Vaccine of wetland virus glycoprotein Gc and application thereof

Abstract

The invention discloses a wetland virus glycoprotein Gc vaccine and application thereof, and belongs to the technical field of biological medicine, the wetland virus glycoprotein Gc is a recombinant wetland virus glycoprotein Gc, and the wetland virus glycoprotein Gc is a glycoprotein truncated body with a truncated C terminal; the amino acid sequence of the wetland virus glycoprotein Gc truncation is a protein shown in the 1st-525th sites of SEQ ID No.1. The invention also discloses a preparation method of the wetland virus glycoprotein Gc truncation. After a mouse is immunized by the vaccine, a high-titer antibody for resisting a WELV glycoprotein Gc truncation can be generated, and a WELV true virus can be neutralized; after the vaccine prepared by the invention strengthens immunization of mice, the mice can be completely protected from death threat under the complete lethal dose of WELV; the recombinant WELV glycoprotein Gc truncated subunit candidate vaccine prepared by the invention has good safety, can effectively stimulate immune response, and can completely protect mice from death threat.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a vaccine of wetland virus glycoprotein Gc 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 disease have been discovered, such as severe fever with thrombocytopenia syndrome virus, Hinterlandia virus, etc. Within the Bunyaviridae family, the genus Orthonavirus contains the major species that cause human infections, including Crimean-Congo haemorrhagic fever, Dugbe virus, Songling virus, etc., all of which are transmitted by tick vectors. With the exception of the pathogenic Crimean-congo haemorrhagic fever virus (CCHFV), the diseases caused by other infections are relatively mild; however, they are of public health significance due to their broad host range and widespread geographic distribution.

[0003] 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 ends of the L, M and S segments have the typical reverse complementary sequences of bunyaviruses, 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. 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 membrane proteins. 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 yet been discovered.

[0004] WELV and CCHFV belong to the Bunyaviridae family. The mortality rate of Crimean-Congo hemorrhagic fever (CCHF) varies from region to region, but is usually between 30% and 50%. 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 CCHF infection, which seriously threatens people's health. The World Health Organization (WHO) has listed CCHFV in the list of infectious pathogens research and development blueprints. The glycoprotein on the surface of the WELV virus 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. The virus's own proteins 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. These proteins of the virus itself are one of the important targets for vaccine design. Inducing the body to produce antibodies against proteins through these protein antigens can effectively prevent viral infection. 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. The wetland virus WELV was first announced in 2024, and the corresponding vaccine and drug research is even more blank. There is an urgent need to develop a WELV vaccine that is both efficient, safe, and low-cost. Summary of the invention

[0005] Based on the problems existing in the prior art, the present invention provides a vaccine of wetland virus glycoprotein Gc and its application, specifically wetland virus (WELV) glycoprotein Gc and an immune composition containing the recombinant protein Gc and its application.

[0006] According to the first aspect of the technical solution of the present invention, a wetland virus glycoprotein Gc is provided, wherein the wetland virus glycoprotein Gc is a recombinant wetland virus glycoprotein Gc, and the wetland virus glycoprotein Gc is a glycoprotein truncation with a C-terminus truncated.

[0007] Preferably, the wetland virus glycoprotein Gc truncation is any of the following proteins: A1, a protein comprising the amino acid sequence as shown in positions 1 to 525 of SEQ ID No. 1; A2, a protein having the same function as that of the amino acid sequence defined in A1 after one or more amino acid residues are substituted and / or deleted and / or added; 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 to that of 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 protein 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, a nucleic acid molecule encoding the above wetland virus glycoprotein Gc; 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.

[0008] According to the third aspect of the technical solution of the present invention, a product containing the above-mentioned wetland virus glycoprotein Gc is provided, wherein the product is an immune composition or a pharmaceutical composition, wherein the immune composition is a vaccine, wherein the vaccine further comprises an adjuvant, wherein the adjuvant is an aluminum adjuvant or a CpG adjuvant, wherein the aluminum adjuvant comprises an aluminum hydroxide adjuvant; wherein the CpG adjuvant is any one of the following: (1) CpG2006 or its thiolated product whose nucleotide sequence is SEQ ID No.5; (2) CpG1018 or its thiolated product whose nucleotide sequence is SEQ ID No.6; (3) CpG684 or its thiolated product whose nucleotide sequence is SEQ ID No. 7; The thiolated product is a full-chain thiolated modified product.

