Nanometer antibody for inhibiting infection of Bandong coke type Ebola virus and Zaleel type Ebola virus

Abstract

The invention discloses a nano antibody for inhibiting infection of a Bandong coke type Ebola virus and a Zaleel type Ebola virus. The invention belongs to the technical field of biology, and particularly relates to a nano antibody for inhibiting infection of Bandong Ebola viruses and Zaleel Ebola viruses. The nano antibody BDBV-Nb02 for targeting the Ebola virus, disclosed by the invention, has three complementary determinants, namely, CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is the 27th to 33rd sites of SEQ ID No: 1, the amino acid sequence of the CDR2 is the 47th to 58th sites of SEQ ID No: 1, and the amino acid sequence of the CDR3 is the 97th to 109th sites of SEQ ID No: 1. The nano antibody BDBV-Nb02 has a neutralizing effect on the Bdisbug type Ebola virus and the Zaire type Ebola virus, and has important application prospects and innovative significance in the medical field.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a nanobody for inhibiting the infection of Bundibugyo and Zaïre Ebola viruses. Background Art

[0002] Due to its high pathogenicity and strong transmissibility, Ebola virus has become a pathogen of great significance to global public health. Among the five Ebola virus subtypes that have been identified to be able to infect humans, the Zaire Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV) have seen their case fatality rates soar to 90% during the epidemics in the past decade, bringing devastating disasters to human health and causing huge impacts on social economy and stability.

[0003] Currently, in the field of research and development of Ebola virus vaccines and therapeutic monoclonal antibodies (mAbs), researchers have achieved many remarkable results. However, at present, the vaccines and monoclonal antibodies (mAbs) approved for use in humans are only limited to products related to the Zaire Ebola virus (EBOV). Given that other Ebola virus species such as the Sudan Ebola virus and Bundibugyo Ebola virus still maintain extremely high fatality rates and continue to pose a serious threat to global public health, and considering that Ebola virus, as an RNA virus, has the significant characteristics of poor sequence conservation and strong mutation ability, developing monoclonal antibodies against the genus Ebola virus with broad-spectrum and high-efficiency characteristics has become an urgent task in current scientific research, and it is necessary to conduct in-depth research and exploration 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), although not widely used due to production limitations, 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 virus invasion by binding to different epitopes (such as glycan caps and core regions) of the viral GP. Clinical trials showed that early use could reduce the mortality rate to 6%. mAb114 is a monoclonal antibody isolated from the body of a survivor in 1995, targeting the receptor-binding domain (RBD) of GP, and directly inhibiting the binding of the virus to host cell receptors (such as NPC1). Its monotherapy has a mortality rate of only 11% and is effective against virus variants. Existing antibodies are limited to products related to the Zaire Ebola virus (EBOV). Given that other Ebola virus species such as the Bundibugyo Ebola virus still maintain extremely high fatality rates and continue to pose a serious threat to global public health, the research on antibodies against other species of Ebola virus still has significance. Summary of the Invention

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

[0006] To solve the above problems, the present invention provides a nanobody targeting Ebola virus or an antigen-binding fragment containing the nanobody.

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

[0008] The above CDRs are sequences defined according to the analysis results of the IMGT system.

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

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

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

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

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

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

[0015] Among the above-mentioned nanobodies or antigen-binding fragments, the nanobody can be any of the following:

[0016] A1) A nanobody with an amino acid sequence as 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 expressed in fusion with the target protein to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein-tag can be a His-tag, Flag-tag, MBP-tag, HA-tag, myc-tag, GST-tag, and / or SUMO-tag, the Fc segment of immunoglobulin G, etc.

[0019] In a specific embodiment, the SEQ ID No:1 can be as follows:

[0020] QVQLVESGGGLVQAGGSLRLSCAASGFIFSINGMGWYRQAPGKERELVASISKGDSTNYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAAQFWLVLYHTRSYDYWGQGTQVTVSS.

[0021] In the above nanobody, the nanobody is composed of the complementary determining region and the framework region.

