A dual-antigen combined subunit vaccine against wetland virus and its application

Abstract

The present invention provides a dual-antigen combined subunit vaccine against wetland virus and its application, which belongs to the field of biomedical technology. The glycoprotein Gn of the wetland virus is a recombinant wetland virus glycoprotein Gn and a truncated glycoprotein Gn truncated at the C-terminus. Moreover, the truncated antigen glycoprotein Gn and / or Gc of the wetland virus (WELV) vaccine are provided, and the amino acid sequence of the truncated glycoprotein Gn of WELV is as shown in SEQ ID No.1. The monovalent recombinant subunit vaccine (Gn) or the dual-antigen combined subunit vaccine (Gn+Gc) prepared by the present invention has good safety, can effectively stimulate the immune response and can completely protect mice from the threat of death.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a wetland virus dual-antigen combined subunit vaccine and its application. Background Art

[0002] Tick-borne virus infections are a global health problem associated with considerable morbidity and mortality. In the past decade, several new tick-borne viruses associated with human diseases have been discovered, such as severe fever with thrombocytopenia syndrome virus, Heartland virus, etc. In the genus Orthonairovirus within the family Nairoviridae of the order Bunyavirales, the genus Orthonairovirus contains multiple viruses that can infect humans, including Crimean-Congo hemorrhagic fever virus, Sandfly fever Naples virus, etc., all of which are transmitted through tick bites. Except for the highly pathogenic Crimean-Congo hemorrhagic fever virus (CCHFV), the diseases caused by other virus infections are relatively mild; however, due to their broad host range and wide geographical distribution, these tick-borne viruses still have public health significance.

[0003] Wetland virus (WELV) is a newly discovered Orthonairovirus with ticks as the transmission vector. In June 2019, a patient developed persistent fever and multiple organ dysfunction after being bitten by a tick in a certain wetland park. High-throughput sequencing analysis of the patient's specimen showed infection with a previously unknown Orthonairovirus. The researchers named it Wetland virus (WELV) through isolation and culture, morphological and molecular biological identification. The virus isolated from the index patient and the tick showed a cytopathic effect in human umbilical vein endothelial cells. Patients infected with WELV presented with non-specific symptoms, including fever, dizziness, headache, fatigue, myalgia, arthritis, and back pain, with petechiae and local lymphadenopathy being less common. Some patients had neurological symptoms. Common abnormal laboratory test results were leukopenia, thrombocytopenia, elevated levels of D-dimer and lactate dehydrogenase. The researchers conducted serological evaluations on the convalescent samples of 8 patients found, and the results showed that the WELV-specific antibody titer was 4 times that of the acute-phase samples; WELV RNA was detected in all 5 species of ticks collected and in sheep, horses, pigs, and plateau zokors. Laboratory transmission tests indicated that Haemaphysalis concinna might be the transmission vector of WELV.

[0004] Wetland virus (WELV) particles are enveloped, spherical and oval, with a diameter ranging from 80 - 160 nm. WELV has a negative-sense single-stranded tripartite RNA genome: small (S), middle (M) and large (L), which encode nucleoprotein (NP), glycoprotein precursor (GPC) and RNA-dependent RNA polymerase (RdRP), respectively. The L, M and S segments have typical terminal inverted complementary sequences, with UCUCAAAGA at the 5' end and AGAGUUUCU at the 3' end. The M segment contains an open reading frame encoding a 1436-amino acid glycoprotein precursor, which is cleaved into two mature membrane glycoproteins, Gn and Gc. Two cleavage sites of subtilisin-kexin isozyme 1 / site 1 protease (SKI-1 / S1P) have been identified at positions RRLM 306↓ and RKLL 791↓. Generally, the replication cycle of nairoviruses starts with the binding of the virus to receptors on the cell surface, which is mediated by the membrane proteins Gn and / or Gc. However, the specific mechanisms by which membrane proteins are involved in virus adsorption, internalization and fusion remain unclear, and the cellular receptors required for nairovirus entry have not been discovered yet.

[0005] WELV belongs to the family Nairoviridae, the same as CCHFV. The case fatality rate of CCHFV infection is as high as 30% - 50%, but it varies slightly in different regions. Humans are generally susceptible to CCHFV, and there are no obvious age and gender differences in susceptibility. Currently, there are no effective preventive and therapeutic measures for CCHFV infection, which seriously threatens people's physical health. The World Health Organization (WHO) has listed CCHFV in the research and development blueprint list of infectious pathogens. Viral surface glycoproteins can specifically recognize receptors on the surface of host cells and initiate the invasion process of the virus into host cells by binding to specific receptors. Viral surface glycoproteins are usually one of the main targets recognized by the immune system and can stimulate the host's immune response, including the production of antibodies and the activation of cellular immunity. These antibodies can neutralize the virus and prevent the virus from infecting host cells, while cellular immunity can directly attack virus-infected cells. Viral surface glycoproteins are important targets for vaccine design. By inducing the body to produce antibodies against glycoproteins, virus infection can be effectively prevented. In recent years, some studies have explored different types of CCHFV candidate vaccines, such as DNA candidate vaccines, recombinant attenuated live vesicular stomatitis virus vaccines, recombinant human adenovirus type 5 CCHFV candidate vaccines, etc. However, so far, no CCHF vaccine that combines high efficiency, safety and low cost has been developed, and no vaccine has been approved for marketing.

[0006] Existing studies have shown that although single Gn or Gc subunit vaccines can induce some neutralizing antibodies, their protective efficacy is limited (animal experiments showed a survival rate < 50%), and they cannot effectively stimulate the Th1 / Th2 balanced immune response.

[0007] Currently, the development of Wetland virus (WELV) vaccines faces the following key problems:

[0008] (1) Antigen design defects: The full-length Gn / Gc proteins have problems such as complex post-translational modifications and low expression levels, which restrict large-scale production;

[0009] (2) Insufficient immune synergy: Single antigens are difficult to cover the entire process of virus invasion, and lack a dual protection mechanism to block "receptor binding - membrane fusion";

[0010] (3) Safety hazards: Traditional vaccine adjuvants (such as aluminum adjuvants) are prone to cause local inflammatory reactions, and have limited effects on activating cellular immunity.

