Nano-antibodies against Ebola virus VP40 protein and their encoding mRNA and applications

Abstract

The present invention discloses a nano antibody against Ebola virus VP40 protein and its encoding mRNA and application, wherein the nano antibody has a unique CDR region and has excellent antigen-antibody binding activity. The present invention also discloses an mRNA molecule encoding the nano antibody, wherein the mRNA molecule loaded by lipid nanoparticles is delivered into cells, and its expression product in the cells can specifically bind to the Ebola virus VP40 in the cells and inhibit the VP40-mediated virus budding, showing the application prospect of the mRNA in the preparation of therapeutic drugs for treating Ebola virus disease.

Description

Technical Field

[0001] The invention discloses a nano antibody and its encoding mRNA, belonging to the field of protein and nucleic acid. Background Art

[0002] Ebola hemorrhagic fever is a highly contagious disease caused by the Ebola virus. Since it was first discovered in 1976, there have been more than 40 Ebola outbreaks worldwide, infecting more than 34,000 people and killing about 15,000 people. The Ebola virus is one of the most deadly viruses in the world, with a very high mortality rate after infection, posing a great threat to human health and national security.

[0003] Ebola virus belongs to the Filoviridae family. It is a non-segmented single-stranded negative-sense RNA virus with an envelope. The genome of Ebola virus is wrapped by the nucleoprotein NP, which together with polymerase L, viral proteins VP35 and VP30 form a nucleocapsid with transcription and replication functions, interacts with VP24, and is wrapped by the matrix protein VP40 attached to the inner membrane of the virus. VP40 is the most abundant protein in Ebola virus and is the most important target in the immunological detection of Ebola virus. There are currently 6 subtypes of Ebola virus reported: Zaire Ebola virus (EBOV), Sudan Ebola virus (SUDV), Bundibugyo Ebola virus (BDBV), Tai Forest Ebola virus (TAFV), Reston Ebola virus (RESTV) and Bombali Ebola virus (BOMV). Except for BOMV, the other five subtypes of Ebola virus are all capable of infecting humans. Among them, EBOV is the subtype that causes the most Ebola outbreaks, with an overall mortality rate of over 65%.

[0004] Antibody drugs have the characteristics of high targeting and high specificity, and have great application prospects in the prevention and diagnosis and treatment of infectious diseases. The vast majority of antiviral antibodies act on the surface proteins of the virus, and achieve neutralization by blocking the recognition, adhesion, and membrane fusion of the virus and host cells. For Ebola virus, most of the antibody drugs under development target the surface glycoprotein GP, ​​but the glycoprotein GP has the characteristics of complex modification and high mutation rate, so this type of antibody drug has defects such as poor broad spectrum and easy to produce drug resistance.

[0005] mRNA vaccines deliver mRNA molecules encoding protective antigens into the body, which then express the corresponding antigens in the body to stimulate the body to produce specific humoral immune responses and produce antibodies. Unlike mRNA vaccines, mRNA antibodies directly produce antibodies by delivering mRNA molecules encoding protective antibodies into the body. Through mRNA antibody technology, not only can the secretion and expression of antibodies achieve the effect of conventional neutralizing antibodies, but also antibodies that act on key targets inside cells can be designed to broaden treatment channels.

[0006] There is an antibody that naturally lacks light chains in the peripheral blood of camelids. The antibody includes a heavy chain variable region (VH) and constant regions 2 (CH2) and 3 (CH3). This structural homodimer is called a heavy chain antibody (HCAb), and its VH domain is the heavy chain antibody variable region fragment (VHH). VHH has a small molecular weight of about 15 kDa, so it is also called a nanobody. The complementary determining region 3 (CDR3) of VHH has a longer amino acid chain than that of human VH, and can form a convex loop-like structure, which can specifically bind to enzyme active sites and special epitopes of viral particles, enhancing the recognition ability of hidden antigen epitopes.