[0022] In the present invention, the term "antibody" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons with the same structural characteristics, which is 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 the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; 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. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0023] In the present invention, "variable" means that certain parts of the variable regions in antibodies are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved parts in the variable regions are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-sheet configuration, connected by three CDRs that form connecting loops and can form partial β-sheet structures in some cases. The CDRs in each chain are held closely together by 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, Volume I, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

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

[0025] The above antigen-binding fragments can be a complete antibody containing the nanobody, a fusion antibody, an antibody-drug conjugate, a Fab fragment, an Fv fragment, a Fab' fragment, an F(ab')2 fragment, a single-chain antibody (ScFv), or a minimal recognition unit (MRU).

[0026] The term "Fab fragment" is a heterodimer formed by the heavy-chain Fd and the complete light chain through a disulfide bond, containing only one antigen-binding site. The above heavy-chain Fd refers to about 1 / 2 of the H chain part in Fab (about 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0027] The term "Fv fragment" means that vectors containing VH and VL genes can be constructed respectively, co-transfected into cells, expressed respectively, and then assembled into a functional Fv antibody; or a stop codon can be set between VH and VL in the vector, two small-molecule protein fragments are expressed respectively, and then an Fv antibody (Fv fragment) is formed by non-covalent binding.

[0028] The term "Fab' fragment" contains a light chain and a part of a heavy chain including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, and thus an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0029] The term "F(ab’)2 fragment" contains two light chains and two heavy chains that contain portions of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, the F(ab’)2 fragment consists of two Fab’ fragments held together by a disulfide bond between the two heavy chains.

[0030] In some embodiments, the nanobody of the present invention can be truncated at the N-terminus or C-terminus so that it only contains a portion of FR1 and / or FR4, or lacks one or both of those framework regions, as long as it substantially retains antigen binding and specificity.

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

[0032] The present invention also provides biomaterials related to the nanobody described above, and the biomaterials can be any of the following:

[0033] B1) A nucleic acid molecule encoding the nanobody or antigen-binding fragment described above;

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

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

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

[0037] B5) A recombinant microorganism containing the nucleic acid molecule described in B1);

[0038] B6) A recombinant microorganism containing the expression cassette described in B2);

[0039] B7) A recombinant microorganism containing the recombinant vector described in B3);

[0040] B8) A recombinant microorganism containing the recombinant vector described in B4).

[0041] Among the above biomaterials, 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, etc.

[0042] Among the above biomaterials, the expression cassette described in B2) refers to DNA that can express the nanobody in a host cell. This DNA can not only include a promoter that initiates transcription of the nanobody-encoding gene, but also include a terminator that terminates transcription of the nanobody-encoding gene. Further, the expression cassette can also include an enhancer sequence.

[0043] Among the above biological materials, the vector can be a plasmid, cosmid, phage or viral vector. A recombinant vector containing the said expression cassette can be constructed using an existing expression vector.

[0044] Among the above biological materials, the recombinant vector can be a recombinant vector obtained by introducing the nucleic acid molecule of B1) into the nanobody fusion protein expression vector pET22b+.

[0045] In the present invention, the recombinant vector can be the recombinant vector pET22b+BDBV-Nb02.

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

[0047] Among the above biological materials, the host cell contains the nucleic acid molecule or vector of the above-mentioned nanobody or antigen-binding fragment. The host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, animal cells (such as mammalian cells, for example, mouse cells, human cells), insect cells and plant cells, etc.

[0048] In the present invention, the cell can be Lemo21(DE3) Escherichia coli cells.

[0049] Among the above biological materials, the nucleic acid molecule of B1) can be a nucleic acid molecule encoding the above-mentioned nanobody. In the nucleic acid molecule, the coding gene of CDR1 is the 79-99th nucleotide of SEQ ID No:2, the coding gene of CDR2 is the 139-174th nucleotide of SEQ ID No:2, and the coding gene of CDR3 is the 289-327th nucleotide of SEQ ID No:2.

