Nanobodies that inhibit infection by bundibugyo and zaire ebola virus

Abstract

The application discloses a kind of nanobody for inhibiting bundibugio and zaire type Ebola virus infection.The application belongs to the field of biotechnology, specifically relates to a kind of nanobody for inhibiting bundibugio and zaire type Ebola virus infection.The nanobody BDBV-Nb02 of the application targeted Ebola virus has three complementarity determining regions CDR1, CDR2 and CDR3;The amino acid sequence of CDR1 is the 27-33 of SEQ ID No:1, the amino acid sequence of CDR2 is the 47-58 of SEQ ID No:1, and the amino acid sequence of CDR3 is the 97-109 of SEQ ID No:1.Nanobody BDBV-Nb02 presents neutralization effect to bundibugio Ebola virus and zaire type Ebola virus, and has important application prospect and innovation significance in medical field.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a nanobody for inhibiting infection of Bundibugyo and Zaire ebola virus. BACKGROUND

[0002] Ebola virus has become a pathogen of great significance to global public health due to its high pathogenicity and strong transmissibility. Among the five subtypes of Ebola virus that have been confirmed to be able to infect humans, the Ebola virus subtype (EBOV), Sudan virus (SUDV) and Bundibugyo virus (BDBV) once had a mortality rate of up to 90% in the past decade, causing a devastating disaster to human health and a huge impact on social economy and stability.

[0003] Currently, in the field of Ebola virus vaccine and therapeutic monoclonal antibody (mAb) research and development, researchers have made many remarkable achievements. However, the vaccines and monoclonal antibodies (mAbs) approved for human use at this stage are only limited to products related to Zaire Ebola virus (EBOV). Given that other Ebola virus species such as Sudan Ebola virus and Bundibugyo Ebola virus still maintain a very high mortality rate, continue to pose a serious threat to global public health, and Ebola virus as an RNA virus has the significant characteristics of poor sequence conservation and strong mutation ability, the development of monoclonal antibodies with broad-spectrum and high efficiency against Ebola virus has become an urgent task for current scientific research, and it is necessary to conduct in-depth research to comprehensively improve the prevention and control efficiency of Ebola virus and build a solid barrier for global public health security.

[0004] The early antibody cocktail therapy ZMapp (optimized from MB-003 and ZMAb) could not be widely used due to production limitations, but its structural research provided a template for subsequent antibody design. For example, its antibody combination strategy was used to improve REGN-EB3. REGN-EB3 is a cocktail therapy composed of three monoclonal antibodies, which blocks viral invasion by binding to different epitopes of the virus GP (such as the glycan cap and core region). Clinical trials show that early use can reduce mortality to 6%. mAb114 is a monoclonal antibody isolated from a survivor in 1995, which targets the receptor binding domain (RBD) of GP and directly inhibits the binding of the virus to host cell receptors (such as NPC1). Its single-drug treatment mortality rate is only 11%, and it is effective against viral variants. Antibodies are limited to products related to Zaire Ebola virus (EBOV). Given that other Ebola virus species such as Bundibugyo Ebola virus still maintain a very high mortality rate, continue to pose a serious threat to global public health, research on antibodies against other species of Ebola virus is still meaningful. SUMMARY

[0005] The main problem to be solved by the present application is how to develop a monoclonal antibody with broad-spectrum and high efficiency against the genus Ebola virus.

[0006] In order to solve the above problems, the present application provides a nanobody targeting Ebola virus or an antigen-binding fragment containing the nanobody.

[0007] The present application provides a nanobody targeting Ebola virus or an antigen-binding fragment containing the nanobody, wherein the nanobody has three complementarity determining regions CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID No: 1 at positions 27-33, the amino acid sequence of the CDR2 is shown in SEQ ID No: 1 at positions 47-58, and the amino acid sequence of the CDR3 is shown in SEQ ID No: 1 at positions 97-109.

[0008] The above CDR is a sequence defined according to the analysis results of the IMGT system.

[0009] The nanobody described herein generally includes a VHH composed of four framework regions (FRs) and three complementarity determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and the antigen-binding fragment contains at least a part of the nanobody, which is sufficient to endow the fragment with the ability to specifically bind to Ebola virus.

[0010] The four framework regions can be FR1, FR2, FR3 and FR4.

[0011] The amino acid sequence of the FR1 is SEQ ID No: 1 at positions 1-26;

[0012] The amino acid sequence of the FR2 is SEQ ID No: 1 at positions 34-46;

[0013] The amino acid sequence of the FR3 is SEQ ID No: 1 at positions 59-96;

[0014] The amino acid sequence of the FR4 is SEQ ID No: 1 at positions 110-122.

