Wetland virus double-antigen combined subunit vaccine and application thereof

Abstract

The invention provides a wetland virus double-antigen combined subunit vaccine and application thereof, and belongs to the technical field of biological medicine, wetland virus glycoprotein Gn is recombinant wetland virus glycoprotein Gn and is a glycoprotein Gn truncated body with a truncated C terminal; the invention also provides an antigen wetland virus glycoprotein Gn truncated body and / or Gc of a wetland virus (WELV) vaccine, and the amino acid sequence of the glycoprotein Gn truncated body of the WELV is as shown in SEQ ID No.1. The prepared monovalent recombinant subunit vaccine (Gn) or double-antigen combined subunit vaccine (Gn + Gc) is good in safety, can effectively stimulate immune response and can completely protect mice from death threats.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a wetland virus double antigen combined subunit vaccine and application thereof. Background Art

[0002] Tick-borne viral 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 and Hinterlandia virus. In the family Nairoviridae of the order Bunyavirales, the genus Orthonairovirus contains a variety of viruses that can infect humans, including Crimean-Congo haemorrhagic fever virus and Pine Ridge virus, all of which are transmitted through tick bites. With the exception of the highly pathogenic Crimean-Congo haemorrhagic fever virus (CCHFV), the diseases caused by these other viral infections are relatively mild; however, these tick-borne viruses are still of public health significance due to their broad host range and widespread geographic distribution.

[0003] Wetland virus (WELV) is a newly discovered tick-transmitted orthonavirus. In June 2019, a patient developed persistent fever and multi-organ dysfunction after being bitten by a tick in a wetland park. High-throughput sequencing analysis of the patient's specimens showed infection with a previously unknown orthonavirus, which the researchers named wetland virus (WELV) through isolation culture, morphological and molecular biological identification. The virus isolated from the index patient and ticks showed cytopathic effect in human umbilical vein endothelial cells. Patients infected with WELV showed nonspecific symptoms, including fever, dizziness, headache, fatigue, myalgia, arthritis and back pain, and less commonly petechiae and regional lymphadenopathy. Some patients developed neurological symptoms. Common abnormal laboratory test results were leukopenia, thrombocytopenia, and elevated D-dimer and lactate dehydrogenase levels. The researchers conducted a serological evaluation of the samples collected from the eight patients during the recovery period, and the results showed that the titer of WELV-specific antibodies was four times that of the samples collected during the acute period. WELV RNA was detected in the five tick species collected, as well as sheep, horses, pigs, and plateau zokors. Laboratory transmission experiments showed that Haemaphysalis gregaris ticks may be the vector of WELV.

[0004] Wetland wetland virus (WELV) particles are enveloped, spherical and ellipsoidal, with diameters ranging from 80 to 160 nm. WELV has a negative-sense single-stranded tripartite RNA genome: small (S), middle (M) and large (L), encoding the nucleoprotein (NP), membrane protein precursor (GPC) and RNA-dependent RNA polymerase (RdRP), respectively. The L, M and S segments have typical terminal reverse complementary sequences, 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 for subtilisin-kexin isozyme 1 / site 1 protease (SKI-1 / S1 P) were identified at positions RRLM 306↓ and RKLL 791↓. Typically, the replication cycle of Nairovirus begins with the binding of the virus to receptors on the cell surface, a process mediated by the membrane proteins Gn and / or Gc. However, the specific mechanisms of membrane proteins involved in virus adsorption, internalization and fusion remain unclear, and the cell receptors required for Nairovirus entry have not yet been discovered.

[0005] WELV and CCHFV belong to the Nairoviridae family. The mortality 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 is no obvious age and gender difference in susceptibility. At present, there is no effective prevention and treatment measures for CCHFV infection, which seriously threatens people's health. The World Health Organization (WHO) has listed CCHFV in the list of infectious pathogens research and development blueprint. Virus surface glycoproteins can specifically recognize receptors on the surface of host cells, and initiate the process of virus invasion of host cells by binding to specific receptors. Virus surface glycoproteins are usually one of the main targets of immune system recognition 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 cells infected by the virus. Virus surface glycoproteins are important targets for vaccine design. By inducing the body to produce antibodies against glycoproteins, viral infection can be effectively prevented. In recent years, some studies have explored different types of CCHFV candidate vaccines, such as DNA candidate vaccines, recombinant vesicular stomatitis virus live attenuated vaccines, and recombinant human adenovirus type 5 CCHFV candidate vaccines. However, a CCHF vaccine that is both efficient, safe, and low-cost has not yet been developed, and no vaccine has been approved for marketing.

[0006] Existing studies have shown that although a single Gn or Gc subunit vaccine can induce partial neutralizing antibodies, its protective efficacy is limited (animal experiments show a survival rate of <50%), and it cannot effectively stimulate a Th1 / Th2 balanced immune response.