[0050] Among the above biological materials, the nucleic acid molecule of B1) can be any of the following:

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

[0052] C2) A DNA molecule that hybridizes with the DNA molecule defined in C1) under stringent conditions and encodes the nanobody;

[0053] A DNA molecule that has a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with any of the DNA sequences defined in C1)-C2) and encodes the nanobody.

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

[0055] CAGGTGCAGCTGGTGGAATCGGGTGGGGGATTGGTTCAGGCGGGGGGAAGTTTACGCTTATCGTGTGCGGCA

[0056] TCTGGTTTCATTTTCTCTATCAACGGTATGGGTTGGTATCGCCAGGCACCAGGTAAAGAACGTGAACTGGTTGC

[0057] CTCTATTTCTAAAGGCGACTCTACCAACTACGCGGACTCAGTGAAAGGCCGCTTCACTATCTCCCGTGATAATG

[0058] CTAAGAACACCGTTTATCTGCAGATGAATTCTTTGAAACCTGAAGACACTGCCGTTTATTATTGCGCAGCTCAGTTCTGGCTGGTTCTGTACCATACTCGTTCTTACGACTACTGGGGTCAGGGAACCCAGGTTACGGTTTCTTCT.

[0059] Among them, the stringent conditions may be as follows: Hybridize at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4, and 1 mM EDTA, and wash in 2×SSC, 0.1% SDS at 50°C; it may also be: Hybridize at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and wash in 1×SSC, 0.1% SDS at 50°C; it may also be: Hybridize at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and wash in 0.5×SSC, 0.1% SDS at 50°C; it may also be: Hybridize at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and wash in 0.1×SSC, 0.1% SDS at 50°C; it may also be: Hybridize at 50°C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and wash in 0.1×SSC, 0.1% SDS at 65°C; it may also be: Hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, and then wash the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS respectively.

[0060] Those of ordinary skill in the art can easily mutate the nucleotide sequence of the gene encoding the nanobody BDBV-NB02 described in B1) of the present invention by using known methods, such as directed evolution and site-directed mutagenesis. Those nucleotides that have been artificially modified and have 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 have the activity of nanobody BDBV-NB02, are all derived from the nucleotide sequence of the present invention and are equivalent to the 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: Introduce the nucleic acid molecule encoding the above-mentioned nanobody into a recipient cell to obtain a transgenic cell expressing the nanobody, and culture 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 above-mentioned nanobody may specifically be any one of the following:

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

[0065] C2) A DNA molecule that hybridizes with the DNA molecule defined in C1) under stringent conditions and encodes the nanobody;

[0066] C3) A DNA molecule that has a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the DNA sequence defined in C1) or C2) and encodes the nanobody.

[0067] Furthermore, the recipient cell can be a prokaryotic cell such as an Escherichia coli cell, and a eukaryotic cell such as a yeast cell, an animal cell (such as a mammalian cell, for example, a mouse cell, a human cell), an insect cell and a plant cell, etc.

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

[0069] The present invention also provides a nanobody fusion protein, which is a fusion of the nanobody or antigen-binding fragment described above with another molecule, and the other molecule can 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-tag protein.

[0071] In a specific embodiment of the present invention, the above-mentioned nanobody fusion protein can be any of the following:

[0072] M1) A fusion protein with an amino acid sequence as shown in SEQ ID No:4;

[0073] M2) A fusion protein obtained by substituting and / or deleting and / or adding amino acid residues to the protein of M1) and having an identity of more than 75% with the protein shown in M1);

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

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

[0076] D1) A DNA molecule having a nucleotide sequence as shown in SEQ ID No: 3: CAGGTGCAGCTGGTGGAATCGGGTGGGGGATTGGTTCAGGCGGGGGGAAGTTTACGCTTATCGTGTGCGGCATCTGGTTTCATTTTCTCTATCAACGGTATGGGTTGGTATCGCCAGGCACCAGGTAAAGAACGTGAACTGGTTGCCTCTATTTCTAAAGGCGACTCTACCAACTACGCGGACTCAGTGAAAGGCCGCTTCACTATCTCCCGTGATAATGCTAAGAACACCGTTTATCTGCAGATGAATTCTTTGAAACCTGAAGACACTGCCGTTTATTATTGCGCAGCTCAGTTCTGGCTGGTTCTGTACCATACTCGTTCTTACGACTACTGGGGTCAGGGAACCCAGGTTACGGTTTCTTCTCTCGAGCACCACCACCACCACCACTGA;

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

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

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

[0080] The present invention also provides an ELISA detection kit targeting Ebola virus, and the kit contains the nanobody, biomaterial or fusion protein described above.