[0015] In the nanobody or antigen-binding fragment described above, the nanobody can be any one of the following:

[0016] A1) a nanobody with an amino acid sequence shown in SEQ ID No: 1;

[0017] A2) a nanobody obtained by connecting a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No: 1.

[0018] The protein tag refers to a polypeptide or protein that is fused with the target protein for expression, detection, tracing, and / or purification. The protein tag may be a His tag, Flag tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, or the Fc fragment of immunoglobulin G, etc.

[0019] In one specific embodiment, SEQ ID No:1 may be as follows:

[0020] QVQLVESGGGLVQAGGSLRLSCAASGFIFSINGMGWYRQAPGKERELVASISKGDSTNYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAAQFWLVLYHTRSYDYWGQGTQVTVSS.

[0021] In the above-mentioned nanobody, the nanobody is composed of the complementary determinant cluster region and the framework region.

[0022] In this invention, the term "antibody" refers to a heterotetraglycoprotein of approximately 150,000 Daltons with identical structural features, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0023] In this invention, "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments in the variable regions of the light and heavy chains, called complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91.3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0024] In this invention, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning, referring to the variable region of the cloning antibody heavy chain. A single-domain antibody (VHH) consisting of only one variable region of the heavy chain is constructed, which is the smallest antigen-binding fragment with complete function.

[0025] The antigen-binding fragments mentioned above may be complete antibodies, fusion antibodies, antibody-drug conjugates, Fab fragments, Fv fragments, Fab' fragments, F(ab')2 fragments, single-chain antibodies (ScFv), or minimum recognition units (MRUs) containing the nanobody.

[0026] The term "Fab fragment" refers to a heterodimer composed of a heavy chain (Fd) and a complete light chain linked by disulfide bonds, containing only one antigen-binding site. The aforementioned heavy chain (Fd) refers to approximately half of the H chain portion of the Fab (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0027] The term "Fv fragment" refers to a vector containing VH and VL genes that can be constructed separately, co-transfected into cells to express them separately, and then assembled into a functional Fv antibody; alternatively, a stop codon can be set between VH and VL in the vector to express two small protein fragments, which can then be bound together by non-covalent bonds to form an Fv antibody (Fv fragment).

[0028] The term "Fab' fragment" contains a portion of a light chain and a heavy chain containing the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of two Fab' fragments to form the F(ab')2 molecule.

[0029] The term "F(ab')2 segment" contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments held together by the disulfide bond between the two heavy chains.

[0030] In some embodiments, the nanobodies of the present invention may be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to lack one or both of those backbone regions, as long as they substantially maintain antigen binding and specificity.

[0031] In this invention, the nanobody is named nanobody BDBV-NB02.

[0032] This invention also provides biomaterials related to the nanobodies described above, wherein the biomaterials may be any of the following:

[0033] B1) Nucleic acid molecules that encode the nanobodies or antigen-binding fragments described above;

[0034] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0035] B3) A recombinant vector containing the nucleic acid molecules described in B1);

[0036] B4) A recombinant vector containing the expression cassette described in B2);

[0037] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0038] B6) Recombinant microorganisms containing the expression cassette described in B2);

[0039] B7) Recombinant microorganisms containing the recombinant vector described in B3);

[0040] B8) Recombinant microorganisms containing the recombinant vector described in B4).

[0041] In the above-mentioned biological materials, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0042] In the aforementioned biological materials, the expression cassette (B2) refers to DNA capable of expressing the nanobody in host cells. This DNA may include not only a promoter to initiate transcription of the nanobody-encoding gene, but also a terminator to terminate transcription of the nanobody-encoding gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0043] In the aforementioned biological materials, the vector can be a plasmid, granule, bacteriophage, or viral vector. Recombinant vectors containing the expression cassette can be constructed using existing expression vectors.

[0044] In the above-mentioned biomaterials, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule described in B1) into the nanoantibody fusion protein expression vector pET22b+.

[0045] In this invention, the recombinant vector may be the recombinant vector pET22b+BDBV-NbO2.

[0046] The structure of the recombinant vector pET22b+BDBV-Nb02 is described as follows: A DNA fragment with the sequence SEQ ID No:2 is inserted between the NcoI and Xhol restriction enzyme sites of the starting vector pET22b+, while keeping the other sequences of the pET22b+ vector unchanged. The pET22b+BDBV-Nb02 vector can express the BDBV-Nb02 encoding gene, whose amino acid sequence is SEQ ID No:4.