[0011] In view of the above problems, there is an urgent need to develop a dual-antigen combined subunit vaccine based on Wetland virus Gn and Gc, which can achieve efficient and safe synergistic immune protection by optimizing the antigen structure and formulation strategy. Summary of the Invention

[0012] Based on the problems existing in the prior art, the present invention provides a dual-antigen combined subunit vaccine of Wetland virus and its application, and further provides the Wetland virus glycoprotein Gn and its related biological materials, as well as the vaccine of Wetland virus (WELV) glycoprotein Gn and its application. Specifically, it is the WELV Gn protein, the immune composition containing this recombinant Gn protein, and their applications.

[0013] According to the first aspect of the technical solution of the present invention, there is provided a Wetland virus glycoprotein Gn, and the Wetland virus glycoprotein Gn is a recombinant Wetland virus glycoprotein Gn, and the Wetland virus glycoprotein Gn is a truncated body of the Wetland virus glycoprotein Gn with the C-terminal truncated.

[0014] Furthermore, the truncated body of the Wetland virus glycoprotein Gn is any one of the following proteins:

[0015] A1) A protein comprising the amino acid sequence shown in positions 1-153 of SEQ ID No.1;

[0016] A2) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence defined in A1 and having the same function;

[0017] A3) A protein having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the amino acid sequence defined in A1 or A2 and having the same function;

[0018] A4) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein defined in any one of A1 - A3.

[0019] According to the second aspect of the technical solution of the present invention, there is provided a biological material, which is any one of the following:

[0020] C1) A nucleic acid molecule encoding the glycoprotein Gn of the wetland virus;

[0021] C2) An expression cassette containing the nucleic acid molecule described in C1;

[0022] C3) A recombinant vector containing the nucleic acid molecule described in C2, or a recombinant vector containing the expression cassette described in C2;

[0023] C4) A recombinant microorganism containing the nucleic acid molecule described in C1, or a recombinant microorganism containing the expression cassette described in C2, or a recombinant microorganism containing the recombinant vector described in C3;

[0024] C5) A recombinant cell containing the nucleic acid molecule described in C1, or a recombinant cell containing the expression cassette described in C2, or a recombinant cell containing the recombinant vector described in C3.

[0025] According to the third aspect of the technical solution of the present invention, there is provided a product containing the above-mentioned glycoprotein Gn of the wetland virus. The product is a wetland virus dual-antigen combined subunit vaccine, and the wetland virus dual-antigen in the wetland virus dual-antigen combined subunit vaccine comprises the glycoprotein Gn and Gc of the wetland virus. The wetland virus dual-antigen combined subunit vaccine further comprises an adjuvant; preferably, the adjuvant is an aluminum adjuvant or a CpG adjuvant, and the aluminum adjuvant includes an aluminum hydroxide adjuvant.

[0026] Furthermore, the CpG adjuvant is any one of the following:

[0027] (1) CpG2006 with a nucleotide sequence of SEQ ID No.9 or its thiolated product;

[0028] (2) CpG1018 with a nucleotide sequence of SEQ ID No.10 or its thiolated product;

[0029] (3) CpG684 with a nucleotide sequence of SEQ ID No.11 or its thiolated product;

[0030] The thiolated product is a full-chain thiolated modification product.

[0031] Furthermore, the wetland virus dual-antigen combined subunit vaccine is composed of the truncated form of the wetland virus glycoprotein Gn, the aluminum adjuvant, and the CpG adjuvant; wherein, the mass ratio of the truncated form of the wetland virus glycoprotein Gn, the aluminum adjuvant, and the CpG adjuvant is 10(2.5) : 100 : 50; the amount of the aluminum adjuvant is calculated based on the content of aluminum therein.

[0032] According to the fourth aspect of the technical solution of the present invention, there is provided a method for preparing the wetland virus glycoprotein Gn as described above, and the preparation method includes the following steps:

[0033] Step S1, expressing the truncated form of the wetland virus glycoprotein Gn in yeast;

[0034] Step S2, expressing the truncated form of the wetland virus glycoprotein Gn in the Expi-293F mammalian cell expression system; expressing the truncated form of the wetland virus glycoprotein Gn in the Expi-293F mammalian cell expression system is achieved by introducing a nucleic acid molecule encoding the truncated form of the wetland virus glycoprotein Gn into the Expi-293F mammalian cell expression system;

[0035] Step S3, performing suspension culture on the Expi-293F mammalian cells, and purifying the truncated form of the wetland virus glycoprotein Gn from the supernatant of the suspension cell culture; the purification includes the step of performing nickel ion affinity chromatography on the supernatant of the suspension cell culture.

[0036] According to the fifth aspect of the technical solution of the present invention, there is provided an application of the wetland virus glycoprotein Gn as described above or the above-mentioned biological material in the preparation of a product that can cause an animal to produce antigen-specific antibodies, and the product is a dual-antigen combined subunit vaccine, and the wetland virus dual antigen includes the wetland virus glycoprotein Gn and Gc.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] 1. The present invention discloses a method for constructing an expression vector of a truncated form of the wetland virus glycoprotein Gn (Gn subunit truncated form), and the constructed expression vector can achieve the expression of the truncated form of the wetland virus glycoprotein Gn (Gn subunit truncated form) in mammalian cells, and has the characteristics of fast growth and high safety.

[0039] 2. The truncated wetland virus glycoprotein Gn (Gn subunit truncation) obtained by recombinant expression of the present invention does not have the problem of over-glycosylation. Immunizing mice with it alone or in combination with wetland virus glycoprotein Gc can produce high-titer specific IgG antibodies against the truncated wetland virus glycoprotein Gn (Gn subunit truncation). After booster immunization, it can protect mice from lethal attacks by WELV. Therefore, the recombinant subunit candidate vaccine of WELV Gn subunit truncation prepared using mammalian cells can provide a good protective response and provide a reference for the development of WELV subunit vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figures 1A - 1B It is a schematic diagram of the structure of the recombinant truncated wetland virus glycoprotein subunit. Figure 1A It is a schematic diagram of the structure of the truncated WELV Gn glycoprotein subunit and a protein structure prediction diagram (the blue part represents the subunit used for protein expression, and the gray part represents the truncated subunit); Figure 1B It is a schematic diagram of the structure of the truncated WELV Gc glycoprotein subunit and a protein structure prediction diagram;

[0041] Figures 2A - 2B It is the identification result of the protein expression of the recombinant truncated wetland virus glycoproteins Gn and Gc subunits changing with time in the Expi 293F expression system; Figure 2A It is the curve of the cell viability changing with time after transfection of wetland virus glycoprotein Gn and the detection result of the Anti-His tag monoclonal antibody (Sigma, A7058) of the Gn protein expression changing with time; Figure 2B It is the curve of the cell viability changing with time after transfection of wetland virus glycoprotein Gc and the detection result of the Anti-His tag monoclonal antibody (Sigma, A7058) of the Gn protein expression changing with time.

