Marburg gp protein mutant, dna molecule, recombinant vector and use thereof

Abstract

The application provides a Marburg GP protein mutant, a DNA molecule, a recombinant vector and application thereof, and belongs to the technical field of Marburg virus vaccine preparation. The Marburg GP protein mutant is obtained by site mutation on the basis of a wild-type Marburg GP protein, and contains at least one mutation selected from R575P, V576P and L585P. The amino acid sequence of the wild-type Marburg GP protein is shown as SEQ ID NO:1. The Marburg GP mutant can provide a stable GP pre-fusion conformation, and can induce stronger binding activity, neutralizing activity and protective antibodies in mice, and is expected to be used for preparation of a Marburg virus vaccine.

Description

Technical Field

[0001] This invention belongs to the field of Marburg virus vaccine preparation technology, and particularly relates to a Marburg GP protein mutant, DNA molecule, recombinant vector and its application. Background Technology

[0002] Marburg virus disease (MVD) is a highly fatal hemorrhagic fever caused by the Marburg virus (MARV), posing a serious threat to public health and safety with the potential to cause large-scale epidemics. Since its initial discovery in 1967, the disease has mainly occurred in localized outbreaks in certain areas, with some large-scale epidemics and a case fatality rate as high as 88%. In recent years, Marburg outbreaks have also occurred in several other locations.

[0003] The World Health Organization has announced four candidate vaccines for Marburg virus (MARV), all of which are currently in clinical or preclinical research stages. Only the chimpanzee adenovirus vector vaccine, ChAd3, has completed Phase I clinical trials. Most reported MARV neutralizing antibodies have been isolated from survivors of MARV infection, and in very small quantities.

[0004] Marburg virus belongs to the Filoviridae family. Under an electron microscope, it appears as a filamentous structure with a genome length of approximately 19 kb, encoding seven structural proteins: NP, VP, GP, and the polymerase fragment L. The GP protein is the only protein exposed on the surface of the Marburg virus, mediating viral adhesion and cell invasion, and is a key focus of research on neutralizing antibodies and vaccines. The GP protein consists of 681 amino acids, which are cleaved by the host furin protease into two subunits, GP1 and GP2, linked by disulfide bonds, existing as a homotrimer outside the viral envelope. During cell invasion, the GP protein undergoes a conformational change. GP1 binds to the host cell receptor, and then GP2 forms a stable six-helix bundle structure, drawing the virus closer to the host cell membrane and causing fusion, thus undergoing a conformational change.

[0005] Currently, the wild-type Marburg virus GP protein has low immunogenicity and poor effectiveness as a vaccine target. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a Marburg GP mutant, a DNA molecule, a recombinant vector and its applications, wherein the Marburg GP mutant can provide a stable pre-fusion conformation of GP and can induce mice to produce stronger binding activity, neutralizing activity and protective antibodies, and is expected to be used in the preparation of Marburg virus vaccines.

[0007] This invention provides a Marburg GP protein mutant, obtained by site mutation based on wild-type Marburg GP protein, comprising at least one mutation selected from R575P, V576P and L585P; the amino acid sequence of the wild-type Marburg GP protein is shown in SEQ ID NO: 1.

[0008] Preferably, relative to wild-type Marburg GP protein, it also includes at least one of the following modifications:

[0009] 1) Remove amino acids 638-681 from the amino acid sequence corresponding to the wild-type Marburg GP protein;

[0010] 2) Remove amino acids 264-425 from the amino acid sequence corresponding to the wild-type Marburg GP protein.

[0011] Preferably, the amino acid sequence of the Marburg GP protein mutant is shown in SEQ ID NO: 2~SEQ ID NO: 4.

[0012] This invention provides a DNA molecule encoding the Marburg GP protein mutant.

[0013] This invention provides an mRNA molecule obtained by transcription of the aforementioned DNA molecule.

[0014] The present invention provides a recombinant vector for expressing the Marburg GP protein mutant, comprising the DNA molecule and the initial vector.

[0015] Preferably, the initial vector is the insect cell expression vector pMT / BiP / V5-His B.

[0016] The present invention provides a recombinant cell, wherein the recombinant vector is transferred into a host cell, wherein the host cell is an S2 insect cell.