[0047] In the aforementioned biomaterials, the host cell contains nucleic acid molecules or carriers of nanobodies or antigen-binding fragments as described above. The host cell includes, but is not limited to, prokaryotic cells such as *E. coli* cells, eukaryotic cells such as yeast cells, animal cells (such as mammalian cells, such as mouse cells and human cells), insect cells, and plant cells.

[0048] In this invention, the cells may be Lemo21(DE3) Escherichia coli cells.

[0049] In the above-mentioned biomaterials, the nucleic acid molecule described in B1) may be a nucleic acid molecule encoding the nanobody described above. In the nucleic acid molecule, the encoding gene of CDR1 is nucleotides 79-99 of SEQ ID No:2, the encoding gene of CDR2 is nucleotides 139-174 of SEQ ID No:2, and the encoding gene of CDR3 is nucleotides 289-327 of SEQ ID No:2.

[0050] In the above-mentioned biological materials, the nucleic acid molecule described in B1) can be any of the following:

[0051] C1) A DNA molecule with a nucleotide sequence as shown in SEQ ID No:2;

[0052] C2) hybridizes with the DNA molecule defined by C1) under stringent conditions and encodes the DNA molecule of the nanobody;

[0053] A DNA molecule encoding the nanobody has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to any of the defined DNA sequences in C3) and C1)-C2).

[0054] In one specific embodiment, SEQ ID No:2 may be as follows:

[0055] CAGGTGCAGCTGGTGGAATCGGGTGGGGGATTGGTTCAGGCGGGGGGAAGTTTACGCTTATCGTGTGCGGCA

[0056] TCTGGTTTCATTTTCTCTATCAACGGTATGGGTTGGTATCGCCAGGCACCAGGTAAAGAACGTGAACTGGTTGC

[0057] CTCTATTTCTAAAGGCGACTCTACCAACTACGCGGACTCAGTGAAAGGCCGCTTCACTATCTCCCGTGATAATG

[0058] CTAAGAACACCGTTTATCTGCAGATGAATTCTTTGAAACCTGAAGACACTGCCGTTTATTATTGCGCAGCTCAGTTCTGGCTGGTTCTGTACCATACTCGTTCTTACGACTACTGGGGTCAGGGAACCCAGGTTACGGTTTCTTCT.

[0059] The stringent conditions can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1 ...7% SDS, 0.5M Na3PO4, and 1mM EDTA; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS. Hybridize in a mixed solution of SDS, 0.5M Na3PO4 and 1mM EDTA, and wash at 65°C with 0.1×SSC and 0.1% SDS; alternatively, hybridize in a solution of 6×SSC and 0.5% SDS at 65°C, and then wash once each with 2×SSC and 0.1% SDS and 1×SSC and 0.1% SDS.

[0060] Those skilled in the art can readily mutate the nucleotide sequence of the encoding gene of the nanobody BDBV-NB02 described in B1) of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides having 75% or more identity with the nucleotide sequence of BDBV-NB02 described in B1) of the present invention, as long as they encode the nanobody and possess the BDBV-NB02 nanobody activity, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0061] The present invention also provides a method for preparing the above-mentioned nanobody, which may include the following steps: introducing a nucleic acid molecule encoding the nanobody described above into a recipient cell to obtain a transgenic cell expressing the nanobody, culturing the transgenic cell to obtain the nanobody.

[0062] Furthermore, the nucleic acid molecule encoding the nanobody is the nucleic acid molecule described above.

[0063] In the above preparation method, the nucleotide sequence of the nucleic acid molecule encoding the nanobody described above can be any of the following:

[0064] C1) A DNA molecule with a nucleotide sequence as shown in SEQ ID No:2;

[0065] C2) hybridizes with the DNA molecule defined by C1) under stringent conditions and encodes the DNA molecule of the nanobody;

[0066] A DNA molecule encoding the nanobody having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in C3) and C1) or C2).

[0067] Furthermore, the recipient cells may be prokaryotic cells such as Escherichia coli cells, eukaryotic cells such as yeast cells, animal cells (such as mammalian cells, such as mouse cells, human cells), insect cells, and plant cells, etc.

[0068] In a specific embodiment of the present invention, the recipient cell may be a Lemo21(DE3) Escherichia coli cell.

[0069] The present invention also provides a nanobody fusion protein, wherein the nanobody fusion protein is formed by fusing the aforementioned nanobody or antigen-binding fragment with another molecule, wherein the other molecule may include the Fc domain of an immunoglobulin, a fluorescent protein, or a VHH with different specificities.