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

[0082] E1) Application of the nanobody or antigen-binding fragment described above in the preparation of a product for inhibiting Ebola virus;

[0083] E2) Application of the biomaterial described above in the preparation of a product for inhibiting Ebola virus;

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

[0085] E4) Use of the kit described above in the preparation of a product for inhibiting Ebola virus;

[0086] E5) Use of the nanobody described above in the preparation of a product that binds to Ebola virus;

[0087] E6) Use of the biomaterial described above in the preparation of a product that binds to Ebola virus;

[0088] E7) Use of the preparation method described above in the preparation of a product that binds to Ebola virus;

[0089] E8) Use of the kit described above in the preparation of a product that binds to Ebola virus;

[0090] E9) Use of the nanobody or antigen-binding fragment described above in the preparation of a drug for preventing and / or treating Ebola virus infection.

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

[0092] In this article, the Ebola virus can be an Ebola pseudovirus.

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

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

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

[0096] The present invention uses the pNL4-3-Luc-R-E vector as the basic backbone to construct three types of Ebola pseudoviruses, which are used as key tools for evaluating the neutralizing activity of nanobodies. Starting from the Ebola pseudovirus model and the screening of the phage library of nanobodies against the GP protein of Bundibugyo Ebola virus, making full use of advanced virological research tools such as Ebola pseudoviruses, and through methods and means of virology, biochemistry, structural biology, etc., nanobodies against Bundibugyo Ebola virus infection are obtained; in addition, in addition to neutralizing Bundibugyo Ebola virus, this antibody also shows a certain degree of neutralizing effect on Zaire Ebola virus. The binding region of this antibody provides potential broad-spectrum neutralizing sites for Ebola virus treatment strategies and has important application prospects and innovative significance in the medical field. Brief Description of the Drawings

[0097] Figure 1 It is the purification result of the nanobody BDBV-Nb02. Among them, lane 1 is the protein marker, and lanes 2-15 are the screened nanobodies BDBV-Nb01-BDBV-Nb015.

[0098] Figure 2 It is the construction of three types of Ebola pseudoviruses.

[0099] Figure 3 It is the inhibition result of BDBV-Nb02 on the invasion of different types of Ebola pseudoviruses into cells. □ represents the inhibition curve of BDBV-NB02 on the infection of EBOV pseudovirus, ▲ represents the inhibition curve of BDBV-NB02 on the infection of BDBV pseudovirus, and ■ represents the inhibition curve of BDBV-NB02 on the infection of TAFV pseudovirus.

[0100] Figure 4 It is the affinity data of BDBV-Nb02 and the GP1 of Bundibugyo Ebola virus. Detailed Description of the Invention

[0101] The present invention will be further described in detail below in combination with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0102] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0103] Unless otherwise specified, the quantitative tests in the following embodiments are all set with three repeated experiments, and the results are averaged.

[0104] The Huh7.5.1 cells in the following examples have been 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 public can obtain this biological material from the applicant, and this biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

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

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

[0107] The luciferase detection system and cell lysate in the following examples were purchased from Promega Corporation, with the product number: E2610.

[0108] pNL4-3-Luc-R-E in the following examples has been 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. The public can obtain this biological material from the applicant, and this biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0109] The pET22b+ vector 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 between the NcoI and Xhol restriction enzyme cleavage 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 a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No: 2 between the NcoI and Xhol restriction enzyme cleavage sites of the starting vector pET22b+ while keeping other sequences of the vector pET28b+ unchanged. The pET22b+BDBV-Nb02 vector can express the BDBV-Nb02 coding gene, and its amino acid sequence is SEQ ID No: 1.