[0009] Furthermore, in the product, the vaccine is composed of a C-terminally truncated glycoprotein truncation, an aluminum adjuvant and a CpG adjuvant; wherein the mass ratio of the C-terminally truncated glycoprotein truncation, the aluminum adjuvant and the CpG adjuvant is 10(2.5):100:50; and the amount of the aluminum adjuvant is calculated based on the aluminum content therein.

[0010] According to the fourth aspect of the technical solution of the present invention, a method for preparing the wetland virus glycoprotein Gc as described above is provided, the method comprising the following steps: Step S1, expressing the wetland virus glycoprotein Gc truncation in yeast; The wetland virus glycoprotein Gc truncation is expressed in Pichia pastoris genetically modified by N-glycosylation modification pathway to obtain a recombinant yeast cell; the Pichia pastoris genetically modified by N-glycosylation modification pathway is Pichia pastoris with inactivated α-1,6-mannosyltransferase; The expression of the wetland virus glycoprotein Gc truncation in the Pichia pastoris genetically modified by the N-glycosylation modification pathway is achieved by introducing a nucleic acid molecule encoding the wetland virus glycoprotein Gc truncation into the Pichia pastoris genetically modified by the N-glycosylation modification pathway; Step S2, fermenting and culturing the recombinant yeast cells, and purifying the wetland virus glycoprotein Gc truncation from the fermentation culture supernatant; Step S3, expressing the wetland virus glycoprotein Gc truncate in the Expi-293F mammalian cell expression system; expressing the wetland virus glycoprotein Gc truncate in the Expi-293F mammalian cell expression system is achieved by introducing a nucleic acid molecule encoding the wetland virus glycoprotein Gc truncate into the Expi-293F mammalian cell expression system; Step S4, culturing the Expi-293F mammalian cells in suspension, and purifying the wetland virus glycoprotein Gc truncation from the suspension cell culture supernatant; the purification includes the step of subjecting the suspension cell culture supernatant to nickel ion affinity chromatography.

[0011] Furthermore, in the preparation method, the nucleic acid molecule encoding the wetland virus glycoprotein Gc truncation is any of the following: a1, a DNA molecule with a nucleotide sequence as shown in SEQ ID No.2 or positions 1 to 1575 of SEQ ID No.2; a2, a DNA molecule that hybridizes with the DNA molecule defined by a1 under stringent conditions and encodes the truncated form of the wetland virus glycoprotein Gc; 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 by a1 or a2 and encodes the wetland virus glycoprotein Gc truncation.

[0012] According to the fifth aspect of the technical solution of the present invention, there is provided an application of the wetland virus glycoprotein Gc as described above in preparing an immune product that can cause an animal to produce antigen-specific antibodies or an application of the above-mentioned biological material in preparing an immune product that can cause an animal to produce antigen-specific antibodies. In the application, the immune product is a vaccine. The vaccine is immunized by intramuscular injection or subcutaneous injection.

[0013] 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 WELV glycoprotein Gc truncation (Gc subunit truncation). The constructed expression vector can realize the expression of WELV glycoprotein Gc truncation (Gc subunit truncation) in mammalian cells and yeast cells, and has the characteristics of fast growth and high safety.