[0070] In a specific embodiment of the present invention, the other molecule is a his-tagged protein.

[0071] In specific embodiments of the present invention, the nanobody fusion protein described above may be any of the following:

[0072] M1) is a fusion protein with the amino acid sequence shown in SEQ ID No:4;

[0073] M2) is a fusion protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in M1), which has more than 75% identity with the protein shown in M1.

[0074] The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No:4 (M3).

[0075] In the above-mentioned nanobody fusion protein, the nucleic acid molecule encoding the fusion protein can be any of the following:

[0076] D1) Nucleotide sequence such as SEQ ID The DNA molecule shown in No:3: CAGGTGCAGCTGGTGGAATCGGGTGGGGGATTGGTTCAGGCGGGGGGAAGTTTACGCTTATCGTGTGCGGCATCTGGTTTCATTTTCTCTATCAACGGTATGGGTTGGTATCGCCAGGCACCAGGTAAAGAACGTGAACTGGTTGCCTCTATTTCTAAAGGCGACTCTACCAACTACGCGGACTCAGTGA AAGGCCGCTTCACTATCTCCCGTGATAATGCTAAGAACACCGTTTATCTGCAGATGAATTCTTTGAAACCTGAAGACACTGCCGTTTATTATTGCGCAGCTCAGTTCTGGCTGGTTCTGTACCATACTCGTTCTTACGACTACTGGGGTCAGGGAACCCAGGTTACGGTTTCTTCTCGAGCACCACCACCACCACCACTGA;

[0077] D2) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined by D1) and encodes the fusion protein;

[0078] A DNA molecule encoding the fusion protein having 99%, 95%, 90%, 85%, or 80% homology with the DNA sequence defined in D3) or D1) or D2).

[0079] In one specific embodiment, the nanobody fusion protein is BDBV-NB02-his.

[0080] The present invention also provides an ELISA detection kit targeting Ebola virus, the kit comprising the nanobodies, biomaterials or fusion proteins described above.

[0081] This invention also provides any of the following applications:

[0082] E1) The application of the nanobodies or antigen-binding fragments mentioned above in the preparation of products for inhibiting Ebola virus;

[0083] E2) The application of the aforementioned biomaterials in the preparation of products for inhibiting Ebola virus;

[0084] E3) Application of the preparation method described above in the preparation of products for inhibiting Ebola virus;

[0085] E4) The application of the aforementioned kit in the preparation of products for inhibiting Ebola virus;

[0086] E5) The application of the nanobodies mentioned above in the preparation of products that bind to the Ebola virus;

[0087] E6) The application of the aforementioned biomaterials in the preparation of products that bind to the Ebola virus;

[0088] E7) Application of the preparation method described above in the preparation of products conjugated with Ebola virus;

[0089] E8) Application of the aforementioned kit in the preparation of products conjugated with Ebola virus;

[0090] E9) The use of the nanobodies or antigen-binding fragments described above in the preparation of drugs for the prevention and / or treatment of Ebola virus infection.

[0091] In this article, the antigen is Ebola virus EBOV-GP protein (GenBank: AF086833.2, updated on 2012-02-13) or / and BDBV-GP protein (GenBank: MK028856.1, updated on 2019-07-23).

[0092] The Ebola virus mentioned in this article may be an Ebola pseudovirus.

[0093] Furthermore, the Ebola pseudovirus may be Zaire-type Ebola pseudovirus or / and Bundibugyo-type pseudovirus.

[0094] The Zaire-type Ebola pseudovirus contains the EBOV-GP protein (GenBank: AF086833.2, updated on 2012-02-13).

[0095] The Bendibugyo pseudovirus contains the BDBV-GP protein (GenBank:MK028856.1, updated on 2019-7-23).

[0096] This invention uses the pNL4-3-Luc-RE vector as the basic framework to construct three Ebola pseudoviruses, serving as a key tool for evaluating the neutralizing activity of nanobodies. Starting with Ebola pseudovirus models and screening phage libraries of nanobodies against the Bundibugyo type Ebola virus GP protein, and fully utilizing advanced virological research tools such as Ebola pseudoviruses, nanobodies against Bundibugyo type Ebola virus infection were obtained through virological, biochemical, and structural biological methods. Furthermore, this antibody, in addition to neutralizing Bundibugyo type Ebola virus, also exhibits a certain degree of neutralizing activity against Zaire type Ebola virus. The antibody binding region provides a potentially broad-spectrum neutralizing site for anti-Ebola virus treatment strategies, demonstrating significant application prospects and innovative value in the medical field. Attached Figure Description

[0097] Figure 1 The image shows the purification results of the nanobody BDBV-Nb02. Lane 1 is the protein marker, and lanes 2-15 are the screened nanobodies BDBV-Nb01-BDBV-Nb015.