[0007] At present, the development of wetland virus (WELV) vaccines faces the following key issues: (1) Antigen design defects: Full-length Gn / Gc proteins have complex post-translational modifications and low expression levels, which restrict large-scale production; (2) Insufficient immune synergy: A single antigen is difficult to cover the entire process of virus invasion, and lacks the dual protection mechanism of blocking “receptor binding-membrane fusion”; (3) Safety risks: Traditional vaccine adjuvants (such as aluminum adjuvants) are prone to induce local inflammatory reactions and have limited effects on cellular immune activation.

[0008] In response to the above problems, it is urgent to develop a combined subunit vaccine based on the wetland virus Gn and Gc dual antigens to achieve efficient and safe synergistic immune protection by optimizing the antigen structure and compatibility strategy. Summary of the invention

[0009] Based on the problems existing in the prior art, the present invention provides a wetland virus double antigen combined subunit vaccine and its application, and further provides wetland virus glycoprotein Gn and related biological materials and wetland virus (WELV) glycoprotein Gn vaccine and its application. Specifically, it is WELV Gn protein and immune composition containing this recombinant Gn protein and its application.

[0010] According to the first aspect of the technical solution of the present invention, a wetland virus glycoprotein Gn is provided. The wetland virus glycoprotein Gn is a recombinant wetland virus glycoprotein Gn. The wetland virus glycoprotein Gn is a truncate of the wetland virus glycoprotein Gn with a C-terminus truncated.

[0011] Furthermore, the wetland virus glycoprotein Gn truncation is any of the following proteins: A1) a protein comprising the amino acid sequence as shown in positions 1 to 153 of SEQ ID No. 1; A2) a protein having the same function as that defined in A1 by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence; A3) a protein having an amino acid sequence of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined in A1 or A2 and having the same function; A4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of any of the proteins defined in A1-A3. According to the second aspect of the technical solution of the present invention, a biomaterial is provided, wherein the biomaterial is any one of the following: C1) Nucleic acid molecule encoding wetland virus glycoprotein Gn; C2) an expression cassette containing the nucleic acid molecule described in C1; C3) a recombinant vector containing the nucleic acid molecule described in C2, 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.

[0012] According to the third aspect of the technical solution of the present invention, a product containing the above-mentioned wetland virus glycoprotein Gn is provided. The product is a wetland virus double antigen combined subunit vaccine, the wetland virus double antigen in the wetland virus double antigen combined subunit vaccine contains wetland virus glycoproteins Gn and Gc, and the wetland virus double antigen combined subunit vaccine also contains an adjuvant; preferably, the adjuvant is an aluminum adjuvant or a CpG adjuvant, and the aluminum adjuvant contains an aluminum hydroxide adjuvant.

[0013] Furthermore, 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.

[0014] Furthermore, the wetland virus dual antigen combined subunit vaccine is composed of the wetland virus glycoprotein Gn truncation, the aluminum adjuvant and the CpG adjuvant; wherein the mass ratio of the wetland virus glycoprotein Gn truncation, the aluminum adjuvant and the CpG adjuvant is 10 (2.5): 100: 50; the amount of the aluminum adjuvant is calculated based on the aluminum content therein.

[0015] According to the fourth aspect of the technical solution of the present invention, a method for preparing the wetland virus glycoprotein Gn as described above is provided, the preparation method comprising 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.

[0016] According to the fifth aspect of the technical solution of the present invention, there is provided a use of the wetland virus glycoprotein Gn as mentioned above or the above-mentioned biological material in the preparation of a product that can induce animals to produce antigen-specific antibodies, wherein the product is a double antigen combined subunit vaccine, and the wetland virus double antigens contain wetland virus glycoproteins Gn and Gc.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The present invention discloses a method for constructing an expression vector of a wetland virus glycoprotein Gn truncate (Gn subunit truncate). The constructed expression vector can realize the expression of the wetland virus glycoprotein Gn truncate (Gn subunit truncate) in mammalian cells, and has the characteristics of fast growth and high safety.

[0018] 2. The wetland virus glycoprotein Gn truncated body (Gn subunit truncated body) obtained by the recombinant expression of the present invention does not have the problem of excessive glycosylation. After immunizing mice with it alone or in combination with wetland virus glycoprotein Gc, it can produce high-titer anti-wetland virus glycoprotein Gn truncated body (Gn subunit truncated body) specific IgG antibodies, and after booster immunization, it can protect mice from the lethal attack of WELV. Therefore, the WELV Gn subunit truncated body recombinant subunit candidate vaccine prepared by mammalian cells can provide a better protective response and provide a reference for the development of WELV subunit vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A-1B Schematic diagram of the truncated structure of the recombinant wetland virus glycoprotein subunit. Figure 1A Schematic diagram of the truncated structure of the WELV Gn glycoprotein subunit and the predicted protein structure (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 truncated structure of the WELV Gc glycoprotein subunit and a predicted protein structure; Figure 2A-2BThe results are the identification results of the protein expression changes of the recombinant wetland virus glycoprotein Gn and Gc subunit truncations over time in the Expi 293F expression system; Figure 2A The curves of cell viability changes over time after wetland virus glycoprotein Gn transfection and the results of Anti-His tag monoclonal antibody (Sigma, A7058) detection of Gn protein expression over time; Figure 2B This is the curve showing the changes in cell viability over time after transfection of wetland virus glycoprotein Gc and the Anti-His tag monoclonal antibody (Sigma, A7058) detection results showing the changes in Gn protein expression over time.