[0042] Figures 3A - 3C They are the nickel affinity chromatography, SDS-PAGE and WB identifications of the recombinant truncated wetland virus glycoprotein Gn subunit samples, where Figure 3A represents the nickel ion affinity chromatography chromatogram of the recombinant truncated wetland virus glycoprotein Gn subunit, Figure 3B represents the SDS-PAGE schematic diagram of the recombinant truncated wetland virus glycoprotein Gn subunit, Figure 3C represents the WB schematic diagram of the recombinant truncated wetland virus glycoprotein Gn subunit.

[0043] Figures 4A - 4C They are the nickel affinity chromatography, SDS-PAGE and WB identifications of the recombinant truncated wetland virus glycoprotein Gc subunit samples. Figure 4A It is the nickel ion affinity chromatography chromatogram; Figure 4B It is the SDS-PAGE of the samples during the purification process;Figure 4C For WB identification of samples in the purification process.

[0044] Figures 5A - 5B For SDS-PAGE of the purified sample of recombinant wetland virus glycoprotein. Figure 5A For SDS-PAGE after purification of the truncated sample of the Gn subunit of wetland virus glycoprotein; Figure 5B For SDS-PAGE after purification of the truncated sample of the Gc subunit of wetland virus glycoprotein.

[0045] Figures 6A - 6B For detection of antibody titers in mice immunized intramuscularly with the truncated protein of recombinant wetland virus glycoprotein GnGc subunit. Figure 6A For the titers of specific binding antibodies against Gn in the sera of mice after primary immunization and booster immunization; Figure 6B For the titers of specific binding antibodies against Gc in the sera of mice after primary immunization and booster immunization.

[0046] Figure 7 For the survival curve of the evaluation of the protective effect of the truncated protein of recombinant wetland virus glycoprotein GnGc subunit immunized intramuscularly in mice in the WELV lethal model.

[0047] Figure 8 For the weight change of mice immunized intramuscularly with the truncated protein of recombinant wetland virus glycoprotein GnGc subunit after challenge with the lethal dose of WELV.

[0048] In each quantitative result graph, ns indicates P >0.05, * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, **** indicates P <0.0001. Specific implementation manners

[0049] The present invention will be further described in detail below in conjunction with specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0050] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0051] The wetland virus (WELV) strain can be obtained from the Academy of Military Medical Sciences.

[0052] The pCAGGS mammalian expression vector was purchased from AUGCT Biotechnology Co., Ltd., with the catalog number HG-VPA0057.

[0053] The pAB plasmid was stored in our laboratory.

[0054] The Expi 293F mammalian expression system was purchased from Thermo Fisher Scientific Inc., with the catalog number A14635CN.

[0055] The Anti-His tag mouse monoclonal antibody was purchased from Sigma, with the catalog number A7058.

[0056] The Chelating Sepharose FF chromatography medium was from Cytiva, with the product catalog number 17057502.

[0057] BALB / c mice (strain code: 211) were purchased from Vital River Laboratory Animal Technology Co., Ltd., Beijing.

[0058] In the following examples, the virus is specifically the Wetland virus (WELV), which is recorded in the non-patent literature "NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov / ): A New Orthonairovirus Associated with Human Febrile Illness". The public can obtain this biological material from the applicant in accordance with the relevant regulations on biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0059] The present invention relates to a dual-antigen combined subunit vaccine against Wetland virus (WELV) and its application, belonging to the field of biomedical technology. The antigens of the WELV vaccine provided by the present invention are the truncated form of the Wetland virus glycoprotein Gn and / or Gc. The amino acid sequence of the truncated form of the WELV glycoprotein Gn is shown in SEQ ID No.1. After immunizing mice with the vaccine of the present invention, high-titer antibodies against the truncated form of the Wetland virus glycoprotein Gn can be produced, and the live WELV can be neutralized. After booster immunization of mice with the monovalent recombinant subunit vaccine (Gn) or the dual-antigen combined subunit vaccine (Gn+Gc) prepared by the present invention, 67% of the mice can be protected from death under the fully lethal dose of WELV; the recombinant Wetland virus glycoprotein Gn truncated form subunit candidate vaccine prepared by the present invention using a mammalian expression system has good safety, can effectively stimulate the immune response, and can completely protect mice from death threat. The present invention can provide a reference for the development of WELV subunit vaccines.

[0060] In one embodiment of the present invention, the present invention claims to protect a wetland virus (WELV) glycoprotein Gn, preferably a recombinant glycoprotein Gn of WELV. More preferably, the antigen in the double-antigen combined subunit vaccine of WELV claimed by the present invention is a truncated form of the glycoprotein Gn of WELV. The recombinant glycoprotein Gn of WELV comprises a polypeptide having the amino acid sequence shown in SEQ ID NO.1. The truncated form of the glycoprotein Gn of WELV is any of the following proteins:

[0061] A1) a protein having an amino acid sequence as shown in SEQ ID No.1 or positions 1-153 of SEQ ID No.1;

[0062] A2) a protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence defined in A1 and having the same function;

[0063] A3) a protein having an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the amino acid sequence defined in A1 or A2 and having the same function;

[0064] A4) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in any of A1-A3.

[0065] Among the above proteins, the tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using in vitro DNA recombination technology for the purpose of facilitating the expression, detection, tracing or purification of the target protein. The tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag or SUMO tag, etc.

[0066] Among the above proteins, the identity of the amino acid sequence can be determined using an identity search site on the Internet, such as the BLAST web page of the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gapexistence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0067] Among the above proteins, the identity of more than 95% may be at least 96%, 97%, 98% identity. The identity of more than 90% may be at least 91%, 92%, 93%, 94% identity. The identity of more than 85% may be at least 86%, 87%, 88%, 89% identity. The identity of more than 80% may be at least 81%, 82%, 83%, 84% identity.

