WO2020043069A1 - Ns1蛋白的结合蛋白 - Google Patents

Ns1蛋白的结合蛋白 Download PDF

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WO2020043069A1
WO2020043069A1 PCT/CN2019/102632 CN2019102632W WO2020043069A1 WO 2020043069 A1 WO2020043069 A1 WO 2020043069A1 CN 2019102632 W CN2019102632 W CN 2019102632W WO 2020043069 A1 WO2020043069 A1 WO 2020043069A1
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Prior art keywords
binding protein
cdr
sequence
binding
antibody
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PCT/CN2019/102632
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English (en)
French (fr)
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崔鹏
何志强
孟媛
钟冬梅
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东莞市朋志生物科技有限公司
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Priority to US17/272,096 priority Critical patent/US20210395347A1/en
Priority to CA3111163A priority patent/CA3111163A1/en
Priority to KR1020217009156A priority patent/KR20210049893A/ko
Publication of WO2020043069A1 publication Critical patent/WO2020043069A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the fields of biotechnology and medical technology, and particularly to a binding protein of NS1 protein.
  • DF Dengue fever
  • DENV-1 serotype viruses
  • DENV-2 deoxyribonucleic acid
  • DENV-3 deoxyribonucleic acid
  • DENV-4 dengue fever
  • Aedes aegypti Aedes albopictus
  • DF is the most widely distributed and most damaging arbovirus disease. It is widely prevalent in more than 100 countries and regions in tropical, subtropical Africa, the Americas, Southeast Asia, and the Western Pacific.
  • DF is a severe flu-like disease.
  • the main manifestations are sudden onset, high fever, severe headache, posterior orbital pain, muscle and joint pain, which may be accompanied by rash, lymphadenopathy, and leukopenia, which can affect all people, but the symptoms may vary depending on the age of the patient.
  • This type of disease is generally called classical dengue fever, and this type spreads rapidly and can cause larger epidemics.
  • Dengue hemorrhagic fever is characterized by high fever, hemorrhage, large liver, and circulatory failure in severe cases. It has a high mortality rate and is a more serious clinical type.
  • Shock syndrome is called dengue shock syndrome.
  • dengue fever There is no specific cure for dengue fever. Without proper treatment, the case fatality rate of dengue hemorrhagic fever can exceed 20%, and after effective treatment methods, the case fatality rate can be lower than 1%.
  • Key points for diagnosis of dengue fever 1) epidemiological data, activities in the 15 days before the onset, whether you have been to the endemic area, and experience of mosquito bites; 2) clinical features, sudden onset, fever, "three pains and three reds", rash; 3 ) Laboratory tests, white blood cells and platelets decreased; serum characteristics were positive for IgM; IgG in the recovery phase increased 4 times compared to the acute phase; virus or specific antigen was isolated.
  • Clinical methods for detecting dengue virus include virus culture, serological detection, and viral nucleic acid detection.
  • NS1 protein is the only glycoprotein among the non-structural proteins of dengue virus. It is highly antigenic and does not trigger antibody-dependent enhanced infection (ADE), so it can be used as a target for immunoassay.
  • the immunoassay requires specific monoclonal antibodies against the NS1 protein. Traditionally, mouse-derived monoclonal antibodies are used in clinical practice.
  • mouse monoclonal antibodies have been widely used in scientific research, clinical diagnosis and treatment.
  • the hybridoma method requires the use of mouse abdominal cavity to produce hybridomas, it is particularly affected by individual mice, the production is unstable, the batch-to-batch variation is large, and because it contains mouse autoantibodies, purification is difficult.
  • the present disclosure is based on a new anti-dengue virus NS1 7D9 monoclonal antibody obtained through new screening. Through cloning, identification and analysis of the genetic structure, the CDR region sequence is determined, and the corresponding antigen-binding domain including the binding to NS1 protein is constructed Isolated binding protein, and established a corresponding eukaryotic cell expression system, produced and purified the binding protein.
  • An isolated binding protein comprising an antigen-binding domain that binds to an NS1 protein provided by the present disclosure, wherein the antigen-binding domain comprises at least one complementarity determining region selected from the following amino acid sequence;
  • the complementarity determining region CDR-VH1 is A-S-G-Y-X1-F-T-G-X2-Y-M-H, where:
  • X1 is S or T and X2 is F, Y or L;
  • CDR-VH2 is R-V-N-P-X1-N-G-G-X2-S-Y-N-Q-K-F-X3-G, where:
  • X1 is Q, D or N, X2 is S or T, X3 is V, I or F;
  • the CDR-VH3 is A-X1-E-G-X2-H-Y-D-R-A, where:
  • X1 is Q, N or R, and X2 is V, F or Y;
  • CDR-VL1 is Q-S-L-L-Y-X1-S-N-X2-K-N-S-L-A, where:
  • X1 is S or T and X2 is N, Q or H;
  • the complementarity determining region CDR-VL2 is W-A-S-T-X1-E-S, where:
  • X1 is Q, R or N;
  • the complementarity determining region CDR-VL3 is Q-Q-Y-X1-T-Y-X2-Y-T, where:
  • X1 is F, Y or V, and X2 is A, P or G.
  • X1 is S
  • X2 is T and X3 is I;
  • X2 is Q
  • X2 is P.
  • X2 is F.
  • X2 is Y.
  • X2 is L.
  • X1 is Q.
  • X1 is D.
  • X1 is N.
  • X1 is Q and X2 is V.
  • X1 is Q and X2 is F.
  • X1 is Q and X2 is Y.
  • X1 is N and X2 is V.
  • X1 is N and X2 is F.
  • X1 is N and X2 is Y.
  • X1 is R and X2 is V.
  • X1 is R and X2 is F.
  • X1 is R and X2 is Y.
  • X1 is S.
  • X1 is T.
  • X1 is Q.
  • X1 is R.
  • X1 is N.
  • X1 is F.
  • X1 is Y.
  • X1 is V.
  • the amino acid at the corresponding position of the complementarity determining region is as follows,
  • the binding protein includes at least 3 CDRs; or, the binding protein includes at least 6 CDRs;
  • the binding protein is one of Nanobody, F (ab ') 2 , Fab', Fab, Fv, scFv, a bispecific antibody, and the smallest recognition unit of an antibody;
  • the binding protein includes the light chain backbone regions FR-L1, FR-L2, FR-L3, and FR-L4 in sequence as shown in SEQ ID NO: 1-4, and / or, and the sequence as shown in SEQ ID NO : 5-8 heavy chain backbone regions FR-H1, FR-H2, FR-H3 and FR-H4.
  • the binding protein further comprises an antibody constant region sequence
  • the constant region sequence is selected from the sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD;
  • the species source of the constant region is cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, marten, chicken, duck, goose, fire Chicken, cockfight or human;
  • the constant region is derived from a mouse
  • the light chain constant region sequence is shown in SEQ ID NO: 9;
  • the heavy chain constant region sequence is shown in SEQ ID NO: 10.
  • the present disclosure also provides a nucleic acid encoding the aforementioned binding protein.