[0020] Figure 3A-3C The recombinant wetland virus glycoprotein Gn subunit truncated sample was identified by nickel affinity chromatography, SDS-PAGE and WB. Figure 3A Nickel ion affinity chromatography chromatogram showing the truncated form of the Gn subunit of the recombinant wetland virus glycoprotein. Figure 3B Schematic diagram of SDS-PAGE showing the truncated form of the recombinant wetland virus glycoprotein Gn subunit. Figure 3C WB schematic diagram showing the truncated form of the recombinant wetland virus glycoprotein Gn subunit.

[0021] Figure 4A-4C The recombinant wetland virus glycoprotein Gc subunit truncated sample was identified by nickel affinity chromatography, SDS-PAGE and WB. Figure 4A is the nickel ion affinity chromatography chromatogram; Figure 4B SDS-PAGE of samples from the purification process; Figure 4C For WB identification of purification process samples.

[0022] Figure 5A-Figure 5B SDS-PAGE of purified samples of recombinant wetland virus glycoprotein. Figure 5A This is the SDS-PAGE after purification of the truncated sample of the wetland virus glycoprotein Gn subunit; Figure 5B This is the SDS-PAGE after purification of the truncated sample of the wetland virus glycoprotein Gc subunit.

[0023] Figure 6A-6B This is the detection of antibody titer after intramuscular immunization of mice with the truncated protein of the GnGc subunit of the recombinant wetland virus glycoprotein. Fig. 6A It is the specific binding antibody titer against Gn in the mouse serum after the first immunization and booster immunization; Figure 6B It is the specific binding antibody titer against Gc in the mouse serum after the first immunization and booster immunization.

[0024] Figure 7 This is a survival curve to evaluate the protective effect of mice immunized intramuscularly with the truncated protein of the recombinant wetland virus glycoprotein GnGc subunit in the WELV lethal model.

[0025] Figure 8 This is the weight change of mice immunized with recombinant wetland virus glycoprotein GnGc subunit truncated protein by intramuscular injection after challenge with a lethal dose of WELV.

[0026] In each quantitative result figure, ns represents P >0.05, * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, **** indicates P <0.0001. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0028] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0029] Wetland Land Virus (WELV) strains can be obtained from the Military Medical Research Institute.

[0030] The pCAGGS mammalian expression vector was purchased from Aonogen, catalog number HG-VPA0057.

[0031] The pAB plasmid was stored in our laboratory.

[0032] The Expi 293F mammalian expression system was purchased from Thermo Fisher Scientific Inc., catalog number A14635CN.

[0033] Anti-His tag mouse monoclonal antibody was purchased from Sigma, catalog number A7058.

[0034] Chelating Sepharose FF chromatography medium was from Cytiva, catalog number 17057502.

[0035] BALB / c mice (strain code: 211) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0036] In the following examples, the virus is specifically a wetland virus (WELV), which is recorded in the non-patent document "NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov / ): A New Orthonairovirus Associated with Human FebrileIllness". The public may obtain the biological material from the applicant in accordance with relevant biosafety regulations. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0037] The present invention relates to a wetland virus double antigen combined subunit vaccine and its application, which belongs to the field of biomedicine technology. The wetland virus (WELV) vaccine antigen wetland virus glycoprotein Gn truncated body and / or Gc, the amino acid sequence of the glycoprotein Gn truncated body of WELV is shown in SEQ ID No. 1. After the vaccine of the present invention is used to immunize mice, high-titer antibodies against wetland virus glycoprotein Gn truncated body can be produced, and the WELV true virus can be neutralized. After the monovalent recombinant subunit vaccine (Gn) or the double antigen combined subunit vaccine (Gn+Gc) prepared by the present invention is used to boost immunization of mice, 67% of the mice can be protected from the threat of death at a completely lethal dose of WELV; the recombinant wetland virus glycoprotein Gn truncated body subunit candidate vaccine prepared by the present invention using a mammalian expression system has good safety, can effectively stimulate immune response, and can completely protect mice from the threat of death. The present invention can provide a reference for the development of WELV subunit vaccines.

[0038] In one embodiment of the present invention, the present invention claims a wetland virus (WELV) glycoprotein Gn, preferably a WELV recombinant glycoprotein Gn, and more preferably the antigen in the wetland virus double antigen combined subunit vaccine claimed by the present invention is a truncated glycoprotein Gn of WELV. The WELV recombinant glycoprotein Gn comprises a polypeptide having an amino acid sequence shown in SEQ ID NO.1. The truncated glycoprotein Gn of WELV is any of the following proteins: A1) a protein with an amino acid sequence as shown in SEQ ID No.1 or positions 1 to 153 of SEQ ID No.1; A2) a protein having the same function as that defined in A1 by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence; A3) a protein having an amino acid sequence of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined in A1 or A2 and having the same function; A4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of any of the proteins defined in A1-A3.