[0068] In another embodiment, a vaccine containing the recombinant WELV glycoprotein Gn, namely the wetland virus dual-antigen combined subunit vaccine, is provided. The wetland virus dual-antigen combined subunit vaccine further includes an adjuvant. Among them, the adjuvant may be an aluminum adjuvant or a CpG adjuvant. The aluminum adjuvant may be an aluminum hydroxide adjuvant. The CpG adjuvant may be any one of the following:

[0069] (1) CpG2006 with a nucleotide sequence of SEQ ID No.9 or its thiolated product;

[0070] (2) CpG1018 with a nucleotide sequence of SEQ ID No.10 or its thiolated product;

[0071] (3) CpG684 with a nucleotide sequence of SEQ ID No.11 or its thiolated product;

[0072] Among them, the thiolated product may be a full-chain thiolated modified product.

[0073] In a specific embodiment of the present invention, the wetland virus dual-antigen combined subunit vaccine is composed of the truncated form of the wetland virus glycoprotein Gn, the aluminum adjuvant, and the CpG adjuvant; among them, the mass ratio of the truncated form of the wetland virus glycoprotein Gn, the aluminum adjuvant, and the CpG adjuvant is (2.5 - 20) : 100 : (25 - 50). The amount of the aluminum adjuvant is calculated based on the content of aluminum therein.

[0074] In another specific embodiment of the present invention, the wetland virus dual-antigen combined subunit vaccine is composed of the truncated form of the wetland virus glycoprotein Gn and the aluminum adjuvant; among them, the mass ratio of the truncated form of the wetland virus glycoprotein Gn and the aluminum adjuvant is (2.5 - 20) : 100.

[0075] Furthermore, the mass ratio of the truncated form of the wetland virus glycoprotein Gn and the aluminum adjuvant is 10 : 100. The amount of the aluminum adjuvant is calculated based on the content of aluminum therein.

[0076] In another embodiment, a preparation method of the recombinant truncated wetland virus glycoprotein Gn as described above is provided. The preparation method includes the following steps:

[0077] Step S1: Express the truncated wetland virus glycoprotein Gn in Pichia pastoris.

[0078] Step S2: Express the truncated wetland virus glycoprotein Gn in the Expi293F mammalian cell expression system to obtain an expression supernatant.

[0079] Furthermore, the expression of the truncated wetland virus glycoprotein Gn in the Expi293F mammalian cell expression system is achieved by introducing a nucleic acid molecule encoding the truncated wetland virus glycoprotein Gn into the Expi293F mammalian cell expression system. Among them, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0080] Step S3: Suspension culture the transfected Expi293F cells, and purify the truncated wetland virus glycoprotein Gn from the supernatant of the suspension cell culture. Furthermore, the expression of the truncated wetland virus glycoprotein Gn is carried out during the process of the transfection culture. In a specific embodiment of the present invention, it is cultured in a shaker at 37 °C and 125 rpm, and samples are collected after 48 hours. Furthermore, the purification includes subjecting the transfection culture supernatant to nickel ion affinity chromatography (Chelating FF column).

[0081] Even further, the nucleic acid molecule encoding the truncated wetland virus glycoprotein Gn can be any one of the following:

[0082] (a1) A DNA molecule with a nucleotide sequence as shown in positions 1-459 of SEQ ID No. 2;

[0083] (a2) A DNA molecule that hybridizes with the DNA molecule defined in a1 under stringent conditions and encodes the truncated wetland virus glycoprotein Gn;

[0084] (a3) A DNA molecule that has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the DNA sequence defined in a1 or a2 and encodes the truncated glycoprotein Gn of the WELV.

[0085] Among the above nucleic acid molecules, the stringent conditions may be as follows: Hybridization is carried out at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4 and 1 mM EDTA, and rinsing is carried out at 50°C in 2×SSC, 0.1% SDS; alternatively: Hybridization is carried out at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing is carried out at 50°C in 1×SSC, 0.1% SDS; alternatively: Hybridization is carried out at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing is carried out at 50°C in 0.5×SSC, 0.1% SDS; alternatively: Hybridization is carried out at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing is carried out at 50°C in 0.1×SSC, 0.1% SDS; alternatively: Hybridization is carried out at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4 and 1 mM EDTA, and rinsing is carried out at 65°C in 0.1×SSC, 0.1% SDS; alternatively: Hybridization is carried out at 65°C in a solution of 6×SSC, 0.5% SDS, and then the membrane is washed once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0086] In another embodiment, there is provided an application of the recombinant wetland virus glycoprotein Gn truncation as described above in the preparation of a product capable of inducing an antigen-specific antibody in an animal, and the recombinant WELV glycoprotein Gn is immunized by intramuscular injection or subcutaneous injection.

[0087] In another embodiment, there is provided an immunization vaccine, which comprises the recombinant wetland virus glycoprotein Gn truncation as described above, or the composition containing the recombinant wetland virus glycoprotein Gn truncation as described above. Preferably, the vaccine is a dual-antigen combined subunit vaccine, and the dual antigens comprise wetland virus glycoproteins Gn and Gc.

[0088] In another embodiment, there is provided a pharmaceutical composition, which comprises the wetland virus glycoprotein Gn truncation as described above, or the composition containing the wetland virus glycoprotein Gn truncation as described above.

[0089] In another embodiment, the present invention claims any of the following applications:

[0090] (D1) The application of the protein or biological material in the preparation of a wetland virus dual-antigen combined subunit vaccine;

[0091] (D2) The application of the wetland virus dual-antigen combined subunit vaccine or the protein or biological material in the preparation of a product for preventing and / or treating Congo hemorrhagic fever / WELV;

[0092] Use of the dual-antigen combined subunit vaccine or protein or biological material of wetland virus in the preparation of a product for relieving hemorrhagic fever / WELV symptoms;

[0093] Use of the dual-antigen combined subunit vaccine or protein or biological material of wetland virus in the preparation of a product for anti-WELV infection;

[0094] Use of the dual-antigen combined subunit vaccine or protein or biological material of wetland virus in the preparation of a product for neutralizing WELV.

[0095] The gene sequence is as follows.

[0096] SEQ ID No.1: Amino acid sequence of Gn truncation (153AA):

[0097] SSESGSNPCSSGTFLGEGSSAQVVGNKNDGPGEHITFCNGTYVSKIKLGKQHGCFTVRRVKAYRNCHPKEASTSCVVDEELRECEGQRCMNIHLDVRGLVKITRGKYVEVITCDKDCLAKIPSGRGDIQIDCPGGKQHYLETNVVDINCPGPE.