[0014] 2. The wetland virus (WELV) glycoprotein Gc truncation (Gc subunit truncation) obtained by the recombinant expression of the present invention does not have the problem of excessive glycosylation, and can produce high titer anti-WELV glycoprotein Gc truncation (Gc subunit truncation) specific IgG antibodies after immunizing mice with it, and can protect mice from the lethal attack of WELV after booster immunization. Therefore, the recombinantly prepared WELV Gc subunit truncation subunit candidate vaccine can provide a better protective response and provide a reference for the development of WELV subunit vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A-1B They are schematic diagrams of the structures of the recombinant WELV glycoprotein Gc subunit truncation (Gc subunit truncation); Figure 1A This is a schematic diagram of the structure of the Gc subunit truncation of WELV (Gc subunit truncation). Figure 1B The three-dimensional structure prediction of the Gc subunit truncated form of WELV.

[0016] Figure 2A-2C They are the identification results of the protein expression changes of the recombinant WELV glycoprotein Gc subunit truncations over time in the Expi 293F expression system; Figure 2A is the curve showing the change of cell viability over time after transfection; Figure 2B SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is the protein expression over time. Figure 2C This is the detection result of Anti-His tag monoclonal antibody (Sigma, A7058).

[0017] Figure 3A-3C They are nickel affinity chromatography, SDS-PAGE and WB identification of the recombinant WELV glycoprotein Gc subunit truncated sample; Figure 3A is the nickel ion affinity chromatography chromatogram; Figure 3B SDS-PAGE of samples from the purification process; Figure 3C For WB identification of samples during purification.

[0018] Figure 4 SDS-PAGE of the purified sample of the truncated recombinant WELV glycoprotein Gc subunit.

[0019] Figure 5This is the detection of antibody titer after intramuscular injection of recombinant WELV glycoprotein Gc subunit truncated protein into mice.

[0020] Figure 6 This is a survival curve to evaluate the protective effect of mice immunized intramuscularly with recombinant WELV glycoprotein Gc subunit truncated protein in the WELV lethal model.

[0021] Figure 7 The figure shows the weight changes of mice immunized with recombinant WELV glycoprotein Gc subunit truncated protein by intramuscular injection after challenge with a lethal dose of WELV.

[0022] In each quantitative result diagram in the above figures, ns represents P >0.05, * indicates P <0.05, ** indicates P <0.01,*** indicates P <0.001, **** indicates P <0.0001. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] The present invention provides a vaccine of wetland virus glycoprotein Gc and its application. Wetland virus (WELV) belongs to a new species of the genus Orthovirus of the family Neoviridae. The wetland virus glycoprotein Gc is a recombinant wetland virus glycoprotein Gc. Further, the present invention also provides a wetland virus (WELV) vaccine, wherein the antigen wetland virus (WELV) glycoprotein Gc is a truncated body; the amino acid sequence of the truncated body of the glycoprotein Gc of the wetland virus (WELV) is shown in SEQ ID No. 1. After immunizing mice with the vaccine of the present invention, high-titer antibodies against the truncated body of the glycoprotein Gc of the wetland virus (WELV) can be produced, and the WELV true virus can be neutralized. After the vaccine prepared by the present invention is used to boost immunization of mice, the mice can be 100% protected from the threat of death at a completely lethal dose of the wetland virus (WELV). The recombinant WELV glycoprotein Gc truncated body subunit candidate vaccine prepared by the present invention 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 wetland virus (WELV) subunit vaccines.

[0026] In one embodiment, the present invention provides a recombinant glycoprotein Gc of wetland virus (WELV). Furthermore, the antigen in the wetland virus (WELV) vaccine is a truncated form of the glycoprotein Gc of WELV. The recombinant glycoprotein Gc comprises a polypeptide having an amino acid sequence shown in SEQ ID NO.1.

[0027] The glycoprotein Gc truncation of the wetland virus (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 525 of SEQ ID No.1; A2, a protein having the same function as that of the amino acid sequence defined in A1 after one or more amino acid residues are substituted and / or deleted and / or added; 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 to that of 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 protein defined in A1-A3.

[0028] In the above proteins, 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 and / 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 and / or a SUMO tag, etc.

[0029] 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 BLAST2.1, 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 obtained.

[0030] 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.

[0031] In another embodiment, a vaccine containing recombinant wetland virus (WELV) glycoprotein Gc is provided, wherein the wetland virus (WELV) vaccine further comprises an adjuvant, wherein the adjuvant may be an aluminum adjuvant and / or a CpG adjuvant.

