B 81.3 Example 7 Preventive effect of the recombinant nucleic acid vaccine based on the present invention in a mouse infection model To verify whether the cellular immune antigen fusion molecule and the humoral immune antigen fusion molecule had immune protection effects, and to verify whether there was a superimposed synergistic protection effect when the two were co-immunized, in this example, immunotherapy experiments were carried out using vaccine A, vaccine B, and a combination of vaccine A and vaccine B.

[0118] Since the malaria parasite used in this experiment was Plasmodium berghei ( Plasmodium berghei ), which hardly infects humans, in the present invention, a genetically modified mutant strain of Plasmodium berghei was used, and this strain could express the circumsporozoite protein (CSP) of Plasmodium falciparum ( Plasmodium falciparum ). CSP is the main pathogenic protein of Plasmodium falciparum and is also a key target for vaccine design. Therefore, this mutant strain provided an ideal experimental model for verifying the efficacy of malaria vaccines.

[0119] In addition, Plasmodium will transform from the hepatic stage development stage to the intraerythrocytic stage (blood cell stage) 48 hours after infection. At this time, it is impossible to accurately quantify the Plasmodium in the hepatic stage. Therefore, in the present invention, dissection and sample collection are carried out 42 hours after challenge to ensure that quantitative detection is completed before Plasmodium fully enters the intraerythrocytic stage. The selection of this time point can more accurately reflect the inhibitory effect of the vaccine on Plasmodium in the hepatic stage, thereby more truly evaluating the protective efficacy of the vaccine.

[0120] A total of 20 six-week-old Balb / c strain mice were selected for the experiment. The specific immunization grouping and treatment process are shown in Table 3. The dose in this table and the following text refers to the amount of the active ingredient (i.e., the recombinant nucleic acid vaccine of the present invention). The immunization and sampling process is as Figure 6 shown.

[0121] Table 3 Immunization and challenge schedule of experimental animals in Example 7

[0122] Note: PBS refers to replacing the vaccine with PBS solution, which is the control group (non-treatment group) and serves as the control for the immunotherapy experiment.

[0123] The mice in each group were immunotherapied twice according to the immunization process shown in Table 3, and a challenge experiment was carried out on the 35th day. The mice were sacrificed by cervical dislocation 42 h after challenge. The whole liver tissue was taken and placed in Trizol, and mechanically ground into a paste. Total RNA of the liver tissue was extracted by the Trizol method, and the concentration was measured by Nanodrop. The concentration of total RNA was adjusted to 500 ng / μl with RNase-free water, and 1 μg of total RNA was taken for reverse transcription to obtain cDNA. 1 μl of cDNA was taken for qPCR to detect the relative expression level of Plasmodium 18S in the liver tissue, which was used to evaluate the Plasmodium load in the liver tissue. The primers used in the qPCR experiment are shown in Table 4.

[0124] Table 4 Primers used in the qPCR experiment of Example 7 18S upstream primer AAGCATTAAATAAAGCGAATACATCCTTAC (SEQ ID NO: 13) 18S downstream primer GGAGATTGGTTTTGACGTTTATGTG (SEQ ID NO: 14) Housekeeping gene upstream primer GTTGTCTCCTGCGACTTCA (SEQ ID NO: 15) Housekeeping gene downstream primer GGTGGTCCAGGGTTTCTTA (SEQ ID NO: 16)

[0125] Note: The internal reference gene in Table 4 is GapDH.

[0126] The detection results are as Figure 7 shown. Immunization with vaccine A alone, vaccine B alone, or a mixture of vaccine A and vaccine B can all provide significant protective effects. Among them, the effect of immunization with vaccine B alone is better than that of immunization with vaccine A alone, and the effect of immunization with a mixture of vaccine A and vaccine B is the best, with almost complete inhibition in 2 / 5 individuals.