[0039] In the above protein, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag or a SUMO tag, etc.

[0040] In 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 page on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gapexistence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be calculated.

[0041] In the above proteins, the above 95% identity or more may be at least 96%, 97%, 98% identity. The above 90% identity or more may be at least 91%, 92%, 93%, 94% identity. The above 85% identity or more may be at least 86%, 87%, 88%, 89% identity. The above 80% identity or more may be at least 81%, 82%, 83%, 84% identity.

[0042] In another embodiment, a vaccine containing recombinant WELV glycoprotein Gn is provided, namely, a wetland virus double antigen combined subunit vaccine, wherein the wetland virus double antigen combined subunit vaccine further comprises an adjuvant. 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: (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; Wherein, the thiolated product may be a full-chain thiolated modified product.

[0043] In a specific embodiment of the present invention, the wetland virus double antigen combined subunit vaccine is composed of the wetland virus glycoprotein Gn truncate, the aluminum adjuvant and the CpG adjuvant; wherein the mass ratio of the wetland virus glycoprotein Gn truncate, 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 aluminum content therein.

[0044] In another specific embodiment of the present invention, the wetland virus dual antigen combined subunit vaccine is composed of the wetland virus glycoprotein Gn truncation and the aluminum adjuvant; wherein the mass ratio of the wetland virus glycoprotein Gn truncation and the aluminum adjuvant is (2.5-20):100.

[0045] Furthermore, the mass ratio of the wetland virus glycoprotein Gn truncate and the aluminum adjuvant is 10: 100. The amount of the aluminum adjuvant is calculated based on the aluminum content therein.

[0046] In another embodiment, a method for preparing the above-mentioned recombinant wetland virus glycoprotein Gn truncate is provided, the preparation method comprising the following steps: Step S1, expressing the wetland virus glycoprotein Gn truncate in Pichia baskisae.

[0047] Step S2, expressing the wetland virus glycoprotein Gn truncated form in the Expi293F mammalian cell expression system to obtain an expression supernatant; Furthermore, the expression of the wetland virus glycoprotein Gn truncated body in the Expi293F mammalian cell expression system is achieved by introducing a nucleic acid molecule encoding the wetland virus glycoprotein Gn truncated body into the Expi293F mammalian cell expression system. 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.

[0048] Step S3, the transfected Expi293F cells are suspended and cultured, and the wetland virus glycoprotein Gn truncation is purified from the suspension cell culture supernatant. Furthermore, the wetland virus glycoprotein Gn truncation is expressed during the transfection culture. In a specific embodiment of the present invention, the culture is carried out at 37°C and 125rpm in a shaking table, and the sample is collected after 48 hours. Furthermore, the purification includes subjecting the transfection culture supernatant to nickel ion affinity chromatography (Chelating FF column).

[0049] Furthermore, the nucleic acid molecule encoding the truncated form of the wetland virus glycoprotein Gn may be any of the following: (a1) a DNA molecule having a nucleotide sequence as shown in positions 1 to 459 of SEQ ID No. 2; (a2) a DNA molecule that hybridizes with the DNA molecule defined in a1 under stringent conditions and encodes a truncated form of the wetland virus glycoprotein Gn; (a3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the DNA sequence defined in a1 or a2 and encoding a truncated form of the glycoprotein Gn of the WELV.

[0050] In the above nucleic acid molecules, the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, 2×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, 1×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, 0.5×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, 0.1×SSC, 0.1% SDS; it may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, rinsed at 50°C, 0.1×SSC, 0.1% SDS; it may also be: 50°C, 7% Hybridize in a mixed solution of SDS, 0.5M Na3PO4 and 1mM EDTA, and rinse in 0.1×SSC, 0.1% SDS at 65°C. Alternatively, hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, and then wash the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0051] In another embodiment, there is provided a use of the above-mentioned recombinant wetland virus glycoprotein Gn truncated form in the preparation of a product capable of inducing an animal to produce antigen-specific antibodies, wherein the recombinant WELV glycoprotein Gn is immunized by intramuscular or subcutaneous injection.

[0052] In another embodiment, an immune vaccine is provided, the vaccine comprising the above-mentioned recombinant wetland virus glycoprotein Gn truncate, or the above-mentioned composition containing the recombinant wetland virus glycoprotein Gn truncate. Preferably, the vaccine is a double antigen combined subunit vaccine, the double antigen comprising wetland virus glycoprotein Gn and Gc.

[0053] In another embodiment, a pharmaceutical composition is provided, which comprises the above-mentioned wetland virus glycoprotein Gn truncation, or the above-mentioned composition containing the wetland virus glycoprotein Gn truncation.