[0032] Furthermore, the aluminum adjuvant may be an aluminum hydroxide adjuvant.

[0033] Furthermore, the CpG adjuvant may be any of the following: (1) CpG2006 or its thiolated product whose nucleotide sequence is SEQ ID No.5; (2) CpG1018 or its thiolated product whose nucleotide sequence is SEQ ID No.6; (3) CpG684 or its thiolated product whose nucleotide sequence is SEQ ID No. 7; Wherein, the thiolated product may be a full-chain thiolated modified product.

[0034] In a specific embodiment of the present invention, the wetland virus (WELV) vaccine is composed of the wetland virus (WELV) glycoprotein Gc truncation, the aluminum adjuvant and the CpG adjuvant; wherein the mass ratio of the WELV glycoprotein Gc truncation, the aluminum adjuvant and the CpG adjuvant is (2.5-20): 100: 50. The amount of the aluminum adjuvant is calculated based on the aluminum content therein.

[0035] In another specific embodiment of the present invention, the wetland virus (WELV) vaccine is composed of the wetland virus (WELV) glycoprotein Gc truncation and the aluminum adjuvant; wherein the mass ratio of the WELV glycoprotein Gc truncation and the aluminum adjuvant is (2.5-20):100.

[0036] Furthermore, the mass ratio of the WELV glycoprotein Gc truncate and the aluminum adjuvant is 10:100.

[0037] Wherein, the amount of the aluminum adjuvant is calculated based on the aluminum content therein.

[0038] According to the third aspect of the technical solution of the present invention, a method for preparing the above-mentioned recombinant WELV glycoprotein Gc truncate is provided, the method comprising the following steps: Step S1, expressing the wetland virus glycoprotein Gc truncation in Pichia pastoris.

[0039] Step S2, expressing the WELV glycoprotein Gc truncation in the Expi-293F mammalian cell expression system to obtain the expression supernatant.

[0040] Furthermore, expressing the WELV glycoprotein Gc truncation in the Expi-293F mammalian cell and Pichia pastoris expression system is achieved by introducing a nucleic acid molecule encoding the WELV glycoprotein Gc truncation into the Expi-293F mammalian cell expression system and Pichia pastoris.

[0041] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0042] Furthermore, the nucleic acid molecule encoding the WELV glycoprotein Gc truncation may be any of the following: a1, a DNA molecule whose nucleotide sequence is shown as positions 1 to 1575 of SEQ ID No. 2; a2, a DNA molecule that hybridizes with the DNA molecule defined by a1 under stringent conditions and encodes the WELV glycoprotein Gc truncation; 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 by a1 or a2 and encodes a truncated form of the Gc protein of the WELV.

[0043] 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.

[0044] In the above nucleic acid molecules, the identity of the nucleotide sequence is determined using an identity search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, 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 nucleotide sequences, the identity value (%) can be obtained.

[0045] In the above nucleic acid molecules, the above 95% identity may be at least 96%, 97%, 98% identity. The above 90% identity may be at least 91%, 92%, 93%, 94% identity. The above 85% identity may be at least 86%, 87%, 88%, 89% identity. The above 80% identity may be at least 81%, 82%, 83%, 84% identity.

[0046] Step S3, fermenting and culturing the recombinant yeast cells, and purifying the glycoprotein Gc truncated form of the WELV from the fermentation culture supernatant.

[0047] Furthermore, during the fermentation culture, methanol is used to induce the expression of the WELV glycoprotein Gc truncate. In a specific embodiment of the present invention, the recombinant yeast cells are inoculated into BMGY medium, cultured in a shaking incubator at 25°C and 200rpm, and after 24-48 hours, 0.5% (V / V) methanol is added every 12 hours for induction for 48 hours.

[0048] Step S4, culturing the transfected Expi-293F mammalian cells in suspension, and purifying the WELV glycoprotein Gc truncation from the culture supernatant.