[0007] Virus budding is of great significance for virus amplification and accelerating the infection process. The key proteins of virus budding are another focus of therapeutic drug development. The Ebola virus VP40 protein has the function of mediating virus budding and release through oligomerization, and is a target with great potential for therapeutic drug development. However, VP40 is an internal protein of the viral envelope. Conventional therapeutic antibodies cannot pass through the envelope due to their large molecular weight, and therefore cannot interact with VP40. Through mRNA antibody technology, antibodies can be expressed in cells, and then interact with VP40 proteins to inhibit virus budding and achieve the effect of blocking virus spread. At present, there are no reports on mRNA molecules encoding anti-VP40 protein antibodies that can provide protection against Ebola virus infection.

[0008] In order to overcome the defects of the prior art, the purpose of the present invention is to provide a nanobody against the VP40 protein of the Ebola virus, and then to provide an mRNA encoding the antibody. By delivering the mRNA into cells, protective nanobodies against the VP40 protein can be expressed in the cells and the budding release of the Ebola virus can be blocked, thereby achieving the effect of blocking the Ebola virus from infecting surrounding cells. Summary of the invention

[0009] Based on the above objectives, the present invention first provides a nanobody against the Ebola virus VP40 protein, the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the nanobody against the Ebola virus VP40 protein are shown in positions 26-33, 51-57 and 96-112 of SEQ ID NO: 1, respectively.

[0010] The nanobody described in the present invention is an antibody with only a heavy chain, wherein the heavy chain includes a variable region and a constant region, wherein the variable region has three complementary determining regions (CDRs): CDR1, CDR2 and CDR3, which are highly variable and diverse. The sequence diversity of the CDR region determines the specificity and affinity of the antibody, because they recognize and bind to specific antigenic determinants by interacting with the antigen.

[0011] In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the nanobody against Ebola virus VP40 protein is shown in SEQ ID NO: 1. In the present invention, the nanobody having this variable region is named "NC056".

[0012] Secondly, the present invention provides a polynucleotide encoding the above-mentioned anti-Ebola virus VP40 protein nanobody. In the present invention, the polynucleotide whose sequence is shown in SEQ ID NO: 2 is a polynucleotide encoding the amino acid sequence of the nanobody NC056 heavy chain variable region shown in SEQ ID NO: 1 according to a specific embodiment. Based on the triplet code rule, any polynucleotide encoding the amino acid sequence of the nanobody NC056 heavy chain variable region shown in SEQ ID NO: 1 belongs to the polynucleotide encoding the above-mentioned anti-Ebola virus VP40 protein nanobody defined in the present invention.

[0013] Third, the present invention provides a vector containing a polynucleotide encoding the heavy chain variable region of the above-mentioned nanobody against the Ebola virus VP40 protein. The vector is used to clone and / or express the coding gene of the nanobody against the Ebola virus VP40 protein. In a specific embodiment of the present invention, the vector is pcDNA3.4. Other vectors known to those skilled in the art, especially eukaryotic cell expression vectors, can also be used for cloning and expression of the coding gene of the present invention.

[0014] Fourth, the present invention provides a host cell containing a vector of a polynucleotide encoding the heavy chain variable region of the above-mentioned nanobody against the Ebola virus VP40 protein. The host cell is used to express the above-mentioned nanobody against the Ebola virus VP40 protein. In a specific embodiment of the present invention, the host cell is an Expi293F cell. Other host cells known to those skilled in the art, especially eukaryotic host cells, can also be used for the expression of the nanobody of the present invention.

[0015] Fifth, the present invention provides the use of the above-mentioned nano-antibodies against the VP40 protein of the Ebola virus in the preparation of drugs for treating and / or preventing Ebola virus disease. The nano-antibodies provided by the present invention have excellent binding activity with the VP40 protein of the Ebola virus, and can specifically target the VP40 protein of the Ebola virus. Therefore, these characteristics of the nano-antibodies are used as specific inhibition of the binding of the Ebola virus to host cells, or, therapeutic drugs are specifically targeted to infected lesions or pathogens to play a role in clinical treatment or prevention of infection. Therefore, the present invention provides the use of the above-mentioned nano-antibodies against the VP40 protein of the Ebola virus in the preparation of drugs for treating and / or preventing Ebola virus disease.