  • the present disclosure also provides a vector including the aforementioned nucleic acid.
  • the present disclosure also provides a host cell including the above-mentioned nucleic acid or the above-mentioned vector.
  • the present disclosure also provides a kit comprising one or more of the aforementioned binding proteins, nucleic acids or vectors.
  • the kit further includes a label for labeling the binding protein.
  • the present disclosure also provides a method for producing the aforementioned binding protein, comprising the steps of preparing the aforementioned nucleic acid or vector;
  • the above-mentioned host cells are cultured in a medium, and the thus-produced binding protein is recovered from the medium or from the cultured host cells.
  • the present disclosure also provides an application of the above-mentioned binding protein in preparing a product for detecting a dengue infection.
  • the present disclosure provides isolated binding proteins that include an antigen binding domain that binds to the NS1 protein, including specific heavy chain CDRs and light chain CDRs.
  • the binding protein can specifically recognize and bind to the NS1 protein, and has high sensitivity and specificity, thereby realizing the detection of dengue virus.
  • it is not necessary to use mouse abdominal cavity to induce hybridoma cells to produce the binding protein the production is less difficult, and the antibody function is more stable.
  • amino acid means a naturally occurring or non-naturally occurring carboxy alpha-amino acid.
  • amino acid as used in this disclosure may include naturally occurring amino acids and non-naturally occurring amino acids.
  • Naturally occurring amino acids include alanine (three-letter code: A1a, single-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), Cysteine (Cys, c), Glutamine (G1n, Q), Glutamic acid (G1u, E), Glycine (G1y, G), Histidine (His, H), Isoleucine (I1e , I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P) , Serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W
  • Non-naturally occurring amino acids include, but are not limited to, ⁇ -aminoadipate, aminobutyric acid, citrulline, homocitrulline, homoleucine, homoarginine, hydroxyproline, norleucine, pyridine Alanine, sarcosine, etc.
  • isolated binding protein is a protein that, due to its derived origin or source, does not bind to a naturally-binding component that accompanies it in its natural state; it is substantially free of the same species Other proteins; expressed by cells from different species; or not present in nature.
  • a protein that is chemically synthesized or synthesized in a cellular system different from the cell of its natural origin will be “isolated” from its naturally associated components.
  • the protein can also be made substantially free of naturally bound components by isolation, for example using protein purification techniques well known in the art.
  • isolated binding protein including an antigen binding domain refers broadly to all proteins / protein fragments that include a CDR region.
  • antibody includes polyclonal and monoclonal antibodies and the antigen-compound-binding fragments of these antibodies, including Fab, F (ab ') 2, Fd, Fv, scFv, bispecific antibodies, and the minimum recognition units for antibodies, and these antibodies And fragments of single-chain derivatives.
  • the type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
  • antibody includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, and related synthetic isoforms (isoforms).
  • antibody is used interchangeably with "immunoglobulin”.
  • variable region refers to the amino-terminal domain of the heavy or light chain of an antibody.
  • variable domain of the heavy chain is called "VH”.
  • variable domain of the light chain is called "VL”.
  • CDRs complementarity determining regions
  • framework region that separates them.
  • the backbone region of an antibody serves to locate and align the CDRs that are primarily responsible for binding to the antigen.
  • bispecific antibody or “bifunctional antibody” refers to an artificial hybrid binding protein having two different pairs of heavy / light chains and two different binding sites. Bispecific binding proteins can be produced by a variety of methods, including fusion hybridomas or linking Fab ' fragments.
  • sequence identity refers to the similarity between at least two different sequences. This percentage identity can be determined by standard algorithms, such as the Basic Local Alignment Search Tool (BLAST); the algorithm of Needleman, etc .; or the algorithm of Meyers, etc.
  • BLAST Basic Local Alignment Search Tool
  • a set of parameters may be a Blosum 62 scoring matrix and a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of Meyers and Miller ((1989) CABIOS 4: 11-17), which has been incorporated Into the ALIGN program (version 2.0), a PAM120 weighted residue table, gap length penalty of 12, and gap penalty of 4 were used. Percent identity is usually calculated by comparing sequences of similar length.
  • affinity refers to the strength of the binding of an antigen-binding domain of a binding protein or antibody to an antigen or epitope. Affinity can be measured by KD value, the smaller the KD value, the greater the affinity.
  • the present disclosure provides an isolated binding protein comprising an antigen-binding domain that binds to the NS1 protein, wherein the antigen-binding domain includes at least one complementarity determining region selected from the following amino acid sequence: or; and the following amino acid sequence
  • the complementarity determining region has at least 80% sequence identity and has an affinity for NS1 protein of KD ⁇ 6.12 ⁇ 10 -8 mol / L;
  • CDR-VH1 is G-F-N-I-K-X1-Y-Y-X2-H, where:
  • X1 is E or D and X2 is V, I or L;
  • CDR-VH2 is W-I-D-P-X1-N-G-K-T-X2-Y-D-P-K-X3-Q-D, where:
  • the complementarity determining region CDR-VH1 is A-S-G-Y-X1-F-T-G-X2-Y-M-H, where:
  • X1 is S or T and X2 is F, Y or L;
  • CDR-VH2 is R-V-N-P-X1-N-G-G-X2-S-Y-N-Q-K-F-X3-G, where:
  • X1 is Q, D or N, X2 is S or T, X3 is V, I or F;
  • the CDR-VH3 is A-X1-E-G-X2-H-Y-D-R-A, where:
  • X1 is Q, N or R, and X2 is V, F or Y;
  • CDR-VL1 is Q-S-L-L-Y-X1-S-N-X2-K-N-S-L-A, where:
  • X1 is S or T and X2 is N, Q or H;
  • the complementarity determining region CDR-VL2 is W-A-S-T-X1-E-S, where:
  • X1 is Q, R or N;
  • the complementarity determining region CDR-VL3 is Q-Q-Y-X1-T-Y-X2-Y-T, where:
  • X1 is F, Y or V, and X2 is A, P or G.
  • X1 appearing in the six CDR regions of the binding protein described in the present disclosure each independently represent an amino acid defined in the present disclosure
  • X2 in each CDR region independently represents an amino acid defined in the present disclosure
  • X3 appearing in the six CDR regions of the binding protein described in this disclosure each independently represent an amino acid defined in the present disclosure.
  • the binding specificity and affinity of antibodies are mainly determined by CDR sequences. According to mature and well-known existing technologies, the amino acid sequence of non-CDR regions can be easily changed to obtain similar biologically active changes. body. Accordingly, the present disclosure also includes "functional derivatives" of the binding protein.
  • a “functional derivative” refers to a variant of amino acid substitution.
  • a functional derivative retains a detectable binding protein activity, preferably the activity of an antibody capable of binding to the NS1 protein.
  • a “functional derivative” may include a "variant” and a "fragment”, which have similar biological activity as the CDR sequence identical to the binding protein described in the present disclosure.