[0098] Figure 2 Construction of three pseudoviruses for Ebola.

[0099] Figure 3 The results show the inhibitory effects of BDBV-Nb02 on the invasion of cells by different types of Ebola pseudoviruses. □ represents the inhibition curve of BDBV-NB02 against EBOV pseudovirus infection, ▲ represents the inhibition curve of BDBV-NB02 against BDBV pseudovirus infection, and ■ represents the inhibition curve of BDBV-NB02 against TAFV pseudovirus infection.

[0100] Figure 4 Affinity data for BDBV-Nb02 with Bundibugyo type Ebola virus GP1. Detailed Implementation

[0101] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0102] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0103] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0104] The Huh7.5.1 cells used in the following examples are described in: Jin Z, Pablo G, Guofeng C, et al. Robust hepatitis C virus infection in vitro. [J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(26): 9294-9299. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of the present invention and shall not be used for any other purpose.

[0105] The 293T cells used in the following examples were obtained from the National Experimental Cell Resource Sharing Platform, resource number: 3111C0001CCC000091.

[0106] The PBS buffer used in the following examples was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: P1020, pH 7.4, 0.01M.

[0107] The luciferase detection system and cell lysis buffer used in the following examples were purchased from Promega, product number: E2610.

[0108] The pNL4-3-Luc-RE described in the following examples is described in: Connor RI, Chen BK, Choe S, Landau NR. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 206:935–944, 1995. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention and may not be used for any other purpose.

[0109] The pET22b+ vector used in the following examples was purchased from EMD Biosciences (Novagen).

[0110] Example 1: Preparation of nanobody BDBV-Nb02

[0111] 1. Gene cloning

[0112] The BDBV-Nb02 gene (nucleotide sequence SEQ ID No:2) was cloned into the NcoI and Xhol restriction sites of the pET28b+ vector containing the kanamycin resistance gene and the T7 protein expression promoter to obtain the recombinant vector pET28b+BDBV-Nb02.

[0113] The structure of the recombinant vector pET28b+BDBV-Nb02 is described as follows: It is obtained by inserting a DNA fragment with the sequence SEQ ID No:2 between the NcoI and Xhol restriction enzyme sites of the starting vector pET22b+, while keeping the other sequences of the pET28b+ vector unchanged. The pET22b+BDBV-Nb02 vector can express the BDBV-Nb02 encoding gene, whose amino acid sequence is SEQ ID No:1.

[0114] 2. Plasmid transformation

[0115] Following the manufacturer's (New England Biolabs) description, the constructed plasmid was transformed into chemically active Lemo21(DE3) Escherichia coli using a 15-second heat shock procedure. Recombinant Escherichia coli DE3 / pET22b+BDBV-Nb02 was obtained.

[0116] 3. Protein expression

[0117] The transformed Escherichia coli DE3 / pET28b+BDBV-Nb02 cells obtained in step 2 were added to LB medium (10g tryptone, 5g yeast extract, 5g sodium chloride, and brought to a final volume of 1L) and incubated with shaking at 37°C and 200 rpm until the OD value was around 0.6. Then 0.2mM IPTG (MERCK I6758) was added.

[0118] 4. Cell processing and protein separation

[0119] After incubation, the bacteria cultured in step 3 were collected by centrifugation at 4000g and resuspended in lysis buffer (150mM NaCl, 50mM Tris-HCl, pH 8.0, with added protease inhibitors and DNase). Cells were lysed using sonication at 20% amplitude with a 3-second on / off cycle for a total of 20 minutes. The lysed mixture was then centrifuged at 16000g to separate soluble substances. Finally, histogram-tagged proteins were isolated from the soluble fraction using IMAC (immunoaffinity chromatography).

[0120] Purification results are as follows Figure 1 As shown: The target protein was obtained at 15 kDa. This target protein was named the nanobody BDBV-Nb02.

[0121] The amino acid sequence of the nanobody BDBV-Nb02 is shown in SEQ ID No:1: it includes a framework region (FR: FR1, FR2, FR3, FR4) and a complementarity-determining region (CDR: CDR1, CDR2, CDR3). The four parts of the framework region are sequentially labeled as positions 1-26, 34-46, 59-96, and 110-122 of SEQ ID No:1. The three parts of the complementarity-determining region are sequentially labeled as positions 27-33, 47-58, and 97-109 of SEQ ID No:1.