[0016] Furthermore, the present invention also provides a detection kit containing the above-mentioned nano-antibody against the VP40 protein of the Ebola virus. Based on the excellent affinity of the nano-antibody provided by the present invention to the VP40 protein of the Ebola virus, it can be used to detect Ebola virus particles with the VP40 protein that may be present in the sample. The detection antigen is a single antibody detection, that is, the nano-antibody specifically binds to the pathogen as a primary antibody, and then the binding is detected by a secondary antibody; it can also be a combination detection of double antibodies.

[0017] Sixth, the present invention provides an mRNA molecule, which contains mRNA transcribed from the nanobody polynucleotide encoding the anti-Ebola virus VP40 protein, and the sequence of the nanobody polynucleotide encoding the anti-Ebola virus VP40 protein is shown in SEQ ID NO:2.

[0018] In a preferred embodiment, the 5'UTR sequence shown in SEQ ID NO:4 is tandemly connected to the 5' end of the mRNA coding fragment, and the 3'UTR sequence shown in SEQ ID NO:5 is tandemly connected to the 3' end of the mRNA coding fragment.

[0019] In a more preferred embodiment, the 5' end of the mRNA molecule is tandemly connected with a promoter sequence as shown in SEQ ID NO: 3, and the 3' end of the mRNA molecule is tandemly connected with a stop codon of TGATAATAG and a poly(A) of 112 nucleotides in length.

[0020] Through the above design, the present invention provides a specific embodiment of the mRNA molecule, and the sequence of the mRNA molecule is shown in SEQ ID NO:6.

[0021] In a specific embodiment of the present invention, the 5' end of the mRNA molecule further contains a Cap3011 cap structure.

[0022] Seventh, the present invention provides a lipid nanoparticle encapsulating the above-mentioned mRNA molecule.

[0023] The lipid nanoparticles are named NC056 mRNA-lipid nanoparticles in the present invention. In a specific embodiment of the present invention, the encapsulation adopts SM102 lipid formula, and the microfluidic device is Nano L+ type. The free mRNA concentration of the NC056-mRNA-lipid nanoparticles is 3.08 μg / mL, the total mRNA concentration is 110.35 μg / mL, the encapsulation rate is 97.21%, and the particle size is 89.42 nm.

[0024] Finally, the present invention provides the use of the mRNA in preparing a therapeutic drug for treating Ebola virus disease.

[0025] After the mRNA encoding the protective antibody NC056 against the VP40 protein of the Ebola virus provided by the present invention is delivered into the cell via lipid nanoparticles, its expression product can specifically bind to the VP40 protein in the cell, thereby inhibiting the VP40-mediated virus budding and blocking the release of the virus outside the cell. The present invention provides a new anti-Ebola virus treatment scheme targeting intracellular targets, which can be used as an important supplement to anti-Ebola virus infection drugs and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the SDS-PAGE identification result of the anti-Ebola virus VP40 antibody NC056, lane 1 is the marker, lane 2 is the antibody NC056;

[0027] Figure 2 This is the ELISA binding test result of anti-Ebola virus VP40 antibody NC056;

[0028] Figure 3 Schematic diagram of the construction of mRNA encoding the anti-Ebola virus VP40 antibody NC056;

[0029] Figure 4 This is the result of co-immunoprecipitation of mRNA antibody and Ebola virus VP40 protein;

[0030] Figure 5 Western blot results showing that mRNA antibodies inhibited the release of VP40 virus-like particles;

[0031] Figure 6 . Relative quantitative analysis results of mRNA antibody inhibiting the release of VP40 virus-like particles. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0033] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] Example 1. Recombinant expression and identification of anti-Ebola virus VP40 antibodies

[0035] 1. Acquisition, expression and purification of anti-Ebola virus VP40 antibodies