[0114] 2. Plasmid transformation

[0115] According to the description of the manufacturer (New England Biolabs), the constructed plasmid was transformed into chemically competent Lemo21(DE3) Escherichia coli using a 15-second heat shock procedure. The 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 (10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride, made up to 1 L), and incubated with shaking at 37 °C and 200 rpm until the OD value was about 0.6, then 0.2 mM IPTG (MERCK I6758) was added.

[0118] 4. Cell treatment and protein separation

[0119] After incubation, the bacteria cultured in step 3 were centrifuged at 4000 g to collect the cell pellets, which were then resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, supplemented with protease inhibitors and DNAse). The cells were lysed by sonication, with the sonication amplitude set at 20%, and a 3-second on / off cycle for a total of 20 minutes of sonication. Subsequently, the lysed mixture was centrifuged at 16000 g to separate the soluble fraction. Finally, the his-tagged protein was separated from the soluble fraction using the IMAC (immunoaffinity chromatography) method.

[0120] The purification result is as Figure 1 shown: The target protein was obtained at 15 KDa. This target protein was named nanobody BDBV-Nb02.

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

[0122] The nucleotide sequence of the coding gene of the above 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 3 Ebola viruses were cloned onto the pCDNA3.1 vector respectively. EBOV-GP (the gene nucleotide sequence is SEQ ID No:5); BDBV-GP (the gene nucleotide sequence is SEQ ID No:6); TAFV-GP (the gene nucleotide sequence is 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 structure of the recombinant vector pCDNA 3.1-GP(EBOV) is described as follows: A DNA fragment with the sequence of SEQ ID No:5 was inserted between the HindIII and XhoI restriction enzyme sites of the starting vector pCDNA 3.1, and other sequences of the vector pCDNA 3.1 were kept unchanged to obtain the recombinant vector. The pCDNA 3.1-GP(EBOV) vector can express the EBOV-GP protein, and its amino acid sequence is the sequence of GenBank: AHX24649.2 with an update date of 18-12-2014. The nucleotide sequence of the recombinant vector pCDNA 3.1-GP(EBOV) is SEQ ID No:8.

[0126] The structure of the recombinant vector pCDNA 3.1-GP(BDBV) is described as follows: A DNA fragment with the sequence of SEQ ID No:6 is inserted between the HindIII and XhoI restriction enzyme cleavage sites of the starting vector pCDNA 3.1, and other sequences of the vector pCDNA 3.1 remain unchanged to obtain the recombinant vector. The pCDNA 3.1-GP(BDBV) vector can express the BDBV-GP protein, and its amino acid sequence is the sequence of GenBank: YP_003815435.1 with the update date of 07-11-2018. The difference between the recombinant vector pCDNA 3.1-GP(BDBV) and pCDNA 3.1-GP(EBOV) is only that: the nucleotide sequence SEQ ID No:5 in pCDNA 3.1-GP(EBOV) is replaced with SEQ ID No:6, and other sequences remain unchanged.

[0127] The structure of the recombinant vector pCDNA 3.1-GP(TAFV) is described as follows: A DNA fragment with the sequence of SEQ ID No:7 is inserted between the HindIII and XhoI restriction enzyme cleavage sites of the starting vector pCDNA 3.1, and other sequences of the vector pCDNA 3.1 remain unchanged to obtain the recombinant vector. The pCDNA 3.1-GP(TAFV)) vector can express the TAFV-GP protein, and its amino acid sequence is the sequence of GenBank: AWK96625.1 with the update date of 23-7-2019. The difference between the recombinant vector pCDNA 3.1-GP(TAFV) and pCDNA 3.1-GP(EBOV) is only that: the nucleotide sequence SEQ ID No:5 in pCDNA 3.1-GP(EBOV) is replaced with SEQ ID No:7, and other sequences remain unchanged.

[0128] 1. Preparation of EBOV pseudovirus

[0129] (1) Seed 293T cells into a 10-cm petri dish containing DMEM medium with 10% FBS and culture for 24 hours.