[0054] In another embodiment, the present invention claims any of the following applications: (D1) Use of the protein or biological material in the preparation of a wetland virus double antigen combined subunit vaccine; (D2) Use of wetland virus dual antigen combined subunit vaccine or protein or biological material in the preparation of products for the prevention and / or treatment of Congo hemorrhagic fever / WELV; (D3) Use of wetland virus dual antigen combined subunit vaccine or protein or biological material in the preparation of products for alleviating symptoms of hemorrhagic fever / WELV; (D4) Use of wetland virus dual antigen combined subunit vaccine or protein or biological material in the preparation of products for preventing WELV infection; (D5) Application of wetland virus dual antigen combined subunit vaccine or protein or biological material in the preparation of products for neutralizing WELV.

[0055] The gene sequences are as follows.

[0056] SEQ ID No.1: Gn truncation amino acid sequence (153AA): SSESSGSNPCSSGTFLGEGSSAQVVGNKNDGPGEHITFCNGTYVSKIKLGKQHGCFTVRRVKAYRNCHPKEASTSCVVDEELRECEGQRCMNIHLDVRGLVKITRGKYVEVITCDKDCLAKIPSGRGDIQIDCPGGKQHYLETNVVDINCPGPE.

[0057] SEQ ID No.2: Coding gene sequence corresponding to Gn truncation (459 bp): TCATCTGAGTCTGGTTCTAATCCTTGTAGTTCTGGAACCTTTTTGGGTGAAGGTTCTTCCGCTCAAGTTGTTGGAAATAAGAATGATGGACCTGGTGAACACATTACTTTTTGTAATGGAACTTATGTTAGTAAGATAAAGTTGGGTAAACAACATGGTTGTTTCACCGTTAGAAGAGTAAAAGCATATAGAAATTGTCATCCAAAGGAAGCTAGTACTAGTTGTGTAGTTGATGAAGAACTTAGAGAATGTGAAGGTCAAAGATGTATGAACATTCACTTAGATGTTAGAGGATTAGTTAAAATTACTAGAGGTAAATATGTTGAAGTTATTACTTGTGATAAGGATTGTTTGGCTAAGATTCCAAGTGGTAGAGGTGACATCCAAATTGATTGTCCAGGTGGTAAACAACACTACTTAGAAACTAACGTTGTTGATATTAACTGTCCAGGTCCAGAA。

[0058] SEQ ID No.3: Amino acid sequence of the Gn truncation + HIS tag (1 - 164 AA): SSESGSNPCSSGTFLGEGSSAQVVGNKNDGPGEHITFCNGTYVSKIKLGKQHGCFTVRRVKAYRNCHPKEASTSCVVDEELRECEGQRCMNIHLDVRGLVKITRGKYVEVITCDKDCLAKIPSGRGDIQIDCPGGKQHYLETNVVDINCPGPEGGGGSHHHHHH。

[0059] SEQ ID No.4: Coding gene sequence corresponding to the Gn truncation + HIS tag (492bp): TCATCTGAGTCTGGTTCTAATCCTTGTAGTTCTGGAACCTTTTTGGGTGAAGGTTCTTCCGCTCAAGTTGTTGGAAATAAGAATGATGGACCTGGTGAACACATTACTTTTTGTAATGGAACTTATGTTAGTAAGATAAAGTTGGGTAAACAACATGGTTGTTTCACCGTTAGAAGAGTAAAAGCATATAGAAATTGTCATCCAAAGGAAGCTAGTACTAGTTGTGTAGTTGATGAAGAACTTAGAGAATGTGAAGGTCAAAGATGTATGAACATTCACTTAGATGTTAGAGGATTAGTTAAAATTACTAGAGGTAAATATGTTGAAGTTATTACTTGTGATAAGGATTGTTTGGCTAAGATTCCAAGTGGTAGAGGTGACATCCAAATTGATTGTCCAGGTGGTAAACAACACTACTTAGAAACTAACGTTGTTGATATTAACTGTCCAGGTCCAGAA GGAGGTGGAGGTTCCCATCATCATCATCATCAT.

[0060] SEQ ID No.5: Amino acid sequence of Gc truncation (525 AA): FFKGLNSAASRMLNAHKLMTSVSIDAPWGAIQVESTYKPKLPVSNIELAWNSIEEQGDKIILSGKSTSILKLEERTGVQWSLGAESASEEKRLLVSVLDYTQVYSSTFQYITGDRTISEWPKATCTGDCPDRCACRTSTCLFKSWPHSRNWRCNPTWCWGVGTGCTCCGVDIERPFNKYFAVKWSTEYVRTDALVCVELTDLERHCDVVEAGSQFVIGPVRVVVSDPQNVQSKLPQEVLTVQKLDKHQHLDLMHVTNIISAKNACKLQSCTHGSPGDMQILHTDNLILNSHDDGANLAESIPEVNTTWMSWEGCDLDYYCTTGSWPSCTFTGVNTENTESFENLLNTEANLIDRYHFHSKRIYAQGATLQMDLKGRPKSGGGELTVLVDIKGLELHSKRVVLKGLEIKALSCTGCYSCSSGMSCTVDVRIEKPDEFTVHLRSTDPNTAISEGSIMARKLSGGPQSKVRAFTALKVSEVCVEIVEKNYCPTCKETDTKKCAKVDLQPPKDILLEHKGTLIKSQNDS。