[0017] This invention provides the use of the Marburg GP protein mutant, the DNA molecule, the mRNA molecule, the recombinant vector, and the recombinant cell in the preparation of agents for the prevention and / or treatment of Marburg virus disease.

[0018] Preferably, the formulation includes a vaccine.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The Marburg GP protein mutant provided by this invention is obtained by site mutation of the wild-type Marburg GP protein, including at least one mutation selected from R575P, V576P, and L585P. The modified mutant can be efficiently expressed in the S2 insect cell expression system and retains important neutralizing and protective antibody epitopes from the pre-fusion conformation. The GP Marburg mutant maintains good binding activity with neutralizing antibodies MR78 and MR191, and protective antibody MR228. Two immunizations of mice with the GP mutant induce high levels of GP-specific binding antibodies and neutralizing antibodies. Competitive ELISA experiments further demonstrate that immunization of mice with the Marburg GP protein mutant induces higher levels of antibodies with epitopes similar to the protective antibody MR228. These findings indicate that the Marburg GP protein mutant of this invention has the potential to serve as an effective candidate antigen for a Marburg virus vaccine. Attached Figure Description

[0021] Figure 1 Design diagram for truncating GP antigen sequence;

[0022] Figure 2 Schematic diagram for designing Marburg GP protein mutants;

[0023] Figure 3 Gel electrophoresis image of the Marburg GP protein mutant;

[0024] Figure 4 The purpose of this study was to detect the binding activity of GP protein mutants to antibodies using an enzyme-linked immunosorbent assay (ELISA), where A is MR228, B is MR78, and C is MR191.

[0025] Figure 5 To detect binding antibodies in the serum of immunized mice by enzyme-linked immunosorbent assay (ELISA), five groups were obtained by immunizing wild-type Marburg GP protein, R575P mutant, V576P mutant, L585P mutant, and PBS, respectively.

[0026] Figure 6 To detect binding antibodies in the serum of immunized mice by flow cytometry, the five groups were obtained by immunizing wild-type Marburg GP protein, R575P mutant, V576P mutant, L585P mutant, and PBS, respectively.

[0027] Figure 7 To assess the neutralizing capacity of mouse serum after immunization, the five groups were immunized with wild-type Marburg GP protein, R575P mutant, V576P mutant, L585P mutant, and PBS, respectively.

[0028] Figure 8The content of antibodies similar to the MR228 epitope in the serum of mice after immunization is shown in the five groups, which are the results of immunization with wild-type Marburg GP protein, R575P mutant, V576P mutant, L585P mutant and PBS, respectively.

[0029] Figure 9 To determine the competitive rate of immunized mouse serum inhibiting MR228 binding at a 1:30 dilution, the five groups were immunized with wild-type Marburg GP protein, R575P mutant, V576P mutant, L585P mutant, and PBS, respectively. Detailed Implementation

[0030] This invention provides a Marburg GP protein mutant, obtained by site mutation of the wild-type Marburg GP protein, comprising at least one mutation selected from R575P, V576P, and L585P; the amino acid sequence of the wild-type Marburg GP protein is shown in SEQ ID NO: 1, and is as follows:

[0031] MKTTCLLISLILIQGVKTLPILEIASNIQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRAGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAF FLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGTLQEYNSTKNQTCAPSKKPLPLPTAHPEVKLTSTSTDATKLNTTDPNSDDEDLTTSGSGSGEQEPYTTSDAATK QGLSSTMPPTPSPQPSTPQQGGNNTNHSQGVVTEPGKTNTTAQPSMPPHNTTTISTNNTSKHNLSTPSVPIQNATNYNTQSTAPENEQTSAPSKTTLLPTENPTTAKSTNSTKSPTTTVPNTTNKYSTSPSPTPNSTAQHLVY FRRKRNILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPKVNENTAHSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQTAKSLELLL RV TTEERTFS LINRHAIDFLLARWGGTCKVLGPDCCIGIEDLSRNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG* (where the bold underlined part indicates the mutation site).

[0032] This invention introduces proline mutations at key conformational change sites in wild-type Marburg GP protein in order to induce higher levels of GP-specific binding antibodies.