[0098] SEQ ID No.2: Coding gene sequence corresponding to the Gn truncation (459bp):

[0099] TCATCTGAGTCTGGTTCTAATCCTTGTAGTTCTGGAACCTTTTTGGGTGAAGGTTCTTCCGCTCAAGTTGTTGGAAATAAGAATGATGGACCTGGTGAACACATTACTTTTTGTAATGGAACTTATGTTAGTAAGATAAAGTTGGGTAAACAACATGGTTGTTTCACCGTTAGAAGAGTAAAAGCATATAGAAATTGTCATCCAAAGGAAGCTAGTACTAGTTGTGTAGTTGATGAAGAACTTAGAGAATGTGAAGGTCAAAGATGTATGAACATTCACTTAGATGTTAGAGGATTAGTTAAAATTACTAGAGGTAAATATGTTGAAGTTATTACTTGTGATAAGGATTGTTTGGCTAAGATTCCAAGTGGTAGAGGTGACATCCAAATTGATTGTCCAGGTGGTAAACAACACTACTTAGAAACTAACGTTGTTGATATTAACTGTCCAGGTCCAGAA。

[0100] SEQ ID No.3: Amino acid sequence of the Gn truncation + HIS tag (1 - 164 AA):

[0101] SSESGSNPCSSGTFLGEGSSAQVVGNKNDGPGEHITFCNGTYVSKIKLGKQHGCFTVRRVKAYRNCHPKEASTSCVVDEELRECEGQRCMNIHLDVRGLVKITRGKYVEVITCDKDCLAKIPSGRGDIQIDCPGGKQHYLETNVVDINCPGPEGGGGSHHHHHH。

[0102] SEQ ID No.4: Coding gene sequence corresponding to the Gn truncation + HIS tag (492bp):

[0103] TCATCTGAGTCTGGTTCTAATCCTTGTAGTTCTGGAACCTTTTTGGGTGAAGGTTCTTCCGCTCAAGTTGTTGGAAATAAGAATGATGGACCTGGTGAACACATTACTTTTTGTAATGGAACTTATGTTAGTAAGATAAAGTTGGGTAAACAACATGGTTGTTTCACCGTTAGAAGAGTAAAAGCATATAGAAATTGTCATCCAAAGGAAGCTAGTACTAGTTGTGTAGTTGATGAAGAACTTAGAGAATGTGAAGGTCAAAGATGTATGAACATTCACTTAGATGTTAGAGGATTAGTTAAAATTACTAGAGGTAAATATGTTGAAGTTATTACTTGTGATAAGGATTGTTTGGCTAAGATTCCAAGTGGTAGAGGTGACATCCAAATTGATTGTCCAGGTGGTAAACAACACTACTTAGAAACTAACGTTGTTGATATTAACTGTCCAGGTCCAGAA GGAGGTGGAGGTTCCCATCATCATCATCATCAT。

[0104] SEQ ID No.5: Amino acid sequence of the Gc truncation (525 AA):

[0105] FFKGLNSAASRMLNAHKLMTSVSIDAPWGAIQVESTYKPKLPVSNIELAWNSIEEQGDKIILSGKSTSILKLEERTGVQWSLGAESASEEKRLLVSVLDYTQVYSSTFQYITGDRTISEWPKATCTGDCPDRCACRTSTCLFKSWPHSRNWRCNPTWCWGVGTGCTCCGVDIERPFNKYFAVKWSTEYVRTDALVCVELTDLERHCDVVEAGSQFVIGPVRVVVSDPQNVQSKLPQEVLTVQKLDKHQHLDLMHVTNIISAKNACKLQSCTHGSPGDMQILHTDNLILNSHDDGANLAESIPEVNTTWMSWEGCDLDYYCTTGSWPSCTFTGVNTENTESFENLLNTEANLIDRYHFHSKRIYAQGATLQMDLKGRPKSGGGELTVLVDIKGLELHSKRVVLKGLEIKALSCTGCYSCSSGMSCTVDVRIEKPDEFTVHLRSTDPNTAISEGSIMARKLSGGPQSKVRAFTALKVSEVCVEIVEKNYCPTCKETDTKKCAKVDLQPPKDILLEHKGTLIKSQNDS。

[0106] SEQ ID No. 6: Encoding gene sequence corresponding to the Gc truncation (1575 bp):

[0107]

[0108] SEQ ID No.7: Amino acid sequence of Gc truncation + HIS tag (1 - 536 AA):

[0109] FFKGLNSAASRMLNAHKLMTSVSIDAPWGAIQVESTYKPKLPVSNIELAWNSIEEQGDKIILSGKSTSILKLEERTGVQWSLGAESASEEKRLLVSVLDYTQVYSSTFQYITGDRTISEWPKATCTGDCPDRCACRTSTCLFKSWPHSRNWRCNPTWCWGVGTGCTCCGVDIERPFNKYFAVKWSTEYVRTDALVCVELTDLERHCDVVEAGSQFVIGPVRVVVSDPQNVQSKLPQEVLTVQKLDKHQHLDLMHVTNIISAKNACKLQSCTHGSPGDMQILHTDNLILNSHDDGANLAESIPEVNTTWMSWEGCDLDYYCTTGSWPSCTFTGVNTENTESFENLLNTEANLIDRYHFHSKRIYAQGATLQMDLKGRPKSGGGELTVLVDIKGLELHSKRVVLKGLEIKALSCTGCYSCSSGMSCTVDVRIEKPDEFTVHLRSTDPNTAISEGSIMARKLSGGPQSKVRAFTALKVSEVCVEIVEKNYCPTCKETDTKKCAKVDLQPPKDILLEHKGTLIKSQNDSGGGGSHHHHHH。

[0110] SEQ ID No.8: Coding gene sequence corresponding to Gc truncation + HIS tag (1608bp):

[0111]

[0112] The following are the CpG sequences of SEQ ID No. 9-11:

[0113] SEQ ID No. 9: tcgtcgtttt gtcgttttgt cgtt;

[0114] SEQ ID No.10: tgactgtgaa cgttcgagat ga;

[0115] SEQ ID No.11: tcgacgttcg tcgttcgtcg ttc;

[0116] In the following examples, unless otherwise specified, gene synthesis, nucleotide synthesis, primer synthesis, sequencing, etc. were carried out by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. and Beijing Tsingke Biotechnology Co., Ltd.

[0117] In the following examples, unless otherwise specified, the first nucleotide of each nucleotide sequence is the 5'-terminal nucleotide of the corresponding DNA, and the last nucleotide is the 3'-terminal nucleotide of the corresponding DNA.