[0061] SEQ ID No. 6: Coding gene sequence corresponding to the Gc truncation (1575 bp):

[0062] SEQ ID No.7: Amino acid sequence of the Gc truncation + HIS tag (1 - 536 AA): FFKGLNSAASRMLNAHKLMTSVSIDAPWGAIQVESTYKPKLPVSNIELAWNSIEEQGDKIILSGKSTSILKLEERTGVQWSLGAESASEEKRLLVSVLDYTQVYSSTFQYITGDRTISEWPKATCTGDCPDRCACRTSTCLFKSWPHSRNWRCNPTWCWGVGTGCTCCGVDIERPFNKYFAVKWSTEYVRTDALVCVELTDLERHCDVVEAGSQFVIGPVRVVVSDPQNVQSKLPQEVLTVQKLDKHQHLDLMHVTNIISAKNACKLQSCTHGSPGDMQILHTDNLILNSHDDGANLAESIPEVNTTWMSWEGCDLDYYCTTGSWPSCTFTGVNTENTESFENLLNTEANLIDRYHFHSKRIYAQGATLQMDLKGRPKSGGGELTVLVDIKGLELHSKRVVLKGLEIKALSCTGCYSCSSGMSCTVDVRIEKPDEFTVHLRSTDPNTAISEGSIMARKLSGGPQSKVRAFTALKVSEVCVEIVEKNYCPTCKETDTKKCAKVDLQPPKDILLEHKGTLIKSQNDSGGGGSHHHHHH。

[0063] SEQ ID No.8: Coding gene sequence corresponding to the Gc truncation + HIS tag (1608bp):

[0064] The following are the CpG sequences of SEQ ID No. 9-11: SEQ ID No. 9: tcgtcgtttt gtcgttttgt cgtt; SEQ ID No.10: tgactgtgaa cgttcgagat ga; SEQ ID No.11: tcgacgttcg tcgttcgtcg ttc; In the following examples, unless otherwise specified, whole gene synthesis, nucleotide synthesis, primer synthesis, sequencing, etc. were performed by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. and Beijing Qingke Biotechnology Co., Ltd.

[0065] In the following examples, unless otherwise specified, the first position of each nucleotide sequence is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

[0066] Example 1. Construction of mammalian expression vector for WELV Gn and Gc subunit truncations.

[0067] (1) Obtaining the truncated genes of wetland virus glycoprotein Gn and Gc subunits and constructing mammalian expression vectors.

[0068] According to the sequence of WELV published in GenBank: OR860396-OR860409, the inventors artificially designed the codon DNA sequence of amino acids 1 to 153 (S1-E153) of the Gn protein, and added GGGGS connecting peptide and 6 HIS tags, which is a DNA fragment with a sequence as shown in SEQ ID No. 4. Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the relevant DNA fragment (from the 5' end to the 3' end, there are AflII restriction site, GGCGTGCAGTGC, the fragment codon DNA sequence shown in SEQ ID No. 4, and BamHI restriction site), 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.

[0069] The structure of the recombinant expression vector pAB-Gn subunit truncation is described as follows: the DNA fragment between the AflII and BamHI recognition sequences of the pAB vector is replaced with the recombinant plasmid of the DNA fragment shown in SEQ ID No. 4. SEQ ID No. 4 is the coding gene sequence obtained after artificial codon optimization based on the Gn subunit truncation (S1-E153), encoding the Gn subunit truncation protein with 6 His tags and GGGGS connecting peptide shown in SEQ ID No. 1. Positions 1-153 of SEQ ID No. 3 are Gc subunit truncation proteins. Figure 1A The figure shows a schematic diagram of the structure of the Gn subunit truncation of WELV (Gn subunit truncation) and a predicted three-dimensional structure of the Gn subunit truncation of WELV (the blue part represents the part involved in the present invention, and the gray part represents truncation).

[0070] 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, the inventors artificially designed the codon DNA sequence, and added the GGGGS connecting peptide and 6 HIS tags, which is a DNA fragment with a sequence as shown in SEQ ID No. 8. Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the relevant DNA fragment (from the 5' end to the 3' end, there are EcoRI restriction site, BM40 signal peptide, fragment codon DNA sequence shown in SEQ ID No. 8, NotI restriction site), and the synthesized fragment was inserted between the EcoRI and NotI restriction sites of the pCAGGS vector to obtain the recombinant expression vector pCAGGS-Gc subunit truncation, that is, the Gc subunit truncation expression vector.

[0071] The structure of the recombinant expression vector pCAGGS-Gc subunit truncation is described as follows: 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 after artificial codon optimization based on the Gc subunit truncation (F1-S525), encoding the Gc subunit truncation protein with 6 His tags and a GGGGS connecting peptide shown in SEQ ID No. 5. Positions 1-536 of SEQ ID No. 8 are the Gc subunit truncation protein. Figure 1B The figure shows a schematic diagram of the structure of the Gc subunit truncation of WELV (Gc subunit truncation) and a predicted three-dimensional structure of the Gc subunit truncation of WELV (the blue part represents the part involved in the present invention, and the gray part represents truncation).