[0033] Preferably, the Marburg GP protein mutant of the present invention, relative to the wild-type Marburg GP protein, further includes at least one of the following changes: 1) removal of amino acids 638 to 681 corresponding to the amino acid sequence of the wild-type Marburg GP protein; 2) removal of amino acids 264 to 425 corresponding to the amino acid sequence of the wild-type Marburg GP protein.

[0034] In this invention, amino acids 638-681 of the wild-type Marburg GP protein sequence constitute a transmembrane region; amino acids 264-425 constitute a partial mucin-like domain. This invention reduces structural complexity and improves the soluble expression of the Marburg GP protein mutant by removing this structure. In this invention, the C-terminus of the Marburg GP protein mutant is preferably fused with a Twin-Strep-tag tag, which facilitates purification.

[0035] In this invention, the preferred amino acid sequence of the Marburg GP protein mutant is shown in SEQ ID NO: 2~SEQ ID NO: 4.

[0036] SEQ ID NO: 2

[0037] MKTTCLLISLILIQGVKTLPILEIASNIQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRAGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGTLQEYNSTKNQTCAPSKKPLPLPTAHPEVKLTSTSTDATKLNTTDPNSDDQHLVYFRRKRNILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPKVNENTAHSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQTAKSLELLL PV TTEERTFS L INRHAIDFLLARWGGTCKVLGPDCCIGIEDLSRNISEQIDQIKKDEQKEGTG

[0038] SEQ ID NO:3

[0039] MKTTCLLISLILIQGVKTLPILEIASNIQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRAGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGTLQEYNSTKNQTCAPSKKPLPLPTAHPEVKLTSTSTDATKLNTTDPNSDDQHLVYFRRKRNILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPKVNENTAHSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQTAKSLELLL RP TTEERTFSL INRHAIDFLLARWGGTCKVLGPDCCIGIEDLSRNISEQIDQIKKDEQKEGTG

[0040] SEQ ID NO: 4

[0041] MKTTCLLISLILIQGVKTLPILEIASNIQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRAGVPPKNVEYTEGEEAKTCYNISVTDPSGK SLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQT NDTGCFGTLQEYNSTKNQTCAPSKKPLPLPTAHPEVKLTSTSTDATKLNTTDPNSDDQHLVYFRRKRNILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPKVNENTAHSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQTAKSLELLL RV TTEERTFS P INRHAIDFLLARWGGTCKVLGPDCCIGIEDLSRNISEQIDQIKKDEQKEGTG

[0042] This invention also provides a DNA molecule encoding the Marburg GP protein mutant. The specific sequence of the DNA molecule is not particularly limited, as long as it can encode the corresponding Marburg GP protein mutant.

[0043] In this invention, the DNA molecule is a DNA molecule optimized with insect cell codons, which is more conducive to the expression of the Marburg GP protein mutant in insect cells.

[0044] The present invention provides an mRNA molecule obtained by transcription of the aforementioned DNA molecule, which can be used to prepare an RNA vaccine.

[0045] This invention provides a recombinant vector for expressing the Marburg GP protein mutant, comprising the aforementioned DNA molecule and an initial vector. The recombinant vector is used to load, replicate, and express the DNA molecule. The initial vector can be a prokaryotic cell expression vector, a eukaryotic cell expression vector, or a viral vector. In one specific embodiment of this invention, the vector is the insect cell expression vector pMT / BiP / V5-His B. A specific construction strategy of this invention involves ligating a codon-optimized DNA molecule encoding the Marburg GP mutant into the pMT / BiP / V5-HisB expression vector via an enzyme restriction site.

[0046] In this invention, the initial vector is preferably the insect cell expression vector pMT / BiP / V5-His B.

[0047] The present invention also provides a recombinant cell into which the recombinant vector is transferred, wherein the host cell includes, but is not limited to, S2 insect cells.

[0048] This invention utilizes the recombinant cells to express the Marburg GP protein mutant in the S2 insect cell expression system. After purification by StrepTrap affinity chromatography and size exclusion chromatography, the Marburg GP protein mutant was successfully prepared.