[0049] Furthermore, the expression of the WELV glycoprotein Gc truncated form during the transfection culture is performed. In a specific embodiment of the present invention, the culture is performed in a shaking incubator at 37°C and 125 rpm, and the samples are collected after 48 hours.

[0050] Furthermore, the purification includes subjecting the transfection culture supernatant to nickel ion affinity chromatography (ChelatingFF column).

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

[0052] In another embodiment, an immune vaccine is provided, wherein the vaccine comprises the above-mentioned recombinant WELV glycoprotein Gc truncation, or the above-mentioned composition containing the recombinant WELV glycoprotein Gc truncation.

[0053] In another embodiment, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises the above-mentioned WELV glycoprotein Gc truncation, or the above-mentioned composition containing the WELV glycoprotein Gc truncation.

[0054] In another embodiment, a biomaterial is provided, wherein the biomaterial is any one of the following: C1, a nucleic acid molecule encoding the above-mentioned WELV glycoprotein Gc truncation; 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.

[0055] In another embodiment, the present invention provides any of the following applications: D1, use of the above protein or the above biological material in preparing the above WELV vaccine; D2. Use of the above-mentioned WELV vaccine or the above-mentioned protein or the above-mentioned biological material in the preparation of a product for preventing and / or treating Congo hemorrhagic fever / WELV; D3, use of the above WELV vaccine or the above protein or the above biological material in the preparation of a product for alleviating hemorrhagic fever / WELV symptoms; D4, use of the above-mentioned WELV vaccine or the above-mentioned protein or the above-mentioned biological material in the preparation of a product for preventing WELV infection; D5. Use of the above-mentioned WEL vaccine or the above-mentioned protein or the above-mentioned biological material in the preparation of a product for neutralizing WELV.

[0056] In the present invention, the WELV strain can be obtained from the New England Journal of Medicine A New Orthonairovirus Associated with Human Febrile Illness. N Engl J Med.2024;391:821-31.

[0057] The pCAGGS mammalian expression vector was purchased from Aonogen, catalog number HG-VPA0057.

[0058] The Expi-293F mammalian expression system was purchased from Thermo Fisher Scientific Inc., catalog number A14635CN.

[0059] Anti-His tag mouse monoclonal antibody was purchased from Sigma, catalog number A7058.

[0060] Chelating Sepharose FF chromatography medium was from Cytiva, catalog number 17057502.

[0061] BALB / c mice (strain code: 211) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0062] In the following embodiments, the virus is specifically a wetland virus (WELV), which is recorded in the document "NCBI (National Center for Biotechnology Information, (https: / / www.ncbi.nlm.nih.gov / ): A New Orthonairovirus Associated with Human Febrile Illness. N Engl J Med. 2024 Sep5;391(9):821-831. doi: 10.1056 / NEJMoa2313722". 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.

[0063] SEQ ID No.1, Gc truncation amino acid sequence (525AA) is as follows: .

[0064] SEQ ID No.2, the coding gene sequence corresponding to the Gc truncation (1575 bp) is as follows:

[0065] SEQ ID No. 3, Gc truncation amino acid sequence + HIS tag (1-536 AA) is as follows: .

[0066] SEQ ID No.4, the coding gene sequence corresponding to the Gc truncation + HIS tag (1608 bp) is as follows:

[0067] The following are the CpG sequences of SEQ ID No. 5-SEQ ID No. 7: SEQ ID No. 5, CpG2006 is as follows: tcgtcgtttt gtcgttttgt cgtt; SEQ ID No. 6, CpG1018 is as follows: tgactgtgaa cgttcgagat ga; SEQ ID No. 7, CpG684 is as follows: tcgacgttcg tcgttcgtcg ttc.

[0068] 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.

[0069] 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.

[0070] Example 1. Construction of mammalian expression vector for Gc subunit truncations.

[0071] (1) Obtaining the WELV glycoprotein Gc truncation (Gc subunit truncation) gene and constructing a mammalian expression vector.