[0036] 1 mg of eukaryotically expressed Zaire Ebola virus VP40 protein (genbank ID: 911825) was mixed with Freund's complete adjuvant at a ratio of 1:1 and immunized alpacas at multiple points on the back subcutaneously. After booster immunization every 14 days, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant, and serum titer was monitored by ELISA. Peripheral blood was collected from alpacas, lymphocytes were separated by density gradient centrifugation, RNA was extracted and reverse transcribed into cDNA. Using cDNA as a template, the heavy chain variable region VHH gene was amplified by PCR, and the phagemid pComb3X-VHH was constructed after enzyme digestion and ligation. The phagemid was transformed into Escherichia coli XL1-Blue by electroporation, and the helper phage VCSM13 was added in the logarithmic phase. The phages were collected after overnight culture to obtain a phage display nanoantibody library against Ebola virus VP40 protein. The solid phase affinity panning method was used to select anti-Ebola virus VP40 protein nanoantibodies from the prepared antibody library. After multiple rounds of panning, a clone was selected for phage-ELISA identification, and the positive clone was sequenced to obtain the VP40-specific nanoantibody sequence named "NC056".

[0037] The sequencing results showed that the amino acid sequence of the heavy chain variable region of Nanobody NC056 was as shown in SEQ ID NO: 1, wherein the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region were as shown in positions 26-33, 51-57 and 96-112 of SEQ ID NO: 1, respectively.

[0038] The nano antibody sequence obtained by sequencing in step 1 was optimized for human codons, and the optimized sequence was shown in SEQ ID NO.2. It was fused with the human Fc protein gene (the sequence was shown in SEQ ID NO.7, wherein the 1-12 bp encoded the linker peptide) at the C-terminus, constructed into the pCDNA3.4 vector, and transfected into Expi293F cells for expression using PEI transfection reagent (Yisheng Bio, 40816ES01). On the second day of expression, 5% protein-free feed (Aupumai, F081918) and 2mM protein expression enhancer sodium valproate (VPA) (MCE, HY-10585A) were added, and the cell supernatant was collected on the sixth day after transfection. The collected supernatant was centrifuged to remove cell debris, and the antibody was purified by protein A affinity chromatography. After purification, the antibody was replaced by ultrafiltration and stored in PBS buffer.

[0039] 2. SDS-PAGE to detect antibody purity

[0040] Mix 5 μg of purified antibody with protein reduction buffer, add PBS to 30 μL, heat at 100°C for 10 minutes to fully denature the protein, and add 9 μL to the prefabricated polyacrylamide gel well. Set the voltage to 80V for 30 minutes and then 140V for 50 minutes for separation. Use a staining and destaining instrument to stain and destain the gel, and then scan the gel image and save it.

[0041] The results of SDS-PAGE were as follows: Figure 1 As shown in the figure, the molecular weights of the two bands corresponding to the antibody NC056 fused with human Fc protein under reducing conditions are both about 40 kDa, which is consistent with the expected protein molecular weight, and no obvious mixed bands appear, indicating good purity.

[0042] Example 2. Binding activity analysis of antibody NC056

[0043] 1. Dilute the Zaire Ebola virus VP40 protein to 1 μg / mL with carbonate coating solution, add 100 μL to each well of the ELISA plate, seal the plate with a sealing film, and place at 4°C overnight;

[0044] 2. Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the ELISA plate dry;

[0045] 3. Add 100 μL of blocking solution (PBST containing 3% BSA by mass) to each well, seal the plate with a sealing film, and incubate at 37°C for 1 hour;

[0046] 4. After blocking, discard the liquid in the wells, wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the ELISA plate dry;

[0047] 5. During this period, add 100 μL of 4-fold diluted NC056 antibody at a concentration of 2 μg / mL in the first well to each well of the ELISA plate and incubate at 37°C for 1 h;

[0048] 6. Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the ELISA plate dry;

[0049] 7. Add 100 μL of diluted anti-human IgG (HRP) antibody to the corresponding wells, seal the plate with a sealing film, and incubate at 37°C for 1 h;

[0050] 8. Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the ELISA plate dry;

[0051] 9. Add 100 μL of color development solution to each well and incubate at room temperature for 15 min;

[0052] 10. Add 50 μL of stop solution to each well and gently shake the ELISA plate until mixed evenly;

[0053] 11. Use an ELISA reader to measure the absorbance of each well at 450 nm and 630 nm, and read the value within 10 min after termination;

[0054] 12. The results of ELISA binding experiments showed that antibody NC056 could specifically bind to Zaire Ebola virus VP40 protein, EC 50 8.6 ng / mL ( Figure 2 ).