[0130] (2) After completing step (1), take the petri dish and transfect the cells therein with pNL4-3-Luc-R-E and pCDNA 3.1-GP(EBOV) (using TransMAXi transfection reagent), then change the culture system to a 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, abbreviated as EBOV virus solution.

[0132] 2. Preparation of BDBV pseudovirus

[0133] Replace pCDNA 3.1-GP(EBOV) with pCDNA 3.1-GP(BDBV), and keep the other steps the same as in Step 1 to obtain a virus solution containing BDBV pseudovirus, abbreviated as BDBV virus solution.

[0134] 3. Preparation of TAFV pseudovirus

[0135] Replace pCDNA 3.1-GP(EBOV) with pCDNA 3.1-GP(TAFV), and keep the other steps the same as in Step 1 to obtain a virus solution containing TAFV pseudovirus, abbreviated as TAFV virus solution.

[0136] The preparation and culture conditions of Ebola pseudoviruses of various species are all: 37°C, 5% CO2, static.

[0137] 4. Preparation of control solution

[0138] (1) Inoculate 293T cells into a 10-cm petri dish containing DMEM medium with 10% FBS and culture for 24 hours.

[0139] (2) After completing Step (1), take the petri dish and transfect the cells therein with pNL4-3-Luc-R-E (using TransMAXi transfection reagent), then change the culture system to a 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 signal

[0142] Detect the fluorescence signals of each virus solution obtained in Steps 1 to 3 and the control solution obtained in Step 4 respectively. The fluorescence signals of each virus solution obtained in Steps 1 to 3 are all more than 100 times that of the control solution.

[0143] The results are as Figure 3 shown: BDBV-Nb02 has good neutralizing activity against BDBV, with an IC50 of approximately 0.02 μg / mL, also has a certain neutralizing effect on EBOV, with an IC50 of approximately 0.25 μg / mL, and has a 60% neutralizing effect on TAFV.

[0144] Example 3. Inhibition of Bundibugyo ebolavirus infection by nanobody BDBV-Nb02

[0145] The pseudoviruses to be tested are: EBOV, BDBV, TAFV. EBOV represents the Zaire ebolavirus pseudovirus, BDBV represents the Bundibugyo ebolavirus pseudovirus, and TAFV represents the Tai Forest ebolavirus pseudovirus. Each of the above pseudoviruses to be tested carries a luciferase reporter gene.

[0146] Before the experiment, prepare sufficient amounts of nanobody BDBV-Nb02, BDBV pseudovirus, EBOV pseudovirus, and TAFV pseudovirus.

[0147] 1. Resuscitate and culture Huh7.5.1 cells. When the cells reach the logarithmic growth phase, digest the cells with trypsin, resuspend the cells with DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture), and adjust the cell concentration to an appropriate density. Add 100 μL of the prepared cell suspension to each well to make the final cell concentration appropriate, and place the 96-well plate in a cell culture incubator at 37°C and 5% CO2 for overnight culture.

[0148] 2. After completing step 1, in a sterile 96-well plate, serially dilute nanobody BDBV-Nb02 from a high concentration of 100 μg / mL by 10-fold, with a total of 10 different concentration gradients, and set 3 - 4 replicates for each concentration, 50 μL per well. In the 96-well plate with added nanobody, add 50 μL of the diluted pseudovirus to each well, and gently mix to ensure full contact between nanobody BDBV-Nb02 and the pseudovirus. Place the 96-well plate in a 37°C cell culture incubator for incubation for 1 hour to allow the nanobody to fully bind to the pseudovirus.

[0149] 3. After completing step 2, add 100 μL of the prepared virus-antibody mixture to each well of the 96-well plate seeded with Huh7.5.1, and culture for 48 - 72 hours. During this period, observe the cell growth status and pathological changes.