[0118] Example 1. Construction of mammalian expression vectors for truncated forms of WELV Gn and Gc subunits.

[0119] (1) Obtaining of truncated genes of wetland virus glycoprotein Gn and Gc subunits and construction of mammalian expression vectors.

[0120] According to the sequence of WELV published in GenBank: OR860396-OR860409, the inventors artificially designed the codon DNA sequence for amino acids 1 to 153 (S1-E153) of the Gn protein, and added a GGGGS linker peptide and 6 HIS tags, which is a DNA fragment with the sequence shown in SEQ ID No. 4. Beijing Tsingke Biotechnology Co., Ltd. was commissioned to synthesize the relevant DNA fragment (from the 5' end to the 3' end, it is the AflII restriction site, GGCGTGCAGTGC, the codon DNA sequence of the fragment shown in SEQ ID No. 4, and the BamHI restriction site in turn), and the synthesized fragment was inserted between the AflII and BamHI restriction sites of the pAB vector to obtain the recombinant expression vector pAB-Gn subunit truncation, that is, the Gn subunit truncation expression vector.

[0121] The structure of the recombinant expression vector pAB-Gn subunit truncated form is described as: a recombinant plasmid in which the DNA fragment between the AflII and BamHI recognition sequences of the pAB vector is replaced with the DNA fragment shown in SEQ ID No. 4. SEQ ID No. 4 is a coding gene sequence obtained by artificial codon optimization based on the Gn subunit truncated form (S1-E153), encoding the Gn subunit truncated form protein carrying 6 His tags and a GGGGS linker peptide shown in SEQ ID No. 1. The 1st to 153rd positions of SEQ ID No. 3 are the Gc subunit truncated form protein. Figure 1A The figure is a schematic diagram of the structure of the Gn subunit truncated form (Gn subunit truncated form) of WELV and the three-dimensional structure prediction of the Gn subunit truncated form of WELV (the blue part represents the part involved in the present invention, and the gray part represents the truncated part).

[0122] According to the sequence of WELV published in GenBank: OR860396-OR860409, the 1st to 525th amino acids (F1-S525) of the Gc protein were selected, and the inventor artificially designed the codon DNA sequence and added a GGGGS linker peptide and 6 HIS tags, which is a DNA fragment with the sequence shown in SEQ ID No. 8. Beijing Tsingke Biotechnology Co., Ltd. was entrusted to synthesize the relevant DNA fragment (from the 5' end to the 3' end are the EcoRI cleavage site, BM40 signal peptide, the codon DNA sequence of the fragment shown in SEQ ID No. 8, and the NotI cleavage site), and the synthesized fragment was inserted between the EcoRI and NotI cleavage sites of the pCAGGS vector to obtain the recombinant expression vector pCAGGS-Gc subunit truncated form, that is, the Gc subunit truncated form expression vector.

[0123] The structure of the recombinant expression vector pCAGGS-Gc subunit truncated form is described as: a recombinant plasmid in which the DNA fragment between the EcoRI and NotI recognition sequences of the pCAGGS vector is replaced with the DNA fragment shown in SEQ ID No. 8. SEQ ID No. 8 is a coding gene sequence obtained by artificial codon optimization based on the Gc subunit truncated form (F1-S525), encoding the Gc subunit truncated form protein carrying 6 His tags and a GGGGS linker peptide shown in SEQ ID No. 5. The 1st to 536th positions of SEQ ID No. 8 are the Gc subunit truncated form protein. Figure 1B The figure is a schematic diagram of the structure of the Gc subunit truncated form (Gc subunit truncated form) of WELV and the three-dimensional structure prediction of the Gc subunit truncated form of WELV (the blue part represents the part involved in the present invention, and the gray part represents the truncated part).

[0124] (2) Extraction of recombinant expression vectors pAB-Gn and truncated plasmid of pCAGGS-Gc subunit (TIANGEN Endotoxin-Free Plasmid Mini Kit, Beijing, DP118-02).

[0125] Example 2: Expression and purification of recombinant truncated glycoproteins of Gn and Gc subunits.

[0126] (2.1) Transfection of Expi293F suspension cells.

[0127] Pick the plasmids identified in Example 1 (i.e., pAB-Gn and truncated plasmid of pCAGGS-Gc subunit) and transfect them into Expi293F suspension cells with a cell density of 3×10^6 / mL. The transfection system is shown in Table 1 below (taking a transfection volume of 100 mL as an example, and the other volumes are scaled up proportionally).

[0128] Table 1 Transfection system

[0129]

[0130] After transfection, culture at 37°C, 8% CO2, and 125 rpm. Monitor the cell viability every 12 h and sample 200 μL for monitoring the protein expression process. Stop the protein expression when the cell viability drops below 60%.

[0131] The results are as Figures 2A - 2B shown, Figure 2A The left figure in... shows the change of cell viability over time after transfection with pAB-Gn plasmid; Figure 2A The right figure in... shows the WB detection of the change of target protein expression over time. Figure 2B The left figure in... shows the change of cell viability over time after transfection with pCAGGS-Gc plasmid; Figure 2B The right figure in... shows the WB detection of the change of target protein expression over time. The results show that the samples are collected 72 h after transfection with the truncated plasmid of Gn subunit and 60 h after transfection with the truncated plasmid of Gc subunit for the next experiment.

[0132] (2.2) Purification of truncated Gn and Gc subunits (the steps are the same).

[0133] 1. Nickel ion affinity chromatography.

[0134] Pretreatment of the sample before purification:

[0135] Dialyze the culture supernatant induced in Step 1 overnight to replace the system. The replacement system is 20 mM pH 7.5 Tris-HCl + 500 mM NaCl.

[0136] Nickel affinity chromatography:

[0137] A: 20 mM pH 7.5 Tris-HCl + 500 mM NaCl + 5 mM imidazole;

[0138] B: 20 mM pH 7.5 Tris-HCl + 500 mM NaCl + 500 mM imidazole.

[0139] After sample loading, equilibrate with A, and then elute with B at 5%-10%-25%-50%-100% respectively.