  • the antigen-binding domain has at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75% of the complementarity determining region of the amino acid sequence described below. , Or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97% , or at least 98%, or at least 99% sequence identity with the NS1 protein and having KD ⁇ 6.12 ⁇ 10 -8 mol / L, for example, may be a KD value 3.13 ⁇ 10 -10 mol / L, 4.45 ⁇ 10 - 10 mol / L, 5.34 ⁇ 10 -10 mol / L, 7.05 ⁇ 10 -10 mol / L, 2.32 ⁇ 10 -9 mol / L, 2.94 ⁇ 10 -9 mol / L, 3.10 ⁇ 10 -9
  • X1 is S
  • X2 is T and X3 is I;
  • X2 is Q
  • X2 is P.
  • X2 is F.
  • X2 in the complementarity determining region CDR-VH1, X2 is Y.
  • X2 is L.
  • X1 is D.
  • X1 is N.
  • X1 is Q and X2 is V.
  • X1 is Q and X2 is F.
  • X1 is Q and X2 is Y.
  • X1 is N and X2 is V.
  • X1 is N and X2 is F.
  • X1 is N and X2 is Y.
  • X1 is R and X2 is V.
  • X1 is R and X2 is F.
  • X1 is R and X2 is Y.
  • X1 is S.
  • X1 is T in the CDR-VL1.
  • X1 is Q.
  • X1 is R in the CDR-VL2.
  • X1 is N in the CDR-VL2.
  • X1 is F in the CDR-VL3.
  • X1 is Y in the CDR-VL3.
  • X1 is V in the CDR-VL3.
  • the binding protein includes at least 3 CDRs; or, the binding protein includes at least 6 CDRs.
  • the binding protein is a whole antibody comprising a variable region and a constant region.
  • the binding protein is one of a Nanobody, F (ab ') 2, Fab', Fab, Fv, scFv, a bispecific antibody, and an antibody minimum recognition unit.
  • the binding protein includes a light chain framework region FR-L1, FR-L2, FR-L3, and FR-L4, which are shown in sequence as SEQ ID NOs: 1-4, and / Alternatively, the sequence is in the order of the heavy chain backbone regions FR-H1, FR-H2, FR-H3, and FR-H4 shown in SEQ ID NOs: 5-8.
  • the binding protein further comprises an antibody constant region sequence.
  • the constant region sequence is selected from the sequence of any one of the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.
  • the species source of the constant region is cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, marten , Chicken, duck, goose, turkey, cockfighting or human.
  • the constant region is derived from a mouse
  • the light chain constant region sequence is shown in SEQ ID NO: 9;
  • the heavy chain constant region sequence is shown in SEQ ID NO: 10.
  • the disclosure includes a nucleic acid sequence comprising the binding protein.
  • a nucleic acid sequence includes variants of its conservative substitutions (e.g., degenerate codon substitutions) and complementary sequences.
  • the terms "nucleic acid” and “polynucleotide” are synonymous and encompass genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.
  • the disclosure includes an expression vector containing a nucleic acid sequence encoding the binding protein, wherein the nucleic acid sequence is operably linked to at least one regulatory sequence.
  • operably linked is meant that a coding sequence is linked to a regulatory sequence in a manner that allows expression of the coding sequence.
  • Regulatory sequence selection is used to direct the expression of the protein of interest in a suitable host cell, and includes promoters, enhancers and other expression regulatory elements.
  • a vector may refer to a molecule or agent comprising a nucleic acid of the present disclosure, or a fragment thereof, capable of carrying genetic information and that can deliver the genetic information into a cell.
  • Typical vectors include plasmids, viruses, phages, cosmids, and minichromosomes.
  • the vector can be a cloning vector (i.e., a vector for transferring genetic information into a cell, the cell can be propagated and the cell can be selected for the presence or absence of the genetic information) or an expression vector (i.e., contains the necessary genetic elements) A vector that allows the genetic information of the vector to be expressed in a cell).
  • the cloning vector may contain a selectable marker and an origin of replication that matches the cell type specified by the cloning vector, while the expression vector contains regulatory elements necessary to affect expression in a given target cell.
  • the nucleic acids of the disclosure or fragments thereof can be inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragments of the disclosure.
  • This new carrier is also part of this disclosure.
  • the vector may include a plasmid, a phage, a cosmid, a minichromosome, or a virus, and also includes naked DNA that is transiently expressed only in a specific cell.
  • the cloning vectors and expression vectors of the present disclosure are capable of spontaneous replication, and thus can provide high copy numbers for high-level expression or high-level replication purposes for subsequent cloning.
  • the expression vector may include a promoter for driving expression of a nucleic acid fragment of the present disclosure, optionally a nucleic acid sequence encoding a signal peptide that secretes or integrates the peptide expression product onto a membrane, a nucleic acid fragment of the present disclosure, and optionally Nucleic acid sequence encoding a terminator.
  • the expression vector When the expression vector is manipulated in a production strain or cell line, the vector may be integrated into the host cell genome when introduced into the host cell, or it may not be integrated into the host cell genome.
  • Vectors typically carry a replication site and a marker sequence capable of providing phenotypic selection in transformed cells.
  • the expression vectors of the present disclosure are used to transform host cells. Such transformed cells are also part of the present disclosure and may be cultured cells or cell lines used to propagate nucleic acid fragments and vectors of the present disclosure, or to recombinantly prepare the polypeptides of the present disclosure.
  • the transformed cells of the present disclosure include microorganisms such as bacteria (such as E. coli, Bacillus, etc.). Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably mammalian cells, such as CHO cells.
  • the transformed cells are capable of replicating the nucleic acid fragments of the disclosure.
  • the expression product may be exported to a culture medium or carried on the surface of the transformed cell.
  • the binding proteins provided by the present disclosure can be used to detect the presence of one or more target molecules in a biological sample.
  • detection includes quantitative or qualitative detection.
  • the biological sample comprises cells or tissue.
  • colloidal gold immunoassay is an immunolabeling technique that uses colloidal gold as a tracer marker to apply to antigens and / or antibodies.
  • Colloidal gold is polymerized by chloroauric acid under the action of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, tannic acid, etc. into gold particles of a specific size, and becomes a stable colloidal state due to electrostatic action.
  • the immunoassay of the present disclosure includes a colloidal gold immunoassay, and also includes ELISA, chemiluminescence, and other tests or methods using an antigen-antibody reaction.
  • the present disclosure provides an article of manufacture (e.g., a kit) that includes a material that can be used to diagnose a dengue virus infection.
  • the article includes a container and a label or package insert on or with the container.
  • Suitable containers include, for example, bottles or syringes and the like.
  • the container may be made of various materials such as glass or plastic.
  • the container holds a composition, either alone or in combination with another composition that can be effectively used to diagnose dengue fever.
  • At least one active agent in the composition is a binding protein provided by the present disclosure.
  • detection kits comprising a binding protein, nucleic acid, or vector described herein.
  • a method for detecting an NS1 protein antigen in a test sample including:
  • the binding protein may be labeled with an indicator that shows signal strength to make the complex easily detectable.