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

[0151] (5) Data analysis

[0152] The wells containing only the pseudovirus and cells (without adding the BDBV-Nb02 nanobody) were used as the positive control, and the wells containing only cells and culture medium were used as the negative control. According to the detection values of each well, the inhibition rate of the nanobody against pseudovirus infection was calculated.

[0153] The formula for calculating the inhibition rate is: Inhibition rate (%) = [(Detection value of the positive control well - Detection value of the experimental well) / Detection value of the positive control well] × 100%. By plotting the dose-response curve and using GraphPad Prism for non-linear regression analysis to calculate the IC 50 value.

[0154] Calculate the half-maximal inhibitory concentration IC 50 value of the BDBV-Nb02 nanobody on the expression of cellular fluorescent molecules caused by different Ebola pseudoviruses. The results are shown in Table 1. In Table 1, BDBV-Nb02 has good neutralizing activity against BDBV, and the IC 50 is approximately 0.02 μg / mL. It also has a certain neutralizing effect on EBOV, and the IC 50 is approximately 0.25 μg / mL. It has a weak neutralizing effect on TAFV.

[0155] The binding force results of BDBV-GP1 and BDBV-Nb02 are as Figure 4 shown: BDBV-Nb02 binds to the BDBV-GP1 protein at a 2-fold gradient from 4 nM to 64 nM, and finally the KD value is measured to be 0.86 nM.

[0156] Table 1. Antiviral activity IC 50

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

[0158] A total of 15 antibodies with good binding activity to the BDBV-GP protein were screened in this study. 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 above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. A nanobody targeting Ebola virus or an antigen-binding fragment containing the nanobody, characterized in that: The nanobody has three complementary determining clusters CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown at positions 27-33 of SEQ ID No: 1, the amino acid sequence of the CDR2 is shown at positions 47-58 of SEQ ID No: 1, and the amino acid sequence of the CDR3 is shown at 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 the following A1) or A2): A1) a Nanobody with an amino acid sequence as shown in SEQ ID No: 1; 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.

3. A biomaterial associated with the Nanobody of claim 1 or 2, wherein the biomaterial is any 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 molecule described in B1); B4) a recombinant vector containing the expression cassette described in B2); B5) a recombinant microorganism containing the nucleic acid molecule described in B1); B6) a recombinant microorganism containing the expression cassette described in B2); B7) a recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4).

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

2.

5. A method for preparing the Nanobody according to claim 1 or 2, comprising the following steps: The nucleic acid molecule encoding the nanobody according to claim 1 or 2 is introduced into a recipient cell to obtain a transgenic cell expressing the nanobody, and the transgenic cell is 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 whose nucleotide sequence is shown in SEQ ID No: 2; C2) a DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in C1) and encodes said Nanobody; C3) A DNA molecule that has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined in C1) or C2) and encodes the Nanobody.

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

8. The nanobody fusion protein according to claim 7, characterized in that The fusion protein is any of the following: M1) a fusion protein with an amino acid sequence as shown in SEQ ID No: 4; M2) A protein obtained by connecting 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 Ebola virus, characterized in that: The kit comprises the nanobody according to claim 1 or 2, the biological material according to any one of claims 3-5, or the fusion protein according to claim 7 or 8.

10. Any of the following applications: E1) Use of the Nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of a product for inhibiting Ebola virus; E2) Use of the biological material according to any one of claims 3 to 5 in the preparation of a product for inhibiting Ebola virus; E3) Use of the preparation method according to claim 6 or 7 in preparing a product for inhibiting Ebola virus; E4) Use of the kit according to claim 9 in the preparation of a product for inhibiting Ebola virus; E5) Use of the Nanobody of claim 1 or 2 in the preparation of a product that binds to Ebola virus; E6) Use of the biological material according to any one of claims 3 to 5 in the preparation of a product combined with Ebola virus; E7) Use of the preparation method according to claim 6 or 7 in the preparation of a product combined with Ebola virus; E8) Use of the kit according to claim 9 in the preparation of a product combined with Ebola virus; E9) Use of the Nanobody or antigen-binding fragment of claim 1 or 2 in the preparation of a medicament for preventing and / or treating Ebola virus infection.

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