[0072] According to the M fragment sequence of WELV published in GenBank: OR860408, the amino acids 1 to 525 (F1-S525) of the Gc protein were selected, a codon DNA sequence was artificially designed, and a GGGGS connecting peptide and 6 HIS tags were added, 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 EcoRI restriction site, BM40 signal peptide, fragment codon DNA sequence shown in SEQ ID No. 4, 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.

[0073] 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. 4. SEQ ID No. 4 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. 1. Positions 1-536 of SEQ ID No. 3 are Gc subunit truncation proteins. Figure 1A This is a schematic diagram of the structure of the Gc subunit truncation of WELV (Gc subunit truncation) (the green part represents the extracellular domain, the gray part represents the transmembrane region, and the white part represents the intracellular domain). Figure 1B The three-dimensional structure prediction of the truncated Gc subunit of WELV (the blue part indicates the part involved in the present invention, and the gray part indicates truncation).

[0074] (2) Extraction of the recombinant expression vector pCAGGS-Gc subunit truncated plasmid (Tiangen Endotoxin-Free Mini-Preparation Medium Kit, Beijing, DP118-02).

[0075] Example 2: Expression and purification of recombinant Gc subunit truncated glycoprotein.

[0076] (2.1) Transfection of Expi293F suspension cells.

[0077] The plasmid identified in Example 1 (i.e., pCAGGS-Gc subunit truncated form) was selected and transfected into cells at a cell density of 3×10 6 cells / mL of Expi293F suspension cells. The transfection system is shown in Table 1 below (taking 100 mL transfection volume as an example, the remaining volumes are scaled up in equal proportions).

[0078] Table 1 Transfection system

[0079] 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%.

[0080] The results are as follows Figure 2A-2C As shown, Figure 2A It indicates the change of cell viability over time after cell transfection; Figure 2B SDS-PAGE showing the expression of the target protein over time. Figure 2C WB detection showing changes in target protein expression over time. Figure 2AThe results showed that 60 hours after cell transfection, the cell viability had dropped to 60%-70%, and protein expression should be stopped at this time. Figure 2B and Figure 2C The expression of Gc protein increased with time. Taking into account factors such as cell viability and protein yield, we decided to select 60 hours after cell transfection as the protein expression endpoint.

[0081] (2.2) Purification of Gc subunit truncation.

[0082] 1. Nickel ion affinity chromatography.

[0083] The culture supernatant from step 1 inducing expression for 60 hours was dialyzed overnight to replace the system, and the replacement system was 20mM pH7.5 Tris-HCl+500mM NaCl pH 7.5.

[0084] A: 20mM pH7.5 Tris-HCl + 500mM NaCl + 5mM imidazole pH 7.5; B: 20 ​​mM pH 7.5 Tris-HCl + 500 mM NaCl + 500 mM imidazole pH 7.5.

[0085] After loading, balance with A, and then elute with B at 5%-10%-25%-50%-100% respectively.

[0086] Purify samples such as Figure 3A-3C shown. Figure 3A represents the nickel ion affinity chromatography chromatogram; Figure 3B Represents SDS-PAGE of samples during purification; Figure 3C Indicates WB identification of samples during purification. Figure 3B and Figure 3C The results showed that the target protein was eluted at imidazole concentrations of 125 and 250 mM, and the target protein was well separated from the impurities (no obvious impurities were observed in the lanes). The fractions containing the target protein (25% B, 50% B) were concentrated by ultrafiltration to replace the system and then the next step of the experiment could be carried out. Figure 4 This is the SDS-PAGE of the purified Gc protein.

[0087] Example 3: Mouse immunization experiment.

[0088] The specific immunization method is 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. The vaccine used was 2.5 μg Gc subunit truncation / 100 μg Al(OH)3 (CRODA, Denmark, the same below) / 50 μg CpG2006. The Gc subunit truncation was the recombinant WELV glycoprotein Gc subunit truncation expressed by Expi-293F prepared above, and the vaccine was prepared with saline in a volume of 100 μl containing 10 μg Gc subunit truncation, 100 μg Al(OH)3 and 50 μg CpG 2006.