[0055] Example 3. Preparation of mRNA-lipid nanoparticles encoding anti-Ebola virus VP40 protein nanobodies

[0056] 1. Molecular design of mRNA

[0057] Based on the amino acid sequence of the heavy chain variable region of the nanoantibody NC056 against the Ebola virus VP40 protein, an mRNA encoding the VP40 protective antibody was designed. The construct includes the following elements in sequence along the positive chain, i.e., the 5'→3' direction: promoter, 5' non-coding region (5'UTR), antibody NC056 heavy chain variable region, 3' non-coding region (3'UTR), and polyadenylic acid sequence (poly A).

[0058] 2. Synthesis of mRNA

[0059] Using the linear double-stranded DNA encoding the NC056 nanobody containing a T7 promoter sequence as a template, and ATP, GTP, and N1-Me-pUTP as substrates, the DNA sequence downstream of the promoter was transcribed by T7 polymerase to synthesize mRNA, which was capped using a one-step chemical method to finally obtain the NC056 nanobody mRNA transcription product, named "NC056-mRNA". The 5' cap structure is m7G(5')ppp(5')(2'OMeA)pG (Shenji Bio, CAP3011), and the mRNA 5'→3' direction includes the following elements in sequence: a promoter as shown in SEQ ID NO:3, a 5' non-coding region (5'UTR) as shown in SEQ ID NO:4, a start codon (M), an antibody NC056 heavy chain variable region as shown in SEQ ID NO:2, a linker (GGGC), an Fc, a Flag tag (DYKDDDDK), a stop codon with a sequence of TGATAATAG, a 3' non-coding region (3'UTR) as shown in SEQ ID NO:5, and a polyadenylic acid sequence (poly A) of 112 nucleotides. The complete sequence of the mRNA is shown in SEQ ID NO:6.

[0060] 3. Encapsulation of mRNA-lipid nanoparticles

[0061] NC056 mRNA-lipid nanoparticle encapsulation uses SM102 lipid formula: SM-102, distearoylphosphatidylcholine, cholesterol, dimyristoylglycerol-polyethylene glycol 2000 form a lipid mixture at a molar ratio of 50:10:38.5:1.5, and the mass ratio of the lipid mixture to the mRNA solution is 1:3. The microfluidic device is Nano L+ type, and the encapsulation efficiency is determined by Ribogreen fluorescent dye method ( Anal Biochem . 1998;265(2):368-374. doi:10.1006 / abio.1998.2914) to accurately quantify the concentration of total and free mRNA.

[0062] Schematic diagram of NC056 mRNA-lipid nanoparticles Figure 3 As shown, the prepared mRNA-lipid nanoparticles had a free mRNA concentration of 3.08 μg / mL, a total mRNA concentration of 110.35 μg / mL, an encapsulation efficiency of 97.21%, and a particle size of 89.42 nm.

[0063] Example 4. Identification of the expression of NC056-mRNA-lipid nanoparticles in cells and their interaction with VP40 protein

[0064] 1. Cell culture and sample preparation

[0065] HEK293T cells were cultured at 5 × 10 5 / mL density, 2 mL per well was inoculated in a 6-well cell culture plate, and cultured statically in a carbon dioxide incubator at 37 °C for 12 h;

[0066] NC056-mRNA-lipid nanoparticles were diluted to 2 μg / mL with 100 μL Opti-MEM medium (Thermo Fisher, 31985070), added dropwise to a 6-well cell culture plate, and cultured in a CO2 incubator at 37 °C for 6 h;

[0067] 2 μg of EBOV VP40 expression plasmid was transfected into the above cells using Lipofectamine 3000 transfection reagent (Thermo Fisher, L3000015) according to the instructions, and cultured statically in a carbon dioxide incubator at 37 °C for 20 h;

[0068] 20 h after transfection, 500 μL of cell lysis buffer was added to each well of cells, and the cells were shaken and lysed at 600 rpm for 15 min at room temperature. The lysate was centrifuged at 12000 rpm for 5 minutes and the supernatant was collected to obtain the cell lysate sample.