[0122] The nucleotide sequence of the encoding gene for the above-mentioned nanobody BDBV-Nb02 is shown in SEQ ID No:2.

[0123] Example 2: Preparation of Ebola pseudovirus

[0124] First, the GP protein gene sequences of three Ebola viruses were cloned into the pCDNA3.1 vector, respectively: EBOV-GP (gene nucleotide sequence SEQ ID No: 5); BDBV-GP (gene nucleotide sequence SEQ ID No: 6); and TAFV-GP (gene nucleotide sequence SEQ ID No: 7). Recombinant vectors pCDNA 3.1-GP (EBOV), pCDNA 3.1-GP (BDBV), and pCDNA 3.1-GP (TAFV) were obtained.

[0125] The recombinant vector pCDNA 3.1-GP(EBOV) is described as follows: It is obtained by inserting a DNA fragment with the sequence SEQ ID No:5 between the HindIII and XhoI restriction sites of the starting vector pCDNA 3.1, while keeping the other sequences of the vector pCDNA 3.1 unchanged. The pCDNA 3.1-GP(EBOV) vector can express the EBOV-GP protein, whose amino acid sequence is available in GenBank: AHX24649.2, updated on 18-12-2014. The nucleotide sequence of the recombinant vector pCDNA 3.1-GP(EBOV) is SEQ ID No:8.

[0126] The recombinant vector pCDNA 3.1-GP(BDBV) is described as follows: It is obtained by inserting a DNA fragment with sequence SEQ ID No:6 between the HindIII and XhoI restriction sites of the starting vector pCDNA 3.1, while keeping the other sequences of the vector pCDNA 3.1 unchanged. The pCDNA 3.1-GP(BDBV) vector can express the BDBV-GP protein, whose amino acid sequence is located in GenBank: YP_003815435.1, updated on 07-11-2018. The only difference between the recombinant vector pCDNA 3.1-GP(BDBV) and pCDNA 3.1-GP(EBOV) is that the nucleotide sequence SEQ ID No:5 in pCDNA 3.1-GP(EBOV) is replaced with SEQ ID No:6, while keeping the other sequences unchanged.

[0127] The recombinant vector pCDNA 3.1-GP (TAFV) is described as follows: It is obtained by inserting a DNA fragment with the sequence SEQ ID No:7 between the HindIII and XhoI restriction sites of the starting vector pCDNA 3.1, while keeping the other sequences of the vector pCDNA 3.1 unchanged. The pCDNA 3.1-GP (TAFV) vector can express the TAFV-GP protein, and its amino acid sequence is available in GenBank: AWK96625.1, updated on 23-7-2019. The only difference between the recombinant vector pCDNA 3.1-GP (TAFV) and pCDNA 3.1-GP (EBOV) is that the nucleotide sequence SEQ ID No:5 in pCDNA 3.1-GP (EBOV) is replaced with SEQ ID No:7, while keeping the other sequences unchanged.

[0128] 1. Preparation of EBOV pseudovirus

[0129] (1) Seed 293T cells into 10cm petri dishes containing DMEM medium containing 10% FBS and cultured for 24 hours.

[0130] (2) After completing step (1), take the plate and transfect the cells in it with pNL4-3-Luc-RE and pCDNA 3.1-GP (EBOV) (using TransMAXi transfection reagent). Then replace the culture system with new DMEM medium containing 2% FBS and culture for 48 hours.

[0131] (3) After completing step (2), centrifuge and collect the supernatant, which is the virus solution containing EBOV pseudovirus, referred to as EBOV virus solution.

[0132] 2. Preparation of BDBV pseudovirus

[0133] Replace pCDNA 3.1-GP (EBOV) with pCDNA 3.1-GP (BDBV), and follow the same procedure as in step 1 to obtain a viral solution containing BDBV pseudovirus, referred to as BDBV viral solution.

[0134] 3. Preparation of TAFV pseudovirus

[0135] Replace pCDNA 3.1-GP (EBOV) with pCDNA 3.1-GP (TAFV), and follow the same procedure as in step 1 to obtain a viral solution containing TAFV pseudovirus, referred to as TAFV viral solution.

[0136] The preparation and culture conditions for various Ebola pseudoviruses were: 37℃, 5% CO2, and static incubation.

[0137] 4. Preparation of control solution

[0138] (1) Seed 293T cells into 10cm petri dishes containing DMEM medium containing 10% FBS and cultured for 24 hours.

[0139] (2) After completing step (1), take the plate and transfect the cells in it with pNL4-3-Luc-RE (using TransMAXi transfection reagent), then replace the culture system with new DMEM medium containing 2% FBS and culture for 48 hours.