  • the immune complex further includes a second antibody, and the second antibody binds to the binding protein
  • the binding protein forms a paired antibody with the second antibody in the form of a first antibody for binding different epitopes of the NS1 protein;
  • the second antibody may be labeled with an indicator showing signal strength, so that the complex can be easily detected.
  • the immune complex further includes a second antibody, and the second antibody binds to the NS1 protein antigen;
  • the binding protein is used as an antigen of the second antibody, and the second antibody may be labeled with an indicator showing signal strength so that the complex can be easily detected.
  • the indicator for displaying signal intensity includes a fluorescent substance, a quantum dot, a digoxin labeled probe, biotin, a radioisotope, a radioactive contrast agent, a paramagnetic ion fluorescent microsphere, and an electron. Any of a dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, colloidal gold, or an enzyme.
  • the fluorescent substances include Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY -FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ′, 5′-dichloro-2 ′, 7′-dimethoxyfluorescein, 5-carboxy-2 ′, 4 ′ , 5 ′, 7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxo-1,
  • the radioisotopes include 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y , 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re , 51 Mn, 52 mMn, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb, and 83 Sr.
  • the enzyme includes any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.
  • the fluorescent microspheres are: polystyrene fluorescent microspheres, which are coated with rare earth fluorescent ion europium inside.
  • the present disclosure provides a kit for determining, for example, the presence of an NS1 protein in a subject infected with dengue fever, the kit comprising at least one binding protein provided by the present disclosure, related Buffer, a reagent required for reacting a liquid sample with the binding protein, and a reagent for determining the presence of a positive or negative binding reaction between the NS1 protein and the binding protein.
  • the kit can, for example, utilize a labeled binding protein as an antibody, where the label can be any suitable label, such as a colloidal gold label.
  • the present disclosure also provides the use of a binding protein as described herein for detecting dengue infection.
  • the present disclosure also provides a method for detecting a dengue infection, including:
  • the presence of the immune complex is indicative of the presence of a dengue infection.
  • the method is based on a fluorescence immunoassay technique, a chemiluminescence immunoassay technique, an immunochromatography technique, a radioimmunoassay, and / or an enzyme-linked immunoassay technique.
  • the method is based on an enzyme-linked immunoassay.
  • the method is based on a colloidal gold immunoassay.
  • the sample is selected from at least one of whole blood, peripheral blood, serum, or plasma.
  • the subject is a mammal, such as a primate, such as a human.
  • the restriction enzyme and Prime Star DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO.
  • SMARTERTM RACE cDNA amplification kit was purchased from Takara.
  • the pMD-18T vector was purchased from Takara.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
  • the hybridoma cell line secreting anti-dengue virus NS1 and 7D9 monoclonal antibodies is a hybridoma cell line newly obtained by the applicant.
  • NUP Nested Universal Primer A
  • Light chain genes were amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mkR primers, and heavy chain genes were amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mHR primers Amplify.
  • the product was purified and recovered by agarose gel electrophoresis. The product was added with rTaq DNA polymerase, and then inserted into the pMD-18T vector, transformed into DH5 ⁇ competent cells. After the bacteria grew, the heavy chain and light chain genes were cloned. Four clones were sent to Invitrogen for sequencing.
  • the gene sequence obtained by the above sequencing was analyzed in the IMGT antibody database, and analysis was performed using VNTI11.5 software to confirm that the heavy chain and light chain primers were correct for the amplified genes, among which the light chain amplified genes In the fragment, the VL gene sequence is 339 bp, belonging to the VkII gene family, with a 57 bp leader peptide sequence in front; among the gene fragments amplified by the heavy chain primer pair, the VH gene sequence is 364 bp, belonging to the VH 1 gene family, with 57 bp in front Leader peptide sequence.
  • the vector is a constructed eukaryotic expression vector of the recombinant antibody.
  • the expression vector has been introduced into polyclonal restriction sites such as HindIII, BamHI, and EcoRI, and is named as pcDNA 3.4A expression vector, which is abbreviated as 3.4A expression vector in the following; according to the aforementioned pMD-18T
  • the results of the sequencing of the antibody gene in China were used to design the heavy- and light-chain gene-specific primers for the anti-dengue virus NS1 7D9 antibody, with HindIII and EcoRI restriction sites and protective bases at each end.
  • the primers are as follows:
  • DN7D9-HF 5’-CCCAAGCTTATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATG-3 ’;
  • DN7D9-HR 5’-CACGAATTCTTACTAACACTCATTCCTGTTGAAGCTCTTGACAATG-3 ’;
  • DN7D9-LF 5’-CATGAAGCTTATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATG-3 ’;
  • DN7D9-LR 5'-CATGGAATTCTTACTAACACTCATTCCTGTTGAAGCTCTTGACA-3 '.
  • 0.72KB light chain gene fragment and 1.4KB heavy chain gene fragment were amplified by PCR amplification method.
  • the heavy and light chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragment and the vector were purified and recovered, the heavy chain and light chain genes were ligated into the 3.4A expression vector, respectively. Recombinant expression plasmids for heavy and light chains were obtained.
  • the plasmid was diluted to 400 ng / ml with ultrapure water, and the Chinese hamster ovary (CHO) cells were adjusted to 1.43 ⁇ 10 7 cells / mL in a centrifuge tube. 100 ⁇ L of the plasmid was mixed with 700 ⁇ L of the cells, transferred into an electric rotor, and transferred into a 10 mL CD containing CD.
  • the CHO AGT medium was cultured in a shaker at 37 ° C (8% CO 2 , vibration amplitude 150); the cell viability was sampled every day, and when the cell viability was less than 50%, the cell culture supernatant was centrifuged to obtain a protein sample.
  • Antigen DN-II-Ag # (Phenix Bio Products) was diluted 1000-fold with CB, 100 ⁇ L of polystyrene enzyme standard block was added to each well, and overnight at 4 ° C; the next day, the washing solution was washed twice with PBST, patted dry; added to block Solution (20% BSA + 80% PBS), 120 ⁇ L per well, 37 ° C, 1h, pat dry; add diluted cell supernatant, 100 ⁇ L / well, 37 ° C, 30min (partial supernatant 1h); wash solution wash 5 Beat dry; add sheep anti-mouse IgG-HRP, 100 ⁇ L per well, 37 ° C, 30 min; wash the washing solution 5 times, pat dry; add coloring solution A (50 ⁇ L / well), add coloring solution B ( 50 ⁇ L / well), 10 min; add stop solution, 50 ⁇ L / well; read the OD value at 450 nm (refer to 630 nm) on a micro
  • the above sample was subjected to affinity purification using a protein A affinity chromatography column, and 500 mg of the recombinant antibody was obtained after purification. 4 ⁇ g of the purified antibody was subjected to reducing SDS-PAGE. Two bands were shown after reducing SDS-PAGE, one was a 28KD light chain (the sequence is shown in SEQ ID NO: 11), and the other was a 50KD heavy chain (the sequence is shown in SEQ ID NO: 12).