[0069] 2. Co-immunoprecipitation

[0070] The lysate was mixed with Anti-Flag tag magnetic beads in an EP tube, placed in a flip rack, and incubated at 4°C overnight;

[0071] The magnetic beads were enriched on the tube wall using a magnetic rack, washed three times with PBS, and eluted with 0.1 M glycine at pH 2.7 for 5 minutes. The eluted supernatant was taken and Tris buffer at pH 9 was added to adjust the pH of the eluted product to neutral.

[0072] 3. Western blot verification of the interaction between NC056 antibody and VP40

[0073] The cell lysate sample and the immunoprecipitation sample were mixed with reducing protein loading buffer and separated by SDS-PAGE. The protein was transferred to the NC membrane using a membrane transfer apparatus. Mouse anti-Flag mAb (GeneTex, GTX629631) was used as the primary antibody and Goat-anti mouse IgG (HRP) pAb (Abcam, ab97265) was used as the secondary antibody to detect the expression of mRNA antibody; Rabbit anti-EBOV VP40 pAb (GeneTex, GTX134034) was used as the primary antibody and Goat-anti rabbit IgG (HRP) pAb (Sino biological, SSA003) was used as the secondary antibody to detect VP40 protein; Rabbit anti-GAPDH pAb (Solarbio, K110496P) was used as the primary antibody and Goat-anti rabbit IgG (HRP) pAb (Sino biological, SSA003) was used as the secondary antibody to detect the internal reference GAPDH. After development with developer, the gel was photographed and analyzed in a gel imager.

[0074] Western Blot results Figure 4 As shown, the WB results of the total cell lysate indicated that the cells transfected with VP40 expressed the VP40 protein intracellularly, and that in the NC056-mRNA-lipid nanoparticle antibody treatment group, there was expression of a specific nanoantibody of size 15 kDa; the results of co-immunoprecipitation indicated that the NC056 antibody was able to specifically interact with the VP40 protein in the cells.

[0075] Example 5. Western blot of mRNA-lipid nanoparticles encoding anti-Ebola virus VP40 protein nanobody on the inhibition of VP40 virus-like particle budding

[0076] 1. Cell culture and sample preparation

[0077] HEK293T cells were cultured at 5 × 10 5 / mL density, 2 mL per well was inoculated in a 6-well cell culture plate, and cultured statically in a carbon dioxide incubator at 37 °C for 12 h;

[0078] The mRNA-lipid nanoparticles were diluted to 2 μg / mL with 100 μL Opti-MEM medium (Thermo Fisher, 31985070), added dropwise to a 6-well cell culture plate, and cultured statically in a carbon dioxide incubator at 37 °C for 6 h;

[0079] 2 μg of EBOV VP40 expression plasmid was transfected into the above cells using Lipofectamine 3000 transfection reagent (Thermo Fisher, L3000015) according to the instructions, and cultured statically in a carbon dioxide incubator at 37 °C for 20 h;

[0080] 20 h after transfection, the cell supernatant was collected, centrifuged at 2000 rpm for 5 min, and the supernatant was aspirated;

[0081] 500 μL of cell lysis buffer was added to each well of cells, and the cells were shaken and lysed at 600 rpm for 15 min at room temperature. The lysate was centrifuged at 12000 rpm for 5 minutes and the supernatant was collected to obtain the cell lysate sample.