[0072] (2) Extraction of recombinant expression vector pAB-Gn and pCAGGS-Gc subunit truncated plasmid (Tiangen Endotoxin-Free Mini-Preparation Medium Kit, Beijing, DP118-02).

[0073] Example 2: Expression and purification of recombinant Gn and Gc subunit truncated glycoproteins.

[0074] (2.1) Transfection of Expi293F suspension cells.

[0075] The plasmids identified in Example 1 (i.e., pAB-Gn, pCAGGS-Gc subunit truncations) were selected and transfected into Expi293F suspension cells with a cell density of 3*10^6 / mL. The transfection system is shown in Table 1 below (taking 100 mL transfection volume as an example, the remaining volumes are enlarged in equal proportions).

[0076] Table 1 Transfection system

[0077] After transfection, the cells were cultured at 37°C, 8% CO2, and 125 rpm. The cell viability was monitored every 12 h and 200 μL of samples were taken to monitor the protein expression process. Protein expression was stopped when the cell viability dropped below 60%.

[0078] The results are as follows Figure 2A-2B As shown, Figure 2A The left figure shows the change of cell viability over time after pAB-Gn plasmid transfection; Figure 2A The right figure in the figure shows the WB detection of the expression change of the target protein over time. Figure 2B The left figure shows the change of cell viability over time after pCAGGS-Gc plasmid transfection; Figure 2B The right figure in the figure shows the WB detection of the expression of the target protein over time. The results show that the Gn subunit truncated plasmid was sampled 72 hours after transfection of the cells, and the Gc subunit truncated plasmid was sampled 60 hours after transfection of the cells for the next experiment.

[0079] (2.2) Purification of Gn and Gc subunit truncations (same steps).

[0080] 1. Nickel ion affinity chromatography.

[0081] Sample pretreatment before purification: The culture supernatant of the induced expression in step 1 was dialyzed overnight against a replacement system of 20 mM pH 7.5 Tris-HCl + 500 mM NaCl.

[0082] Nickel affinity chromatography: A: 20mM pH7.5 Tris-HCl+500mM NaCl+5mM imidazole; B: 20 ​​mM pH 7.5 Tris-HCl + 500 mM NaCl + 500 mM imidazole.

[0083] After loading, balance with A, and then elute with B at 5%-10%-25%-50%-100% respectively.

[0084] Purify samples such as Figure 3A-3C shown. Figure 3A Nickel ion affinity chromatography chromatogram showing the truncated form of the Gn subunit of the recombinant wetland virus glycoprotein. Figure 3B Schematic diagram of SDS-PAGE showing the truncated form of the recombinant wetland virus glycoprotein Gn subunit. Figure 3C The WB schematic diagram of the recombinant wetland virus glycoprotein Gn subunit truncated form; Figure 4 shows the nickel ion affinity chromatography chromatogram, SDS-PAGE, and WB of the Gc subunit truncated form. The results show that the target protein Gn subunit truncated form was eluted at imidazole concentrations of 125 and 250 mM, and the target protein Gc subunit truncated form was eluted at imidazole concentrations of 125 and 250 mM, and the target protein was well separated from the impurities. After concentration, the next step of the experiment can be carried out.

[0085] Figure 5A SDS-PAGE of the purified Gn protein; Figure 5B This is the pure SDS-PAGE of the purified Gc protein.

[0086] Example 3: Mouse immunization experiment.

[0087] The immunization method has been disclosed in many documents, such as "Replication of Animal Models of Human Diseases, edited by Li Cai, published by People's Medical Publishing House". The details are as follows: 40 female BALB / C mice aged 6-8 weeks were randomly divided into the following 4 groups: Immunization group 1 (n = 6): 100 μl of vaccine was injected intramuscularly on days 0 and 21, respectively. The vaccine used was 10 μg Gn subunit truncated / 10 μg Gc subunit truncated / 100 μg Al(OH)3 (CRODA, Denmark, the same below) / 50 μg CpG2006. Among them, Gn, Gc subunit truncated is the recombinant wetland virus glycoprotein Gn, Gc subunit truncated expressed by Expi293F prepared above, and the vaccine was prepared with normal saline in a volume of 100 μl, containing 10 μg Gn and 10 μg Gc subunit truncated, 100 μg Al(OH)3 and 50 μg CpG 2006.

[0088] Immunization group 2 (n = 6): 100 μl of vaccine was injected intramuscularly on days 0 and 21, respectively. The vaccine used was 2.5 μg Gn subunit truncated / 2.5 μg Gc subunit truncated / 100 μg Al(OH)3 / 50 μg CpG2006. Among them, Gn, Gc subunit truncated is the recombinant wetland virus glycoprotein Gn, Gc subunit truncated expressed by Expi293F prepared above, and the vaccine was prepared in a volume of 100 μl containing 2.5 μg Gn and 2.5 μg Gc subunit truncated, 100 μg Al(OH)3 and 50 μg CpG2006 with normal saline.