[0140] (3) After completing step (2), centrifuge and collect the supernatant, which is the control solution.

[0141] 5. Detection of fluorescence signals

[0142] The fluorescence signals of each virus solution obtained in steps 1 to 3 and the control solution obtained in step 4 were detected respectively. The fluorescence signals of each virus solution obtained in steps 1 to 3 were more than 100 times that of the control solution.

[0143] The results are as follows Figure 3 As shown, BDBV-NbO2 has good neutralizing activity against BDBV, with an IC50 of approximately 0.02 ug / mL. It also has a slight neutralizing effect on EBOV, with an IC50 of approximately 0.25 ug / mL, and a 60% neutralizing effect on TAFV.

[0144] Example 3: Nanobody BDBV-Nb02 inhibits Bundibugyo type Ebola virus infection

[0145] The pseudoviruses to be tested were: EBOV, BDBV, and TAFV. EBOV represents the Zaire-type Ebola pseudovirus, BDBV represents the Bundibugyo-type pseudovirus, and TAFV represents the Tai Forest-type pseudovirus. All of the above pseudoviruses carried a luciferase reporter gene.

[0146] Before the experiment begins, prepare sufficient quantities of nanobodies BDBV-Nb02, BDBV pseudovirus, EBOV pseudovirus, and TAFV pseudovirus.

[0147] 1. Resuscitate and culture Huh7.5.1 cells until they reach the logarithmic growth phase. Digest the cells with trypsin, resuspend them in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture, and adjust the cell concentration to a suitable density. Add 100 μL of the prepared cell suspension to each well to achieve the appropriate final cell concentration. Incubate the 96-well plate overnight in a cell culture incubator at 37°C and 5% CO2.

[0148] 2. After completing step 1, in a sterile 96-well plate, the nanobody BDBV-Nb02 was sequentially diluted 10-fold from a high concentration of 100 μg / mL, creating 10 different concentration gradients. Each concentration was used in 3-4 replicates, with 50 μL per well. In each well of the 96-well plate containing the nanobody, 50 μL of diluted pseudovirus was added and gently mixed to ensure sufficient contact between the BDBV-Nb02 nanobody and the pseudovirus. The 96-well plate was then incubated at 37°C for 1 hour to allow for complete binding of the nanobody and pseudovirus.

[0149] 3. After completing step 2, add 100 μL of the prepared virus antibody mixture to each well of a 96-well plate containing Huh7.5.1, and incubate for 48-72 hours, observing cell growth and lesion conditions during this period.

[0150] 4. After completing step 3, use the appropriate detection reagent to detect the pseudovirus based on the reporter gene (luciferase gene) it carries. For example, for pseudoviruses carrying the luciferase gene, after culturing, aspirate the culture medium from the 96-well plate, wash the cells 2-3 times with PBS, and then add 1× cell lysis buffer (50 μL / well) and lyse at room temperature for 20 minutes. Transfer the liquid phase from each well of the 96-well plate to an opaque 96-well plate in a one-to-one correspondence. Then add LuciferaseAssay Reagent (40 μL / well) to each well and detect the fluorescence intensity. The fluorescence intensity reflects the activity of luciferase, thus indicating the level of pseudovirus infection.

[0151] (5) Data Analysis

[0152] Wells containing only pseudoviruses and cells (without BDBV-Nb02 nanobody) served as positive controls, while wells containing only cells and culture medium served as negative controls. The inhibition rate of the nanobody against pseudovirus infection was calculated based on the detection values ​​of each well.

[0153] The inhibition rate is calculated using the formula: Inhibition rate (%) = [(Positive control well detection value - Experimental well detection value) / Positive control well detection value] × 100%. The IC was calculated using nonlinear regression analysis performed with GraphPad Prism after plotting dose-response curves. 50 value.

[0154] Calculate the half-maximal inhibitory concentration (IC50) of the BDBV-Nb02 nanobody against the expression of fluorescent molecules induced by different Ebola pseudoviruses in cells. 50 The values ​​are shown in Table 1. In Table 1, BDBV-NbO2 exhibits good neutralizing activity against BDBV, with an IC50 value of [value missing]. 50 At approximately 0.02 ug / mL, it also has a slight neutralizing effect on EBOV, IC50. 50 It is approximately 0.25 ug / mL. It has a weak neutralizing effect on TAFV.

[0155] The bonding force results between BDBV-GP1 and BDBV-NbO2 are as follows: Figure 4 As shown, BDBV-Nb02 binds to BDBV-GP1 protein in a 2-fold gradient from 4nM to 64nM, and the final measured KD value is 0.86nM.