[0089] Immunization group 2 (n = 6): 100 μl of vaccine was injected intramuscularly on days 0 and 21. The vaccine used was 10 μg Gc subunit truncation / 100 μg Al(OH)3 / 50 μg CpG2006. The Gc subunit truncation was the recombinant WELV glycoprotein Gc subunit truncation expressed by Expi-293F prepared above, and the vaccine was prepared with saline in a volume of 100 μl containing 2.5 μg Gc subunit truncation, 100 μg Al(OH)3 and 50 μg CpG2006.

[0090] 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.

[0091] Immunization group 4 (n = 6), i.e., blank control group: 100 μl of normal saline was injected intramuscularly on days 0 and 21, respectively.

[0092] The nucleotide sequence of CpG2006 is SEQ ID No. 5 and the whole chain is thiolated. If necessary, CpG2006 can also be replaced by any of the following: CpG1018 or its thiolated product having a nucleotide sequence of SEQ ID No. 6; CpG684 or its thiolated product having a nucleotide sequence of SEQ ID No. 7 (such as whole chain thiolated).

[0093] 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.

[0094] The specific IgG antibody titer against the Gc subunit truncated form 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.

[0095] The results are as follows Figure 5 As shown: the antibody titers of the first three-week immunization group and the second three-week immunization group were significantly higher than those of the adjuvant control group ( P <0.01). There was no statistical difference in the specific antibody titer between different doses of Gc antigen after each immunization. However, there was a significant difference in the specific antibody titer between the first and second immunizations at a dose of 2.5 μg Gc antigen ( P <0.05), there was a significant difference in the specific antibody titer in mice three weeks after the first immunization and three weeks after the second immunization with a dose of 10 μg Gc antigen ( P <0.01). At the same time, three weeks after the second immunization, the specific antibody titers of all antigen immune groups were greater than 1:100000, and the subunit vaccine effectively stimulated the immune response in mice.

[0096] Example 4, real virus challenge test.

[0097] 1) Vero cells in DMEM medium containing 10% FBS were mixed and counted and plated into 24-well plates, with 1×10 cells per well. 5 cells / well. After the cells have attached to the wall for 12 h, they are inoculated with viruses; 2) The cell culture medium was discarded, and the serum of the mouse to be tested (the serum obtained three weeks after the three immunizations in Example 3) and WELV virus were diluted with DMEM medium. The diluted virus (preferably, the titer is 5×10 3 FFU / mL) 100 μL / well and 100 μL / well of diluted serum were mixed and incubated at 37°C for 1.5 h. The process disclosed in “A New Orthonairovirus Associated with Human Febrile Illness. N Engl J Med. 2024;391:821-31” can be used; 3) 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 minutes to fix the cells. Wash the formaldehyde 4-5 times with 1×PBST; 6) Discard the 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. After immunizing rabbits with WELV NP protein, obtain anti-NP protein antiserum (preferably diluted with 0.05% PBST, dilution is 1:2000), incubate for 1.5 hours, and wash three times with 0.05% PBST; 9) Incubate with anti-rabbit HRP-IgG antibody (preferably diluted in 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.

[0098] The results are as follows Figure 6 , Figure 7 shown. Figure 6 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 Gc dose group and the 10 μg Gc dose group were able to completely protect mice under the lethal dose of WELV. Figure 7 The following is the weight change curve of mice in each group after virus attack. The results showed that compared with the 2.5 μg Gc dose group, the weight fluctuation of mice in the 10 μg Gc dose group was smaller, less than 5%. This suggests that high-dose immunization with Gc antigen can better protect mice from WELV attack.

[0099] 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 Gc, characterized in that: The wetland virus glycoprotein Gc is a recombinant wetland virus glycoprotein Gc, and the wetland virus glycoprotein Gc is a glycoprotein truncation with a C-terminus truncated.