[0127] Mouse sera were collected 42 h after inoculation with the virulent agent, and the CSP-specific IgG antibody titers in the sera were detected. The full-length Plasmodium falciparum CSP protein expressed in vitro was used as an antigen to coat an ELISA plate, which was blocked with 1% casein. The primary antibody was the mouse serum after inoculation with the virulent agent (diluted 1:100 with 1% casein), with two replicates for each sample. The secondary antibody was goat anti-mouse IgG-HRP (diluted 1:100 with 1% casein). The test results are as Figure 8 shown. Vaccine A is a cellular immune vaccine and does not contain the CSP antigen, so it induced the lowest CSP antibody titer; Vaccine B is a humoral immune vaccine and contains the C-terminal domain of the CSP antigen, inducing a high level of specific IgG antibodies; the combined immunization with Vaccine A and Vaccine B also induced a high antibody titer.

[0128] The results of the above two experiments showed that the humoral immune vaccine B of the present invention could induce mice to produce a high level of specific antibodies, thus playing a significant protective role in the early stage of Plasmodium infection; while the cellular immune vaccine A of the present invention could effectively activate the cellular immune response of mice. Although it could not completely inhibit the proliferation of Plasmodium in the early stage of infection, when used in combination with the humoral immune vaccine B, it could produce a significant synergistic effect and achieve the superposition of protective efficacy.

[0129] In addition, since the antigen sequences used in the vaccine design were completely derived from Plasmodium falciparum ( Plasmodium falciparum ), and the inoculation experiment used genetically modified Plasmodium berghei ( Plasmodium berghei , expressing the Plasmodium falciparum CSP protein), the inhibitory effect shown by the cellular immune vaccine further proved that the vaccine of the present invention could provide good cross-immune protection. This characteristic laid an important foundation for the development of a broad-spectrum malaria vaccine that could prevent multiple diseases with a single vaccine, and had important reference value and application prospects.

[0130] In summary, the antigen and fusion molecular architecture provided by the present invention could induce effective immunity in the model animal mice and had a good preventive effect against Plasmodium infection. Therefore, the present invention could be applied to the production and research and development of animal immune drugs, filling the gap in the current malaria vaccine research and development field, and had extremely high commercial value and broad application prospects.

[0131] The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, rather than limitations on the implementation manners of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present disclosure shall be included within the protection scope of the claims of the present disclosure.

Claims

1. A fusion protein, characterized in that is a fusion protein selected from any one of the following: Fusion protein A, wherein the fusion protein A comprises actin-depolymerizing factor 1 (ADF1) antigen, heat shock protein DNAJ-like Pfj4 (Pfj4) antigen, 14-3-3 protein antigen, and translation initiation factor SUI1 (SUI1) antigen; The fusion protein B comprises a thrombospondin related anonymous protein (TRAP) antigen and a circumsporozoite protein (CSP) antigen; preferably, the fusion protein further comprises a combination of the fusion protein A and the fusion protein B.

2. The fusion protein according to claim 1, characterized in that The antigen is derived from Plasmodium; preferably, the antigen is derived from Plasmodium falciparum ( Plasmodium falciparum ), Plasmodium vivax ( Plasmodium vivax ovale ( Plasmodium ovale ), Plasmodium malariae ( Plasmodium malariae ) or Plasmodium knowlesi ( Plasmodium knowlesi ) or more; More preferably, the antigen is from Plasmodium falciparum ( Plasmodium falciparum ).

3. The fusion protein according to claim 1 or 2, characterized in that Optionally, the fusion protein A or fusion protein B adds functional elements at the N-terminus and / or C-terminus to promote fusion protein expression or epitope presentation.

4. The fusion protein according to claim 1 or 2, characterized in that The fusion protein A comprises the actin depolymerizing factor 1 antigen, the DNAJ-like heat shock protein Pfj4 antigen, the 14-3-3 protein antigen, and the translation initiation factor SUI1 antigen in sequence from the N-terminus to the C-terminus; optionally, the antigens are connected by a linker.

5. The fusion protein according to claim 4, characterized in that For fusion protein A, the amino acid sequence of the actin depolymerizing factor 1 antigen is shown in SEQ ID NO: 1, the amino acid sequence of the DNAJ-like heat shock protein Pfj4 antigen is shown in SEQ ID NO: 2, the amino acid sequence of the 14-3-3 protein antigen is shown in SEQ ID NO: 3, the amino acid sequence of the translation initiation factor SUI1 antigen is shown in SEQ ID NO: 4, and the amino acid sequence of the linker is shown in SEQ ID NO:

12.