[0049] This invention provides the use of the Marburg GP protein mutant, the DNA molecule, the mRNA molecule, the recombinant vector, and the recombinant cell in the preparation of agents for the prevention and / or treatment of Marburg virus disease.

[0050] In this invention, the formulation includes a vaccine; the present invention uses the Marburg GP protein mutant, aluminum adjuvant and CpG in combination to immunize mice, which can induce higher levels of GP-specific binding antibodies.

[0051] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1

[0053] Marburg virus GP protein comprises two subunits, GP1 and GP2. The pre-fusion conformational variant of the Marburg GP protein described in this invention introduces proline mutations at key conformational change sites in the GP antigen structure, based on the wild-type Marburg GP protein. The mutation sites include: R575P, V576P, and L585P proline mutations. Figure 1 ).

[0054] This embodiment selects the Angola strain GP antigen, which has the highest virulence and lethality, as the research object. The amino acid sequence of Angola-GP (QIPD50) was designed, and its transmembrane region (amino acids 638-681) was removed to facilitate protein soluble expression. Its C-terminus is equipped with a Twin-Strep-tag for easy purification. Given that the mucin-like domain contains abundant glycosylation sites, an antigen plasmid with a truncated portion of the mucin-like domain (amino acids 264-425 removed) was designed and named MARV GPΔTM ΔMuc (…). Figure 2 ).

[0055] Based on the GPΔTM ΔMuc sequence, after optimization using insect cell codons, the optimized DNA sequence was constructed into the pMT / BiP / V5-His B expression vectors to obtain the GPΔTM ΔMuc expression plasmids pMT-R575P, pMT-V576P, and pMT-L585P, respectively.

[0056] After obtaining the recombinant expression plasmid, the GP antigen was expressed using the S2 insect cell expression system. The protein was purified using a strepTrap affinity chromatography column selected based on the Twin-Strep-tag label, and further purified by size exclusion chromatography (SEC) using a Superdex200 Increase 10 / 300 GL column (Cytiva). SDS-PAGE analysis showed that all mutants exhibited GP bands. Figure 3 This indicates that the GP mutant protein was successfully expressed and purified.

[0057] Example 2

[0058] To assess the impact of mutation introduction on GP antigen epitope exposure, this embodiment used specific neutralizing antibodies MR78 and MR191 with different binding angles and protective antibody MR228 (which targets the unique "Wing" region of Marburg virus and provides complete protection in mouse infection experiments) to detect the level of specific antibodies in mouse immune serum by enzyme-linked immunosorbent assay.

[0059] All experiments were conducted in parallel with wild-type and mutant mice. Mice were immunized with wild-type Marburg GP protein, R575P mutant protein, V576P mutant protein, L585P mutant protein, and PBS, respectively. Among them, R575P mutant protein, V576P mutant protein, and L585P mutant protein were proteins prepared in Example 1 with the transmembrane region (amino acids 638-681) removed and a portion of the mucin-like domain (amino acids 264-425 removed). The amino acid sequences are shown in SEQ ID NO: 2~SEQ ID NO: 4.

[0060] First, the wild-type GP antigen was diluted to 10 μg / mL using carbonate-bicarbonate buffer (pH 9.6). 100 μL of protein solution was added to each well of a 96-well high-binding microplate (Corning, USA) for coating, and the plate was incubated overnight at 4°C. The 96-well plate was washed three times with PBST (PBS + 0.1% Tween-20), and 100 μL of blocking buffer (PBST containing 2% BSA) was added to each well. The plate was incubated at 37°C for 1 h. The 96-well plate was washed three times with PBST, and diluent (PBST containing 0.2% BSA) was added. 148.5 μL was added to the first well, and 100 μL to the remaining wells. 1.5 μL of serum from a single immunized mouse was added to each well and mixed well. 50 μL of this solution was then added to the remaining wells, and the serum was serially diluted three-fold. The plates were incubated at 37°C for 1 h. After washing the 96-well plate three times, 100 μL of HRP-conjugated goat anti-mouse IgG (Abcam, UK) diluted 10,000 times in diluent was added to each well. The plate was incubated at 37°C for 1 hour, then washed three times. 100 μL of TMB single-component chromogenic buffer (Solepro Science & Technology Co., Ltd., Beijing, China) was added, and the plate was incubated at 37°C for 3 minutes. Finally, 50 μL of ELISA stop buffer (Solepro Science & Technology Co., Ltd., Beijing, China) was added to each well to stop the chromogenic process. Emissions were then measured at 450 nm and 630 nm (SPECTRAMAX 190). The OD450-630 value of the negative control was used as the standard, and a 2.1-fold increase in OD450-630 value compared to the negative serum was used as the criterion for positive serum. The experimental results were analyzed using GraphPad Prism 8.4.2, and the antibody titer in the serum of each mouse was calculated. The results showed that all mutants could effectively bind the above-mentioned antibodies (…). Figure 4 This indicates that the mutation did not disrupt the proper folding and exposure of the GP antigen neutralizing epitope and the protective epitope.