2. The wetland virus glycoprotein Gc according to claim 1, characterized in that: The wetland virus glycoprotein Gc truncation is any of the following proteins: A1, a protein comprising the amino acid sequence as shown in positions 1 to 525 of SEQ ID No. 1; A2, a protein having the same function as that of the amino acid sequence defined in A1 after one or more amino acid residues are substituted and / or deleted and / or added; 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 to that of 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 protein 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 Gc 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 Gc according to any one of claims 1-2.

5. The product according to claim 4, characterized in that: The product is an immune composition or a pharmaceutical composition.

6. The product according to claim 5, characterized in that: The immunological composition is a vaccine, or the vaccine further comprises an adjuvant.

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

8. The product according to claim 7, characterized in that: The aluminum adjuvant comprises aluminum hydroxide adjuvant; or the CpG adjuvant is any one of the following: (1) CpG2006 or its thiolated product whose nucleotide sequence is SEQ ID No.5; (2) CpG1018 or its thiolated product whose nucleotide sequence is SEQ ID No.6; (3) CpG684 or its thiolated product whose nucleotide sequence is SEQ ID No. 7; The thiolated product is a full-chain thiolated modified product.

9. The product according to claim 6, characterized in that: The vaccine is composed of a C-terminally truncated glycoprotein truncation, an aluminum adjuvant and a CpG adjuvant; wherein the mass ratio of the C-terminally truncated glycoprotein truncation, the aluminum adjuvant and the CpG adjuvant is 10(2.5):100:50; and the amount of the aluminum adjuvant is calculated based on the aluminum content therein.

10. A method for preparing wetland virus glycoprotein Gc according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: Step S1, expressing the wetland virus glycoprotein Gc truncation in yeast; The wetland virus glycoprotein Gc truncation is expressed in Pichia pastoris genetically modified by N-glycosylation modification pathway to obtain a recombinant yeast cell; the Pichia pastoris genetically modified by N-glycosylation modification pathway is Pichia pastoris with inactivated α-1,6-mannosyltransferase; The expression of the wetland virus glycoprotein Gc truncation in the Pichia pastoris genetically modified by the N-glycosylation modification pathway is achieved by introducing a nucleic acid molecule encoding the wetland virus glycoprotein Gc truncation into the Pichia pastoris genetically modified by the N-glycosylation modification pathway; Step S2, fermenting and culturing the recombinant yeast cells, and purifying the Gc protein truncation of the wetland virus from the fermentation culture supernatant; Step S3, expressing the wetland virus glycoprotein Gc truncate in the Expi-293F mammalian cell expression system; expressing the wetland virus glycoprotein Gc truncate in the Expi-293F mammalian cell expression system is achieved by introducing a nucleic acid molecule encoding the wetland virus glycoprotein Gc truncate into the Expi-293F mammalian cell expression system; Step S4, culturing the Expi-293F mammalian cells in suspension, and purifying the wetland virus glycoprotein Gc truncation from the suspension cell culture supernatant; the purification includes the step of subjecting the suspension cell culture supernatant to nickel ion affinity chromatography.

11. The preparation method according to claim 10, characterized in that: The nucleic acid molecule encoding the wetland virus glycoprotein Gc truncation is any of the following: a1, a DNA molecule with a nucleotide sequence as shown in SEQ ID No.2 or positions 1 to 1575 of SEQ ID No.2; a2, a DNA molecule that hybridizes with the DNA molecule defined by a1 under stringent conditions and encodes the truncated form of the wetland virus glycoprotein Gc; 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 by a1 or a2 and encodes the wetland virus glycoprotein Gc truncation.

12. Use of the wetland virus glycoprotein Gc as described in any one of claims 1 to 2 in the preparation of an immune product that can induce animals to produce antigen-specific antibodies, or use of the biological material as described in claim 3 in the preparation of an immune product that can induce animals to produce antigen-specific antibodies.

13. The use according to claim 12, characterized in that: The immune product is a vaccine.

14. The use according to claim 13, characterized in that: The vaccine is administered by intramuscular or subcutaneous injection.

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