[0156] Table 1. Antiviral activity IC50 of BDBV-Nb02 against different Ebola pseudoviruses 50

[0157] Pseudovirus IC 50 ]]> EBOV 0.25 μg / mL BDBV 0.02 μg / mL TAFV 60% neutralization

[0158] In this screening, a total of 15 antibodies with good binding activity to BDBV-GP protein were selected. After screening with BDBV pseudovirus and EBOV pseudovirus, BDBV-Nb01 and BDBV-Nb02 with neutralizing ability were obtained. After increasing the antibody dosage, it was found that BDBV-Nb02 had better neutralizing activity.

[0159] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A nanobody targeting Bundibugyo and Zaire Ebola viruses, or an antigen-binding fragment containing said nanobody, characterized in that, The nanobody has three complementary determinant clusters CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 is shown in positions 27-33 of SEQ ID No:1, the amino acid sequence of CDR2 is shown in positions 47-58 of SEQ ID No:1, and the amino acid sequence of CDR3 is shown in positions 97-109 of SEQ ID No:

1.

2. The nanobody or antigen-binding fragment according to claim 1, characterized in that, The nanobody is either A1) or A2) below: A1) Nanobody with an amino acid sequence as shown in SEQ ID No:1; A2) Nanobodies obtained by attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No:

1.

3. A biomaterial relating to the nanobody of claim 1 or 2, wherein the biomaterial is any one of the following: B1) A nucleic acid molecule encoding the nanobody or antigen-binding fragment of claim 1 or 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecules described in B1); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1); B5) Recombinant microorganisms containing the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that, B1) The nucleic acid molecule is a nucleic acid molecule encoding the nanobody of claim 1 or 2, wherein the encoding gene of CDR1 is nucleotide 79-99 of SEQ ID No:2, the encoding gene of CDR2 is nucleotide 139-174 of SEQ ID No:2, and the encoding gene of CDR3 is nucleotide 289-327 of SEQ ID No:

2.

5. A method for preparing the nanobody according to claim 1 or 2, comprising the following steps: Nucleic acid molecules encoding the nanobody of claim 1 or 2 are introduced into recipient cells to obtain transgenic cells expressing the nanobody, and the transgenic cells are cultured to obtain the nanobody.

6. The biomaterial according to claim 3 or 4, or the preparation method according to claim 5, characterized in that, B1) The nucleic acid molecule is any of the following: C1) A DNA molecule with a nucleotide sequence as shown in SEQ ID No:2; C2) hybridizes with the DNA molecule defined by C1) under stringent conditions and encodes the DNA molecule of the nanobody; A DNA molecule encoding the nanobody having 99%, 95%, 90%, 85%, or 80% homology with the DNA sequence defined by C3) and C1) or C2).

7. A nanobody fusion protein, characterized in that, The nanobody fusion protein is formed by fusing the nanobody or antigen-binding fragment described in claim 1 or 2 with another molecule, wherein the other molecule is the Fc domain of an immunoglobulin, a fluorescent protein, or a VHH with different specificities.

8. The nanobody fusion protein according to claim 7, characterized in that, The fusion protein is any one of the following: M1) is a fusion protein with the amino acid sequence shown in SEQ ID No:4; M2) is a protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No:

4.

9. An ELISA detection kit targeting Bundibugyo and Zaire Ebola viruses, characterized in that, The kit comprises the nanobody of claim 1 or 2, the biomaterial of claim 3 or 4, or the fusion protein of claim 7 or 8.

10. Any of the following applications: E1) The use of the nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of products for inhibiting Bundibugyo and Zaire Ebola viruses; E2) Use of the biomaterial of claim 3 or 4 in the preparation of products for inhibiting Bundibugyo and Zaire Ebola viruses; E3) The application of the preparation method of claim 5 or 6 in the preparation of products for inhibiting Bundibugyo and Zaire Ebola viruses; E4) The use of the nanobody of claim 1 or 2 in the preparation of products conjugated with Bundibugyo and Zaire Ebola viruses; E5) Use of the biomaterial of claim 3 or 4 in the preparation of products in combination with Bundibugyo and Zaire Ebola viruses; E6) The application of the preparation method of claim 5 or 6 in the preparation of products in combination with Bundibugyo and Zaire Ebola viruses; E7) Use of the kit of claim 9 in the preparation of products conjugated with Bundibugyo and Zaire Ebola viruses; E8) The use of the nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of medicaments for the prevention and / or treatment of Bundibugyo and Zaire Ebola virus infections.

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