[0061] To evaluate the immunogenicity of the GP mutant, mice were immunized using the GP mutant as the antigen. Mice were randomly divided into 6 groups of 6 mice each. Each mouse was injected with 200 μL of sample (sample preparation specifications: 50 μg antigen + 500 μg aluminum adjuvant + 25 μg CPG), and 100 μL was injected intramuscularly into the left and right limbs, respectively. Mice were immunized at weeks 0 and 2. Blood was collected from the tail vein before the second immunization and two weeks after the second immunization. Serum was obtained by centrifugation at room temperature. The binding antibodies in the mouse serum were detected by ELISA using the same method described above. The results showed that the serum binding antibodies induced by the R575P, V576P, and I586P mutants were only slightly higher than those induced by the unmutated GP antigen. Figure 5 ).

[0062] To further investigate the binding ability of mouse serum to the native conformation of the GP antigen, flow cytometry was used. 4 µg of full-length pcDNA3.1-GP (native conformation) was transfected with Opti-MEM (Gibco) serum-depleted cell culture medium using TranslT-VirusGEN (Thermo Fisher) transfection reagent. After incubation at room temperature for 15 min, the transfected medium was slowly added to the cell culture medium and gently mixed. After 6 h, the medium was replaced with fresh DMEM (Gibco) complete medium. Cells were cultured at 37°C in a 5% CO2 incubator for 24 h. Cells were then collected in 50 mL centrifuge tubes, centrifuged at 300×g for 5 min, the supernatant was discarded, and the cells were resuspended in PBS containing 2% FBS. Cells were filtered through a cell strainer to remove cell clumps and counted. Cells were diluted to 5×10⁶ cells per tube. 5 100 μL of cells were transferred to flow cytometry tubes (BD). Cells were washed with 2 mL of PBS containing 2% FBS in each tube, centrifuged at 300×g for 5 min, and the supernatant was carefully discarded. 100 μL of mouse serum was added to each flow cytometry tube at a 1:100 dilution and incubated at room temperature for 1 h. Cells were washed with 2 mL of PBS containing 2% FBS in each tube, centrifuged at 300×g for 5 min, and the supernatant was carefully discarded. APC Goat Anti-Mouse IgG antibody was diluted according to the manufacturer's instructions, 100 μL per tube, and incubated at room temperature in the dark for 1 h. Cells were washed with 2 mL of PBS containing 2% FBS in each tube, centrifuged at 300×g for 5 min, and the supernatant was carefully discarded. Cells were resuspended in 200 μL of PBS containing 2% FBS in each tube and analyzed using a FACSCanto II flow cytometer. 10,000 cells were recorded per tube. The results showed that the serum of mice immunized with R575P, V576P, and I586P mutants had a better binding ability to the native conformation of GP antigen displayed on the cell membrane surface (Figure 6). The overall trend was consistent with ELISA, indicating that these proline mutations improved the ability of mice to induce GP-specific antibodies.