  • the purified antibody was diluted to 10 ⁇ g / ml with PBST, and the recombinant protein of DN-II quality control product (produced by the company) was diluted with PBST in a gradient: 500nmol / ml, 250nmol / ml, 125nmol / ml, 62.5nmol / ml, 31.3nmol / ml, 15.6nmol / ml, 7.81nmol / ml, 0nmol / ml;
  • Example 2 Although the antibody obtained in Example 2 (having the light and heavy chains shown in SEQ ID NOs: 11 and 12) has the ability to bind to the NS1 protein, the affinity and antibody activity are not ideal. Therefore, the applicant's The light chain CDR and the heavy chain CDR are mutated.
  • CDR-VH1 is A-S-G-Y-T (X1) -F-T-G-F (X2) -Y-M-H;
  • CDR-VH2 is R-V-N-P-Q (X1) -N-G-G-S (X2) -S-Y-N-Q-K-F-V (X3) -G;
  • CDR-VH3 is A-Q (X1) -E-G-V (X2) -H-Y-D-R-A;
  • CDR-VL1 is Q-S-L-L-Y-S (X1) -S-N-N (X2) -K-N-S-L-A;
  • CDR-VL2 is W-A-S-T-Q (X1) -E-S;
  • CDR-VL3 is Q-Q-Y-F (X1) -T-Y-A (X2) -Y-T.
  • X1, X2, and X3 are all mutation sites.
  • Example 2 After the mutation, the method provided in Example 2 was used to detect the antibody activity, and some of the results were as follows:
  • mutation 1 has the best activity effect, so mutation 1 is used as a backbone sequence to screen mutation sites with better titers.
  • the overall affinity of the antibody based on mutation 1 is higher than that of WT on the premise of ensuring antibody activity.
  • the present disclosure provides isolated binding proteins that include an antigen binding domain that binds to the NS1 protein, including specific heavy chain CDRs and light chain CDRs.
  • the binding protein can specifically recognize and bind to the NS1 protein, and has high sensitivity and specificity, thereby realizing the detection of dengue virus.
  • it is not necessary to use mouse abdominal cavity to induce hybridoma cells to produce the binding protein the production is less difficult, and the antibody function is more stable.

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Abstract

提供包括与NS1蛋白结合的抗原结合结构域的分离的结合蛋白，包括特定的重链CDR和轻链CDR。该结合蛋白能够特异性地识别并结合NS1蛋白，具有较高的灵敏度和特异性，从而实现对登革热病毒的检测。并且，无需利用小鼠腹腔诱生杂交瘤细胞来生产该结合蛋白，生产难度小的同时，抗体功能更加稳定。

Description

NS1蛋白的结合蛋白
相关申请的交叉引用
本申请要求于2018年08月28日提交中国专利局的申请号为201810999042.9、名称为“NS1蛋白的结合蛋白”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及生物技术和医学技术领域，尤其是涉及一种NS1蛋白的结合蛋白。
背景技术
登革热(dengue fever,DF)是由4个血清型病毒(DENV-1、DENV-2、DENV-3、DENV-4)引起的急性蚊媒传染病，主要通过埃及伊蚊和白纹伊蚊传播。DF是分布最广，发病最多，危害较大的一种虫媒病毒性疾病，广泛流行于全球热带和亚热带的非洲、美洲、东南亚和西太平洋地区的100多个国家和地区。
在临床上，DF是一种严重的流感样的疾病。主要表现为起病突然、高热、剧烈头痛、眼眶后痛、肌肉和关节痛，可伴有皮疹、淋巴腺肿和白血球减少，可波及所有人群，但症状可因病人的年龄不同而不同。一般将这种病型称为古典登革热，此类型传播迅速，可引起较大规模的流行。在登革热流行期，易感人群的罹患率通常为40％-50％，可高达80％-90％，但病死率很低。登革热出血热是以高热、出血、肝大，严重病例循环衰竭为特征，病死率高，是较为严重的一种临床类型。伴有休克综合症的称为登革休克综合症。
登革热没有特效的治疗方法。如果没有合适的治疗，登革出血热的病死率可超过20％，经过有效的治疗方法，病死率可低于1％。登革热诊断要点：1)流行病学资料，发病前15天的活动情况，有否去过流行区，蚊虫叮咬经历；2)临床特征，突然发病，发热，“三痛三红”，皮疹；3)实验室检查，白细胞、血小板下降；检测血清特性IgM阳性；恢复期IgG比急性期有4倍增长；分离到病毒或特异性抗原。临床上用于登革病毒的检测方法有病毒培养、血清学检测、病毒核酸检测等。病毒分离所需时间较长，达不到快速诊断的目的，而常规的血清学诊断又因存在广泛的交叉反应而受到干扰。胶体金标记的免疫层析方法具有快速、简便、不需要依赖特殊装备、能够实现现场检测等特点，成为目前传染病快速诊断中研究的热点。NS1蛋白是登革病毒非结构蛋白中唯一的糖蛋白，抗原性极强且不引发 抗体依赖的增强感染作用(ADE)，所以可作为免疫检测的靶标。而免疫检测需要针对NS1蛋白的特异性单克隆抗体，传统临床用的都是鼠源性的单克隆抗体。长期以来，鼠单克隆抗体被广泛的应用于科研、临床诊断和治疗。但由于杂交瘤方式需要采用小鼠腹腔来生产杂交瘤，因此受小鼠个体影响特别大，生产不稳定、批间差大、并且由于含小鼠自身抗体，因此纯化难度大。
发明内容
本公开是基于新筛选所获得的抗登革病毒NS1 7D9单克隆抗体，通过克隆、鉴定与基因结构的分析，确定了其CDR区序列，构建了相应的包括与NS1蛋白结合的抗原结合结构域的分离的结合蛋白，并建立了相应的真核细胞表达系统，生产并纯化了该结合蛋白。