[0089] Immunization group 3 (n = 6), i.e. adjuvant control group: 100 μl of vaccine was injected intramuscularly on days 0 and 21. The adjuvant used was 100 μg Al(OH)3+50 μg CpG2006. The vaccine was prepared with saline solution containing 100 μg Al(OH)3 and 50 μg CpG2006 in a volume of 100 μl.

[0090] Immunization group 4 (n = 6), i.e., blank control group: 100 μl of normal saline was injected intramuscularly on days 0 and 21, respectively.

[0091] The nucleotide sequence of CpG2006 is SEQ ID No. 9 and the whole chain is thiolated. If necessary, CpG2006 can also be replaced by any of the following: CpG1018 with a nucleotide sequence of SEQ ID No. 10 or its thiolated product; CpG684 with a nucleotide sequence of SEQ ID No. 11 or its thiolated product (such as whole chain thiolated).

[0092] Blood was collected from the suborbital venous plexus of two pairs of immunized mice in each group before immunization, two weeks after the first immunization, and after the second immunization.

[0093] The specific antibody titer against Gn and Gc subunit truncations in the serum of each group of mice was measured by indirect ELISA. The operation steps can be found in the Concise Molecular Biology Experiment Guide [M]. Science Press, 2008.

[0094] The results are as follows Figure 6A-6B As shown: Fig. 6A The specific binding antibody titers against Gn in the mouse serum after primary and booster immunizations are shown; the antibody titers of the immunization groups after primary and booster immunizations 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 between the 10 μg GnGc and 2.5 μg GnGc antigen dose groups after primary immunization and booster immunization ( P <0.0001).

[0095] 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.

[0096] Example 4, real virus challenge test.

[0097] 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; 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; 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; 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. 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; 6) Discard formaldehyde, add PBS containing 0.3% Triton-X100 and 5% skim milk for blocking and permeabilization, and shake gently on a horizontal shaker for 1.5 h; 7) Discard the blocking and permeabilization solution, wash with 0.05% PBST, shake gently on a horizontal shaker for 5 min, and repeat 3 times; 8) 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, antiserum against NP protein was obtained) (diluted with 0.05% PBST, the dilution was 1:2000) and incubated for 1.5h, and washed three times with 0.05% PBST; in addition, rabbit anti-WELV NP polyclonal antibody can also be synthesized and purified by (Beijing) Biolong Immunotechnology Co., Ltd.

[0098] 9) Incubate with anti-rabbit HRP-IgG antibody (0.05% PBST, dilution 1:1000) for 1 h, and wash three times with 0.05% PBST; 10) Develop with DAB colorimetric solution, discard the colorimetric solution, wash three times with 0.05% PBST, air-dry the plate, and count the spots.

[0099] The results are as follows Figure 7 and Figure 8 shown. Figure 7 is the survival curve of mice in each group after challenge. Under the lethal dose attack, all mice in the saline + double adjuvant group died on the second day after challenge, and mice in the saline group began to die on the second day after challenge and died completely on the fourth day (n = 6). Both the 2.5 μg GnGc dose group and the 10 μg GnGc dose group were able to protect 67% of mice from death threats under the lethal dose of WELV (survival n = 4).

[0100] Figure 8 The following is the weight change curve of mice in each group after virus attack. The results showed that the weight loss of mice in the 10 μg GnGc dose group and the 2.5 μg GnGc dose group was within 10%. This suggests that GnGc antigen immunization of mice can protect mice from the threat of death to a certain extent under the completely lethal dose of WELV.

[0101] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

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 truncated wetland virus glycoprotein Gn with a C-terminal truncated form.

2. The wetland virus glycoprotein Gn according to claim 1, characterized in that The wetland virus glycoprotein Gn truncation is any of the following proteins: A1) a protein comprising the amino acid sequence as shown in positions 1 to 153 of SEQ ID No. 1; A2) a protein having the same function as that defined in A1 by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence; A3) a protein having an amino acid sequence of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined in A1 or A2 and having the same function; A4) A fusion protein obtained by connecting a tag to the N-terminus or C-terminus of any of the proteins defined in A1-A3.

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 C2, 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 dual antigen combined subunit vaccine.

6. The product according to claim 5, characterized in that The wetland virus double antigens in the wetland virus double antigen combined subunit vaccine contain wetland virus glycoproteins Gn and Gc.

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

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

9. The product according to claim 8, 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.

10. The product according to claim 8, characterized in that The wetland virus double antigen combined subunit vaccine is composed of the wetland virus glycoprotein Gn truncation, the aluminum adjuvant and the CpG adjuvant; wherein the mass ratio of the wetland virus glycoprotein Gn truncation, the aluminum adjuvant and the CpG adjuvant is 10 (2.5): 100: 50; the amount of the aluminum adjuvant is calculated based on the aluminum content therein.

11. 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.

12. 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 double antigen combined subunit vaccine, and the wetland virus double antigen comprises wetland virus glycoproteins Gn and Gc.

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