[0082] 2. Western blot verification of Ebola virus VP40 virus-like particles in the supernatant

[0083] The prepared virus-like particle packaging supernatant was mixed with reducing protein loading buffer, separated by SDS-PAGE, and the protein was transferred to the NC membrane using a membrane transfer apparatus. Rabbit anti-EBOV VP40 pAb (GeneTex, GTX134034) was used as the primary antibody and Goat anti-rabbit IgG (HRP (Sino biological, SSA003)) was used as the secondary antibody for antibody incubation to detect EBOV-VP40 in the supernatant; Rabbit anti-GAPDH pAb (Solarbio, K110496P) was used as the primary antibody and Goat-antirabbit IgG (HRP) pAb (Sino biological, SSA003) was used as the secondary antibody to detect the internal reference GAPDH. After development with developer, the gel was photographed and analyzed in a gel imager.

[0084] Western Blot results Figure 5 As shown, the number of VP40 virus-like particles in the supernatant of the control mRNA antibody treatment group and the VP40 blank transfection group was equivalent, while the VP40 virus-like particles in the supernatant of the NC056 mRNA antibody treatment group were significantly reduced. The grayscale values ​​of the VP40 bands in each group were compared with the internal reference GAPDH band using ImageJ software. The VP40 / GAPDH (Fold change to control) values ​​of each group were 2.75, 128.04, 22.11, and 140.66, respectively. Compared with the control mRNA antibody, the NC056 mRNA antibody treatment group was able to reduce the VP40 in the supernatant by more than 6 times (see Figure 6). This indicates that after NC056 mRNA antibody treatment, the antibody interacted with Ebola virus VP40 in the cell and inhibited the VP40-mediated virus budding process.

[0085] The above data indicate that NC056 mRNA lipid nanoparticles can bind to Ebola virus VP40, block the virus budding process inside cells, and thus reduce the level of virus-like particles in the cell supernatant, and have the potential to be developed as therapeutic drugs against Ebola virus infection.

Claims

1. A nanobody against Zaire Ebola virus VP40 protein, characterized in that: The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the anti-Zaire Ebola virus VP40 protein nanobody are shown in positions 27-33, 51-57 and 96-112 of SEQ ID NO: 1, respectively.

2. The nanobody against Zaire Ebola virus VP40 protein according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the nanobody against the Zaire Ebola virus VP40 protein is shown in SEQ ID NO:

1.

3. A polynucleotide encoding the nanobody against the Zaire Ebola virus VP40 protein according to claim 1 or 2. A vector comprising the polynucleotide according to claim 3.

5. A host cell containing the vector according to claim 4.

6. Use of the nanobody against Zaire Ebola virus VP40 protein according to claim 1 or 2 in the preparation of a therapeutic drug or diagnostic reagent for Zaire Ebola virus disease.

7. An mRNA molecule, characterized in that The mRNA molecule contains mRNA transcribed from the polynucleotide encoding the anti-Zaire Ebola virus VP40 protein nanobody according to claim 3, and the sequence of the anti-Zaire Ebola virus VP40 protein nanobody polynucleotide encoding is shown in SEQ ID NO:

2.

8. The mRNA molecule according to claim 7, characterized in that The 5'UTR sequence shown in SEQ ID NO:4 is connected in series with the 5' end of the mRNA transcribed from the polynucleotide, and the 3'UTR sequence shown in SEQ ID NO:5 is connected in series with the 3' end of the mRNA transcribed from the polynucleotide.

9. The mRNA molecule according to claim 8, characterized in that The 5' end of the mRNA molecule is connected in series with a promoter sequence as shown in SEQ ID NO: 3, and the 3' end of the mRNA molecule is connected in series with a stop codon of TGATAATAG and a poly (A) of 112 nucleotides.

10. The mRNA molecule according to claim 9, characterized in that The 5' end of the mRNA molecule also contains a Cap3011 cap structure.

11. A lipid nanoparticle encapsulating the mRNA molecule described in any one of claims 7-10.

12. Use of the mRNA molecule according to any one of claims 7 to 10 in the preparation of a drug for treating Zaire Ebola virus disease.

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