[0140] The purified sample is as Figures 3A - 3C shown. Figure 3A It represents the nickel ion affinity chromatography of the truncated recombinant wetland virus glycoprotein Gn subunit, Figure 3B It represents the SDS-PAGE schematic diagram of the truncated recombinant wetland virus glycoprotein Gn subunit, Figure 3C It represents the WB schematic diagram of the truncated recombinant wetland virus glycoprotein Gn subunit; Figure 4 represents the nickel ion affinity chromatography, SDS-PAGE, and WB of the truncated Gc subunit. The results show that the truncated target protein Gn subunit was eluted at imidazole concentrations of 125 and 250 mM, and the truncated target protein Gc subunit was eluted at imidazole concentrations of 125 and 250 mM with good separation from the impurity proteins. It can be used for the next experiment after concentration.

[0141] Figure 5A It is the SDS-PAGE of the pure product of the purified Gn protein; Figure 5B It is the SDS-PAGE of the pure product of the purified Gc protein.

[0142] Example 3: Mouse immunization experiment.

[0143] The immunization method has been published in many literatures, such as "Replication of Animal Models of Human Diseases, edited by Li Cai, published by People's Medical Publishing House". Specifically as follows: Take 40 female BALB / C mice aged 6-8 weeks, and randomly divide them into the following 4 groups:

[0144] Immunization group 1 (n = 6): Intramuscularly inject 100 μl of the vaccine on days 0 and 21 respectively. The vaccine used is 10 μg truncated Gn subunit / 10 μg truncated Gc subunit / 100 μg Al(OH)3 (CRODA, Denmark, the same below) / 50 μg CpG2006. Among them, the truncated Gn and Gc subunits are the recombinant wetland virus glycoprotein Gn and Gc subunits expressed by Expi293F prepared above. The vaccine is formulated with physiological saline according to 100 μl volume containing 10 μg Gn and 10 μg Gc truncated subunits, 100 μg Al(OH)3 and 50 μg CpG 2006.

[0145] Immune group 2 (n = 6): 100 μl of the vaccine was intramuscularly injected on days 0 and 21 respectively. The vaccine used was 2.5 μg truncated Gn subunit / 2.5 μg truncated Gc subunit / 100 μg Al(OH)3 / 50 μg CpG2006. Among them, the truncated Gn and Gc subunits are the recombinant wetland virus glycoprotein Gn and truncated Gc subunits expressed by Expi293F prepared above. The vaccine was formulated with physiological saline according to 100 μl volume containing 2.5 μg Gn and 2.5 μg truncated Gc subunits, 100 μg Al(OH)3 and 50 μg CpG2006.

[0146] Immune group 3 (n = 6), namely the adjuvant control group: 100 μl of the vaccine was intramuscularly injected on days 0 and 21 respectively. The adjuvant used was 100 μg Al(OH)3 + 50 μg CpG2006. The vaccine was formulated with physiological saline according to 100 μl volume containing 100 μg Al(OH)3 and 50 μg CpG2006.

[0147] Immune group 4 (n = 6), namely the blank control group: 100 μl of physiological saline was intramuscularly injected on days 0 and 21 respectively.

[0148] The nucleotide sequence of the above CpG2006 is SEQ ID No.9 and is fully phosphorothioated. As needed, CpG2006 can also be replaced with any of the following: CpG1018 with the nucleotide sequence of SEQ ID No.10 or its phosphorothioated product; CpG684 with the nucleotide sequence of SEQ ID No.11 or its phosphorothioated product (such as full-chain phosphorothioation).

[0149] Blood was collected from the infraorbital venous plexus of immunized mice in each group before immunization, two weeks after the first immunization, and two weeks after the second immunization.

[0150] The specific antibody titers against the truncated Gn and Gc subunits in the sera of mice in each group were measured by indirect ELISA. The operation steps refer to the Compendium of Molecular Biology Experimental Guide [M]. Science Press, 2008.

[0151] The results are as Figures 6A - 6B shown: Figure 6A showed the specific binding antibody titers against Gn in the sera of mice after primary immunization and booster immunization; the antibody titers in the immunized groups after primary immunization and booster immunization were extremely significantly higher than those in the adjuvant control group ( P <0.0001). There were extremely significant differences in the specific antibody titers in the bodies of mice in the 10 μg GnGc and 2.5 μg GnGc antigen dose groups after primary immunization and booster immunization ( P <0.0001).

[0152] Figure 6B The specific binding antibody titers against Gc in the mouse serum after the primary immunization and booster immunization are shown. The antibody titers of the immunization group after the primary immunization and booster immunization were significantly higher than those of the adjuvant control group ( P <0.0001). There was a significant difference in the specific antibody titer in mice after the first immunization and booster immunization with a dose of 10 μg GnGc antigen ( P <0.05), and there was a significant difference in the specific antibody titer in mice after the first immunization and booster immunization with a dose of 2.5 μg GnGc antigen ( P <0.001). At the same time, after booster immunization, the specific antibody titers of all antigen immune groups were greater than 10^5. The subunit vaccine has effectively stimulated the immune response in mice, and the next step is to conduct a mouse challenge test.

[0153] Example 4, real virus challenge test.

[0154] 1) Vero cells in DMEM medium containing 10% FBS were mixed and counted and then plated into 24-well plates, with 1×10 cells per well. 5 Cells were inoculated with virus after 12 hours of cell attachment;

[0155] 2) DMEM medium was used to dilute the mouse serum to be tested (the serum obtained three weeks after the three immunizations in Example 3) and WELV virus (described in the article “A New Orthonairovirus Associated with Human Febrile Illness. N Engl J Med. 2024; 391: 821-31”). After dilution, the virus (titer was 5×10 3 After mixing 100 μL / well of 50 μL / well diluted serum and incubating at 37°C for 1.5 hours, discard the cell culture medium for virus attack;

[0156] 3) After 1.5 hours, discard the virus solution, add PBS, shake slowly to clean the cell surface, then aspirate, add DMEM medium containing 1.25% methylcellulose, seal the cell plate with sealing film, and culture at 37°C for 3.5 days;

[0157] 4) Add 3.7% formaldehyde and place on a horizontal shaker for 30 min. Aspirate the formaldehyde and culture medium mixture and discard it. Add formaldehyde, shake well and aspirate. Repeat 2-3 times.