[0063] To detect the neutralizing activity of mouse serum immunized with mutant proteins, the neutralizing antibody content was determined using a pseudovirus neutralization assay two weeks after booster immunization. Mouse serum was inactivated in a 56°C metal bath for 30 min. The diluted serum was added to 96-well flat-bottom cell culture plates (Corning) and serially diluted with DMEM complete medium (Gibco) containing 10% FBS. After serum dilution, 50 μL of pseudovirus solution was added to each well, and the 96-well plates were incubated at 37°C in a 5% CO2 incubator for 1 h. HEK293T cells at a density of 70–80% were digested, resuspended, and counted. The digested cell suspension was then diluted at 2.5 × 10⁶ cells per well. 4 Cells were seeded into 96-well cell culture plates. After 48 h of culture, the cell culture supernatant was aspirated, fluorescent substrate was added, and the plates were incubated at room temperature in the dark for 2 min. 150 μL of the fluorescent cell lysate suspension was then added to a white flat-bottomed 96-well plate (Corning), and the fluorescence value was automatically read using a fluorescence detector. The results showed that the level of neutralizing antibodies in the serum of mice immunized with the I586P mutant protein was increased to some extent compared to WT, indicating that the I586P mutation can, to some extent, enhance the ability of GP antigen to induce the production of neutralizing antibodies in mice. Figure 7 ).

[0064] Competitive ELISA was performed on the serum of mice immunized two weeks after the second immunization. To detect the level of protective antibodies against epitopes similar to MR228 in mouse serum, HRP-labeled MR228 was used as the detection antibody in a competitive ELISA experiment. The MR228 antibody was HRP-labeled with horseradish peroxidase. First, the MR228 antibody was diluted to 2 mg / mL with PBS. 5 μL of IgG modification solution was added to 50 μL of the antibody to be labeled, and the mixture was repeatedly pipetted to ensure thorough mixing (avoiding air bubbles). The modified IgG was added to an HRP lyophilized tube (M-HRP to IgG mass ratio of 1:1), thoroughly mixed, and incubated at 25°C in the dark for 3 h. The stop solution was dissolved in water to a concentration of 2.5 mg / mL, and 5.5 μL of the stop solution was added to the lyophilized tube. After thorough mixing, the mixture was incubated at 25°C in the dark for 30 min. After termination, an appropriate amount of 5× antibody preservation solution was added to bring the final solution concentration to 1×, thoroughly mixed, and the labeled antibody was stored at 4°C for subsequent experiments. The competitive ELISA procedure was similar to that described above. Results showed that the immune sera from GP mutant mice competed for the binding of MR228 to the GP mutant protein in a dose-dependent manner. The results also showed that the immune sera from the three groups of GP antigens blocked the binding of GP to MR228 to varying degrees in a dose-dependent manner (Figure 8).

[0065] The competitive rate of serum blocking MR228 binding in each group was calculated based on the ELISA results. Compared with the WT group, the I586P group showed a stronger ability to competitively bind the protective antibody MR228 in the serum of immunized mice (Figure 9), indicating that immunization of mice with the I586P mutant group can induce higher levels of protective antibodies similar to the MR228 epitope.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Marburg GP protein mutant, characterized in that, The wild-type Marburg GP protein was obtained by site mutation, including one mutation selected from R575P, V576P and L585P; the amino acid sequence of the wild-type Marburg GP protein is shown in SEQ ID NO: 1; The amino acid sequences of the Marburg GP protein mutants are shown in SEQ ID NO: 2 to SEQ ID NO:

4.

2. A DNA molecule encoding the Marburg GP protein mutant of claim 1.

3. The mRNA molecule obtained by transcription of the DNA molecule according to claim 2.

4. A recombinant vector expressing the Marburg GP protein mutant of claim 1, characterized in that, Includes the DNA molecule and initial vector as described in claim 2.

5. The recombinant vector according to claim 4, characterized in that, The initial vector was the insect cell expression vector pMT / BiP / V5-His B.

6. A recombinant cell, characterized in that, The recombinant vector of claim 4 or 5 is introduced into a host cell, wherein the host cell is an S2 insect cell.

7. The use of the Marburg GP protein mutant of claim 1, the DNA molecule of claim 2, the mRNA molecule of claim 3, the recombinant vector of claim 4 or 5, and the recombinant cell of claim 6 in the preparation of agents for the prevention and / or treatment of Marburg virus disease.

8. The application according to claim 7, characterized in that, The preparations include vaccines.

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