本公开提供的包括与NS1蛋白结合的抗原结合结构域的分离的结合蛋白，其中所述抗原结合结构域包括选自下述氨基酸序列的至少一个互补决定区；
或与下述氨基酸序列的互补决定区具有至少80％的序列同一性且与NS1蛋白具有KD≤6.12×10 -8mol/L的亲和力；
互补决定区CDR-VH1为A-S-G-Y-X1-F-T-G-X2-Y-M-H，其中，
X1是S或T，X2是F、Y或L；
互补决定区CDR-VH2为R-V-N-P-X1-N-G-G-X2-S-Y-N-Q-K-F-X3-G，其中，
X1是Q、D或N，X2是S或T，X3是V、I或F；
互补决定区CDR-VH3为A-X1-E-G-X2-H-Y-D-R-A，其中，
X1是Q、N或R，X2是V、F或Y；
互补决定区CDR-VL1为Q-S-L-L-Y-X1-S-N-X2-K-N-S-L-A，其中，
X1是S或T，X2是N、Q或H；
互补决定区CDR-VL2为W-A-S-T-X1-E-S，其中，
X1是Q、R或N；
互补决定区CDR-VL3为Q-Q-Y-X1-T-Y-X2-Y-T，其中，
X1是F、Y或V，X2是A、P或G。
例如，
所述互补决定区CDR-VH1中，X1是S；
所述互补决定区CDR-VH2中，X2是T，X3是I；
所述互补决定区CDR-VL1中，X2是Q；
所述互补决定区CDR-VL3中，X2是P。
例如，所述互补决定区CDR-VH1中，X2是F。
例如，所述互补决定区CDR-VH1中，X2是Y。
例如，所述互补决定区CDR-VH1中，X2是L。
例如，所述互补决定区CDR-VH2中，X1是Q。
例如，所述互补决定区CDR-VH2中，X1是D。
例如，所述互补决定区CDR-VH2中，X1是N。
例如，所述互补决定区CDR-VH3中，X1是Q，X2是V。
例如，所述互补决定区CDR-VH3中，X1是Q，X2是F。
例如，所述互补决定区CDR-VH3中，X1是Q，X2是Y。
例如，所述互补决定区CDR-VH3中，X1是N，X2是V。
例如，所述互补决定区CDR-VH3中，X1是N，X2是F。
例如，所述互补决定区CDR-VH3中，X1是N，X2是Y。
例如，所述互补决定区CDR-VH3中，X1是R，X2是V。
例如，所述互补决定区CDR-VH3中，X1是R，X2是F。
例如，所述互补决定区CDR-VH3中，X1是R，X2是Y。
例如，所述互补决定区CDR-VL1中，X1是S。
例如，所述互补决定区CDR-VL1中，X1是T。
例如，所述互补决定区CDR-VL2中，X1是Q。
例如，所述互补决定区CDR-VL2中，X1是R。
例如，所述互补决定区CDR-VL2中，X1是N。
例如，所述互补决定区CDR-VL3中，X1是F。
例如，所述互补决定区CDR-VL3中，X1是Y。
例如，所述互补决定区CDR-VL3中，X1是V。
在一种或多种实施方式中，所述互补决定区对应位点的氨基酸如下，
Figure PCTCN2019102632-appb-000001
Figure PCTCN2019102632-appb-000002
例如，所述结合蛋白中包括至少3个CDRs；或者，所述结合蛋白包括至少6个CDRs；
例如，所述结合蛋白为纳米抗体、F(ab’) 2、Fab’、Fab、Fv、scFv、双特异抗体和抗体最小识别单位中的一种；
例如，所述结合蛋白包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
在一种或多种实施方式中，所述结合蛋白还包含抗体恒定区序列；
例如，所述恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列；
例如，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人；
例如，所述恒定区来源于小鼠；
轻链恒定区序列如SEQ ID NO:9所示；
重链恒定区序列如SEQ ID NO:10所示。
本公开还提供了一种核酸，所述核酸编码上述的结合蛋白。
本公开还提供了一种载体，所述载体包括上述的核酸。
本公开还提供了一种宿主细胞，所述宿主细胞包括上述的核酸或上述的载体。
本公开还提供了一种试剂盒，所述试剂盒包括上述的结合蛋白、核酸或载体中的一种或多种。
在一种或多种实施方式中，所述试剂盒还包括用于标记所述结合蛋白的标记。
本公开还提供了一种生产上述结合蛋白的方法，包括制备上述的核酸或载体的步骤；
可选的，包括如下步骤：
在培养基中培养上述的宿主细胞，从培养基中或从所培养的宿主细胞中回收如此产生的结合蛋白。
另外，本公开还提供了上述的结合蛋白在制备用于检测登革热感染的产品中的应用。
本公开提供的包括与NS1蛋白结合的抗原结合结构域的分离的结合蛋白，包括特定的重链CDR和轻链CDR。该结合蛋白能够特异性地识别并结合NS1蛋白，具有较高的灵敏度和特异性，从而实现对登革热病毒的检测。并且，无需利用小鼠腹腔诱生杂交瘤细胞来生产该结合蛋白，生产难度小的同时，抗体功能更加稳定。
具体实施方式
除非本文另有定义，连同本公开使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。术语的含义和范围应当清晰，然而，在任何潜在不明确性的情况下，本文提供的定义优先于任何字典或外来定义。在本公开中，除非另有说明，“或”的使用意味着“和/或”。此外，术语“包括”及其他形式的使用是非限制性的。
一般地，连同本文描述的细胞和组织培养、分子生物学、免疫学、微生物学、遗传学以及蛋白和核酸化学和杂交使用的命名法和其技术是本领域众所周知和通常使用的那些。除非另有说明，本公开的方法和技术一般根据本领域众所周知，且如各种一般和更具体的 参考文献中所述的常规方法来进行，所述参考文献在本说明书自始至终引用和讨论。酶促反应和纯化技术根据制造商的说明书、如本领域通常实现的或如本文所述来进行。连同本文描述的分析化学、合成有机化学以及医学和药物化学使用的命名法、以及其实验室程序和技术是本领域众所周知和通常使用的那些。
为了本公开可以更容易地理解，选择的术语在下文定义。
术语“氨基酸”表示天然存在或非天然存在的羧基α-氨基酸。术语“氨基酸”用在本公开中可以包括天然存在的氨基酸和非天然存在的氨基酸。天然存在的氨基酸包括丙氨酸(三字母密码：A1a，单字母密码：A)，精氨酸(Arg，R)，天冬酰胺(Asn，N)，天冬氨酸(Asp，D)，半胱氨酸(Cys，c)，谷氨酰胺(G1n，Q)，谷氨酸(G1u，E)，甘氨酸(G1y，G)，组氨酸(His，H)，异亮氨酸(I1e，I)，亮氨酸(Leu，L)，赖氨酸(Lys，K)，甲硫氨酸(Met，M)，苯丙氨酸(Phe，F)，脯氨酸(Pro，P)，丝氨酸(Ser，S)，苏氨酸(Thr，T)，色氨酸(Trp，W)，酪氨酸(Tyr，Y)，和缬氨酸(Va1，V)。非天然存在的氨基酸包括但不限于α-氨基己二酸，氨基丁酸，瓜氨酸，高瓜氨酸，高亮氨酸，高精氨酸，羟基脯氨酸，正亮氨酸，吡啶基丙氨酸，肌氨酸等等。
术语“分离的结合蛋白”是这样的蛋白：其由于衍生起源或来源不与天然结合的组分结合，所述天然结合的组分在其天然状态下与其伴随；基本上不含来自相同物种的其他蛋白；由来自不同物种的细胞表达；或在自然界中不存在。因此，化学合成或在不同于其天然起源的细胞的细胞系统中合成的蛋白将是与其天然结合的组分“分离的”。还可以通过分离，例如使用本领域众所周知的蛋白纯化技术，使得蛋白基本上不含天然结合的组分。
术语“包括抗原结合结构域的分离的结合蛋白”泛指包含CDR区的一切蛋白/蛋白片段。“抗体”此用语包括多克隆抗体及单克隆抗体以及这些抗体的抗原化合物结合片段，包括Fab、F(ab’)2、Fd、Fv、scFv、双特异抗体和抗体最小识别单位，以及这些抗体和片段的单链衍生物。抗体的类型可以选择IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD。此外，“抗体”此用语包括天然存在的抗体以及非天然存在的抗体，包括例如嵌合型(chimeric)、双功能型(bifunctional)和人源化(humanized)抗体，以及相关的合成异构形式(isoforms)。“抗体”此用语可和“免疫球蛋白”互换使用。