6. The fusion protein according to claim 1, characterized in that For fusion protein B, the TRAP antigen is the full-length sequence or the N-terminal domain of TRAP, and the CSP antigen is the full-length sequence or the C-terminal domain of CSP; preferably, the N-terminus of the fusion protein B further comprises a signal peptide and / or an Fc domain; more preferably, the N-terminus of the fusion protein B further comprises a signal peptide and an Fc domain in sequence; most preferably, the signal peptide is a signal peptide of human blue protein, and the Fc domain is a human IGHG1 protein Fc domain; optionally, the TRAP antigen and the Fc domain are connected by a spacer sequence.

7. The fusion protein according to claim 6, characterized in that For fusion protein B, the TRAP antigen is the N-terminal domain of TRAP, and the amino acid sequence of the N-terminal domain of TRAP is shown in SEQ ID NO: 5; the CSP antigen is the C-terminal domain of CSP, and the amino acid sequence of the C-terminal domain of CSP is shown in SEQ ID NO: 6; the amino acid sequence of the signal peptide is shown in SEQ ID NO: 9; the amino acid sequence of the Fc domain is shown in SEQ ID NO: 10; the amino acid sequence of the spacer sequence is shown in SEQ ID NO:

11.

8. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein A is shown in SEQ ID NO: 7, and the amino acid sequence of the fusion protein B is shown in SEQ ID NO:

8.

9. A recombinant nucleic acid molecule, characterized in that Comprising a nucleic acid encoding the fusion protein according to any one of claims 1 to 8.

10. A recombinant gene expression cassette, characterized in that: Comprising the recombinant nucleic acid molecule of claim 9.

11. A recombinant vector, characterized in that: Comprising the recombinant nucleic acid molecule according to claim 9, or the recombinant gene expression cassette according to claim 10.

12. A recombinant host cell, characterized in that Comprising the recombinant nucleic acid molecule of claim 9, or the recombinant gene expression cassette of claim 10, or the recombinant vector of claim 11.

13. An immunogenic composition or a pharmaceutical composition, characterized in that: Comprising one or more fusion proteins according to any one of claims 1 to 8, and / or one or more recombinant nucleic acid molecules according to claim 9, and / or one or more recombinant gene expression cassettes according to claim 10, and / or one or more recombinant vectors according to claim 11, and / or one or more recombinant host cells according to claim 12; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

14. The immunogenic composition or pharmaceutical composition according to claim 13, characterized in that The immunogenic composition or pharmaceutical composition comprises the fusion protein A and the fusion protein B.

15. A recombinant vaccine, characterized in that: Comprising one or more fusion proteins according to any one of claims 1 to 8, and / or one or more recombinant nucleic acid molecules according to claim 9, and / or one or more recombinant gene expression cassettes according to claim 10, and / or one or more recombinant vectors according to claim 11, and / or one or more recombinant host cells according to claim 12, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 13 or 14; preferably, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine; more preferably, the recombinant vaccine is a nucleic acid vaccine.

16. Use of one or more fusion proteins according to any one of claims 1 to 8, and / or one or more recombinant nucleic acid molecules according to claim 9, and / or one or more recombinant gene expression cassettes according to claim 10, and / or one or more recombinant vectors according to claim 11, and / or one or more recombinant host cells according to claim 12, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 13 or 14, and / or one or more recombinant vaccines according to claim 15 in the preparation of vaccines or biological immunity drugs for prevention, treatment and / or vaccination.

17. The use according to claim 16, characterized in that The medicament is used for preventing and / or treating malarial parasite infection; preferably, the malarial parasite is Plasmodium falciparum ( Plasmodium falciparum ), Plasmodium vivax ( Plasmodium Vivax ovale ( Plasmodium ovale ), Plasmodium malariae ( Plasmodium malariae ) or Plasmodium knowlesi ( Plasmodium knowlesi ) or more; More preferably, the malarial parasite is Plasmodium falciparum ( Plasmodium falciparum ).

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