[0158] 5) After cleaning the methylcellulose, add formaldehyde and place it on a horizontal shaker to gently shake for 30 min to fix the cells. Wash the formaldehyde 4-5 times with 1×PBST;

[0159] 6) Discard the formaldehyde, add PBS blocking and permeabilizing solution containing 0.3% Triton-X100 and 5% skim milk, and gently shake on a horizontal shaker for 1.5 h;

[0160] 7) Discard the blocking and permeabilizing solution, wash with 0.05% PBST, gently shake on a horizontal shaker for 5 min, and repeat 3 times;

[0161] 8) Incubate with Anti-rabbit WELV NP antibody (see Li H, Zhang LK, Li SF, et al. Calcium channel blockers reduce severe fever with thrombocytopenia syndrome virus related fatality. Cell Res. 2019;29(9):739 - 753. After immunizing rabbits with WELV NP protein, the antiserum against NP protein was obtained) (diluted with 0.05% PBST at a dilution of 1:2000) for 1.5 h, and wash three times with 0.05% PBST; In addition, the rabbit anti-WELV NP polyclonal antibody can also be synthesized and purified by (Beijing) Bioron Immunotechnology Co., Ltd.

[0162] 9) Incubate with anti-rabbit HRP-IgG antibody (diluted with 0.05% PBST at a dilution of 1:1000) for 1 h, and wash three times with 0.05% PBST;

[0163] 10) Develop color with DAB chromogenic solution, discard the chromogenic solution, wash 3 times with 0.05% PBST, air-dry the plate, and perform spot counting.

[0164] The results are as Figure 7 and Figure 8 shown. Figure 7 is the survival curve of mice in each group after virus challenge. Under the lethal dose challenge, all mice in the normal saline + double adjuvant group died on the 2nd day after virus challenge, and mice in the normal saline group began to die on the 2nd day after virus challenge and all died on the 4th day (n = 6). Both the 2.5 μg GnGc dose group and the 10 μg GnGc dose group were able to protect 67% of the mice from death threat under the WELV lethal dose challenge (n = 4 survived).

[0165] Figure 8 is the body weight change curve of mice in each group after virus challenge. The results showed that in the 10 μg GnGc dose group and the 2.5 μg GnGc dose group, the body weight of mice decreased within the range of 10%. It is suggested that immunizing mice with GnGc antigen can protect mice from death threat to a certain extent under the full lethal dose challenge of WELV.

[0166] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that further improvements can be made to the present invention. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A wetland virus glycoprotein Gn, characterized in that: The wetland virus glycoprotein Gn is a recombinant wetland virus glycoprotein Gn, and the wetland virus glycoprotein Gn is a C-terminally truncated wetland virus glycoprotein Gn truncation; the wetland virus glycoprotein Gn truncation is a protein whose amino acid sequence is shown in positions 1-153 of SEQ ID No.

1.

2. The wetland virus glycoprotein Gn according to claim 1, characterized in that A fusion protein is obtained by connecting a tag to the N-terminus or C-terminus of a protein with an amino acid sequence such as SEQ ID No.

1.

3. A biomaterial, characterized in that: The biological material is any of the following: C1) A nucleic acid molecule encoding the wetland virus glycoprotein Gn according to any one of claims 1-2; C2) an expression cassette containing the nucleic acid molecule described in C1; C3) a recombinant vector containing the nucleic acid molecule described in C1, or a recombinant vector containing the expression cassette described in C2; C4) a recombinant microorganism containing the nucleic acid molecule described in C1, or a recombinant microorganism containing the expression cassette described in C2, or a recombinant microorganism containing the recombinant vector described in C3; C5) A recombinant cell containing the nucleic acid molecule described in C1, or a recombinant cell containing the expression cassette described in C2, or a recombinant cell containing the recombinant vector described in C3.

4. A product containing the wetland virus glycoprotein Gn according to any one of claims 1-2.

5. The product according to claim 4, characterized in that: The product is a wetland virus double antigen combined subunit vaccine, in which the wetland virus double antigens contain wetland virus glycoproteins Gn and Gc, the wetland virus glycoprotein Gn truncation is a protein with an amino acid sequence as shown in SEQ ID No.1, and the wetland virus glycoprotein Gc truncation is a protein with an amino acid sequence as shown in SEQ ID No.

5.

6. The product according to claim 5, characterized in that The wetland virus double antigen combined subunit vaccine also contains an adjuvant.

7. The product according to claim 6, characterized in that: The adjuvant is an aluminum adjuvant and / or a CpG adjuvant, and the aluminum adjuvant comprises an aluminum hydroxide adjuvant.

8. The product according to claim 7, characterized in that: The CpG adjuvant is any one of the following: (1) CpG2006 or its thiolated product whose nucleotide sequence is SEQ ID No.9; (2) CpG1018 or its thiolated product whose nucleotide sequence is SEQ ID No.10; (3) CpG684 or its thiolated product whose nucleotide sequence is SEQ ID No. 11; The thiolated product is a full-chain thiolated modified product.

9. The product according to claim 7, characterized in that The wetland virus double antigen combined subunit vaccine is composed of the wetland virus glycoprotein Gn truncation, the wetland virus glycoprotein Gc truncation, the aluminum adjuvant and the CpG adjuvant; wherein the mass ratio of the wetland virus glycoprotein Gn truncation, the wetland virus glycoprotein Gc truncation, the aluminum adjuvant and the CpG adjuvant is 10:10:100:50 or 2.5:2.5:100:50; the amount of the aluminum adjuvant is calculated based on the aluminum content therein.

10. A method for preparing wetland virus glycoprotein Gn according to any one of claims 1-2, characterized in that: The preparation method comprises the following steps: Step S1, expressing the wetland virus glycoprotein Gn truncated form in yeast; Step S2, expressing the wetland virus glycoprotein Gn truncate in the Expi-293F mammalian cell expression system; expressing the wetland virus glycoprotein Gn truncate in the Expi-293F mammalian cell expression system is achieved by introducing a nucleic acid molecule encoding the wetland virus glycoprotein Gn truncate into the Expi-293F mammalian cell expression system; Step S3, culturing the Expi-293F mammalian cells in suspension, and purifying the wetland virus glycoprotein Gn truncate from the suspension cell culture supernatant; the purification includes the step of subjecting the suspension cell culture supernatant to nickel ion affinity chromatography.

11. An application of the wetland virus glycoprotein Gn as described in any one of claims 1-2 or the biological material as described in claim 3 in the preparation of a product that can induce animals to produce antigen-specific antibodies, wherein the product is a wetland virus double antigen combined subunit vaccine, the wetland virus double antigen comprises wetland virus glycoproteins Gn and Gc; the wetland virus glycoprotein Gn truncate is a protein with an amino acid sequence as shown in SEQ ID No.1, and the wetland virus glycoprotein Gc truncate is a protein with an amino acid sequence as shown in SEQ ID No.5.

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