抗体的“可变区”或“可变结构域”是指抗体的重链或轻链的氨基端结构域。重链的可变结构域被称为“VH”。轻链的可变结构域被称为“VL”。这些结构域通常是抗体的最可变的部分，并含有抗原结合位点。轻链或重链可变区由被三个称为“互补决定区”或“CDR”的高变区以及将其分隔开的骨架区(framework region)构成。抗体的骨架区起到定位和对齐CDR的作用，所述CDR主要负责与抗原的结合。
如本文所用，术语“双特异性抗体”或“双功能性抗体”指具有两对不同重/轻链和两个不同结合位点的人造杂合结合蛋白。双特异性结合蛋白可通过多种方法来生成，包括融合杂交瘤或连接Fab’片段。
如本文所用，术语“序列同一性”指至少两种不同序列之间的相似性。此百分比同一性可通过标准算法来确定，例如基本局部比对搜索工具(Basic Local Alignment Search Tool，BLAST)；Needleman等的算法；或Meyers等的算法。在一种或多种实施方式中，一组参数可以是Blosum 62评分矩阵及缺口罚分12、缺口延伸罚分4、和移码缺口罚分5。在一种或多种实施方式中，两种氨基酸或核苷酸序列之间的百分比同一性还可以使用Meyers和Miller((1989)CABIOS 4：11-17)的算法来确定，该算法已经掺入ALIGN程序(2.0版)，使用PAM120权重残基表、缺口长度罚分12、和缺口罚分4。百分比同一性通常通过比较相似长度的序列来计算。
如本文所用，术语“亲和力”是指结合蛋白或抗体的抗原结合结构域与抗原或抗原表位的结合强度。亲和力可以用KD值来衡量，KD值越小代表亲和力越大。
本公开提供了一种包括与NS1蛋白结合的抗原结合结构域的分离的结合蛋白，其中所述抗原结合结构域包括选自下述氨基酸序列的至少一个互补决定区：或；与下述氨基酸序列的互补决定区具有至少80％的序列同一性且与NS1蛋白具有KD≤6.12×10 -8mol/L的亲和力；
互补决定区CDR-VH1为G-F-N-I-K-X1-Y-Y-X2-H，其中，
X1是E或D，X2是V、I或L；
互补决定区CDR-VH2为W-I-D-P-X1-N-G-K-T-X2-Y-D-P-K-X3-Q-D，其中，
互补决定区CDR-VH1为A-S-G-Y-X1-F-T-G-X2-Y-M-H，其中，
X1是S或T，X2是F、Y或L；
互补决定区CDR-VH2为R-V-N-P-X1-N-G-G-X2-S-Y-N-Q-K-F-X3-G，其中，
X1是Q、D或N，X2是S或T，X3是V、I或F；
互补决定区CDR-VH3为A-X1-E-G-X2-H-Y-D-R-A，其中，
X1是Q、N或R，X2是V、F或Y；
互补决定区CDR-VL1为Q-S-L-L-Y-X1-S-N-X2-K-N-S-L-A，其中，
X1是S或T，X2是N、Q或H；
互补决定区CDR-VL2为W-A-S-T-X1-E-S，其中，
X1是Q、R或N；
互补决定区CDR-VL3为Q-Q-Y-X1-T-Y-X2-Y-T，其中，
X1是F、Y或V，X2是A、P或G。
在一种或多种实施方式中，出现在本公开所述的结合蛋白的六个CDR区中的X1各自彼此独立地代表本公开所限定的氨基酸；出现在本公开所述的结合蛋白的六个CDR区中的X2各自彼此独立地代表本公开所限定的氨基酸；出现在本公开所述的结合蛋白的六个CDR区中的X3各自彼此独立地代表本公开所限定的氨基酸。
本领域公知，抗体的结合特异性及亲合力均主要由CDR序列决定，根据成熟、公知的现有各项技术可轻易地将非CDR区域的氨基酸序列改变而获得具有相类似的生物活性的变体。因此，本公开也包括该结合蛋白的“功能性衍生物”。“功能性衍生物”是指氨基酸替换的变体，一个功能性衍生物保留有可检测的结合蛋白活性，优选为能结合NS1蛋白的抗体的活性。“功能性衍生物”可以包含“变体”和“片段”，因其具有与本公开所述的结合蛋白完全相同的CDR序列，因此具有相类似的生物活性。
在一些实施方式中，所述抗原结合结构域与下述氨基酸序列的互补决定区具有至少50％，或至少55％，或至少60％，或至少65％，或至少70％，或至少75％，或至少80％，或至少85％，或至少90％，或至少91％，或至少92％，或至少93％，或至少94％，或至少95％，或至少96％，或至少97％，或至少98％，或至少99％的序列同一性且与NS1蛋白具有KD≤6.12×10 -8mol/L，例如，KD值也可以为3.13×10 -10mol/L、4.45×10 -10mol/L、5.34×10 -10mol/L、7.05×10 -10mol/L、2.32×10 -9mol/L、2.94×10 -9mol/L、3.10×10 -9mol/L、3.54×10 -9mol/L、3.90×10 -9mol/L或6.12×10 -8mol/L的亲和力；或者3.13×10 -10mol/L≤KD≤6.12×10 -8mol/L。
在一种或多种实施方式中，
所述互补决定区CDR-VH1中，X1是S；
所述互补决定区CDR-VH2中，X2是T，X3是I；
所述互补决定区CDR-VL1中，X2是Q；
所述互补决定区CDR-VL3中，X2是P。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是F。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是Y。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是D。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X1是N。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是Q，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是Q，X2是F。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是Q，X2是Y。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是N，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是N，X2是F。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是N，X2是Y。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是R，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是R，X2是F。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是R，X2是Y。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是S。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是T。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X1是Q。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X1是R。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X1是N。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是F。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是Y。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是V。
在一种或多种实施方式中，所述结合蛋白中包括至少3个CDRs；或者，所述结合蛋白包括至少6个CDRs。
在一种或多种实施方式中，所述结合蛋白为包含可变区和恒定区的完整抗体。
在一种或多种实施方式中，所述结合蛋白为纳米抗体、F(ab’)2、Fab’、Fab、Fv、scFv、双特异抗体和抗体最小识别单位中的一种。
在一种或多种实施方式中，所述结合蛋白包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
在一种或多种实施方式中，所述结合蛋白还包含抗体恒定区序列。
在一种或多种实施方式中，所述恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列。
在一种或多种实施方式中，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在一种或多种实施方式中，所述恒定区来源于小鼠；
轻链恒定区序列如SEQ ID NO:9所示；
重链恒定区序列如SEQ ID NO:10所示。
在一种或多种实施方式中，本公开包括含有编码该结合蛋白的核酸序列。在本文中，核酸序列包含其保守置换的变体(例如简并密码子的置换)和互补序列。术语“核酸”和“多核苷酸”是同义的，包含基因、cDNA分子、mRNA分子以及它们的片段例如寡核苷酸。
在一种或多种实施方式中，本公开包括含有编码该结合蛋白的核酸序列的表达载体，其中的核酸序列与至少一种调节序列可操作连接。“可操作连接”指的是编码序列以允许编码序列的表达的方式与调节序列连接。调节序列选择用来在合适的宿主细胞中指导目的蛋白质的表达，包含启动子、增强子和其它的表达调控元件。
在本文中，载体可以指包含本公开的核酸或其片段的、能够携带遗传信息并且可以将遗传信息递送到细胞中的分子或试剂。典型的载体包括质粒、病毒、噬菌体、黏粒和微型染色体。载体可以是克隆载体(即用于将遗传信息转移到细胞中的载体，可以繁殖所述细胞并且可以选择存在或不存在所述遗传信息的所述细胞)或表达载体(即包含必要的遗传元件从而允许所述载体的遗传信息在细胞中表达的载体)。因此，克隆载体可以包含选择标记，以及与所述克隆载体所指定的细胞类型相匹配的复制起点，而表达载体则包含对于影响指定靶细胞中的表达必要的调节元件。
本公开的核酸或其片段可以插入到合适的载体中以形成携带本公开核酸片段的克隆载体或表达载体。这种新载体也是本公开的一部分。所述载体可以包括质粒、噬菌体、黏粒、微型染色体或病毒，也包括只在特定细胞中瞬时表达的裸DNA。本公开克隆载体和表达载体能够自发的复制，因此能够为用于随后克隆的高水平表达或高水平复制目的提供高拷贝数。表达载体可以包括用于驱动本公开的核酸片段表达的启动子，可选的编码使所述肽表达产物分泌或整合到膜上的信号肽的核酸序列，本公开的核酸片段，以及可选的编码终止子的核酸序列。当在生产菌株或细胞系中操作表达载体时，载体引入到宿主细胞中时可以整合到宿主细胞的基因组中，也可以不能被整合到宿主细胞基因组中。载体通常携带复制位点，以及能够在转化细胞中提供表型选择的标记序列。
本公开的表达载体用于转化宿主细胞。这种转化细胞也是本公开的一部分，可以是用于增殖本公开的核酸片段和载体、或用于重组制备本公开的多肽的培养细胞或细胞系。本公开的转化细胞包括微生物如细菌(如大肠杆菌、芽孢杆菌等)。宿主细胞也包括来自多细胞生物如真菌、昆虫细胞、植物细胞或哺乳动物细胞，优选来自哺乳动物的细胞，例如CHO细胞。所述转化细胞能够复制本公开的核酸片段。当重组制备本公开的肽组合时，所述表达产物可以输出到培养基中或携带在所述转化细胞的表面。
在一种或多种实施方式中，本公开提供的结合蛋白可以用于检侧一种或多种靶分子在生物样品中的存在。术语“检测”用于本文中时，包括定量或定性检测。在一种或多种实施方式中，生物样品包含细胞或组织。
如本文所用，术语“胶体金免疫测定”是以胶体金作为示踪标志物应用于抗原和/或抗体的一种免疫标记技术。胶体金是由氯金酸在还原剂，诸如白磷、抗坏血酸、枸橼酸钠、鞣酸等作用下，聚合成为特定大小的金颗粒，并由于静电作用成为一种稳定的胶体状态。
本公开的免疫测定包括胶体金免疫测定，还包括ELISA,化学发光以及其它利用抗原抗体反应的试验或方法。
在一种或多种实施方式中，本公开提供一种制品(例如试剂盒)，所述制品包含可用于诊断登革热病毒感染的材料。该制品包括容器和在容器上或与容器一起的标签或包装说明书。适合的容器包括，例如，瓶子或注射器等。所述容器可以由各种材料如玻璃或塑料制成。容器装有组合物，所述组合物是单独地或与可有效用于诊断登革热的另一种组合物结合。组合物中至少一种活性试剂是本公开提供的结合蛋白。
在一种或多种实施方式中，本文提供含有本文描述的结合蛋白、核酸或载体的检测试剂盒。
检测测试样品中的NS1蛋白抗原的方法，其包括：
A)在足以发生抗体/抗原结合反应的条件下，使所述测试样品中的NS1蛋白抗原与如上所述的结合蛋白接触以形成免疫复合物；和
B)检测所述免疫复合物的存在，所述复合物的存在指示所述测试样品中所述NS1蛋白抗原的存在；
在一种或多种实施方式中，所述结合蛋白可以标记由显示信号强度的指示剂，以使得所述复合物容易被检测。
在一种或多种实施方式中，在步骤A)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述结合蛋白结合；
在一种或多种实施方式中，所述结合蛋白以第一抗体的形式与所述第二抗体形成配对抗体，用于结合NS1蛋白的不同抗原表位；
所述的第二抗体可以标记由显示信号强度的指示剂，以使得所述复合物容易被检测。
在一种或多种实施方式中，在步骤A)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述NS1蛋白抗原结合；
在一种或多种实施方式中，所述结合蛋白作为所述第二抗体的抗原，所述的第二抗体可以标记由显示信号强度的指示剂，以使得所述复合物容易被检测。
在一种或多种实施方式中，所述显示信号强度的指示剂包括荧光物质、量子点、地高辛标记探针、生物素、放射性同位素、放射性造影剂、顺磁离子荧光微球、电子致密物质、化学发光标记物、超声造影剂、光敏剂、胶体金或酶中的任一种。
在一种或多种实施方式中，所述荧光物质包括Alexa 350、Alexa 405、Alexa 430、Alexa 488、Alexa 555、Alexa 647、AMCA、氨基吖啶、BODIPY 630/650、BODIPY 650/665、BODIPY-FL、BODIPY-R6G、BODIPY-TMR、BODIPY-TRX、5-羧基-4′,5′-二氯-2′,7′-二甲氧基荧光素、5-羧基-2′,4′,5′,7′-四氯荧光素、5-羧基荧光素、5-羧基罗丹明、6-羧基罗丹明、6-羧基四甲基罗丹明、Cascade Blue、Cy2、Cy3、Cy5、Cy7、6-FAM、丹磺酰氯、荧光素、HEX、6-JOE、NBD(7-硝基苯并-2-氧杂-1，3-二唑)、Oregon Green 488、Oregon Green 500、Oregon Green514、Pacific Blue、邻苯二甲酸、对苯二甲酸、间苯二甲酸、甲酚固紫、甲酚蓝紫、亮甲酚蓝、对氨基苯甲酸、赤藓红、酞菁、偶氮甲碱、花青、黄嘌呤、琥珀酰荧光素、稀土金属穴状化合物、三双吡啶基二胺铕、铕穴状化合物或螯合物、二胺、双花青苷、La Jolla蓝染料、别藻蓝蛋白、allococyanin B、藻蓝蛋白C、藻蓝蛋白R、硫胺、藻红青蛋白、藻红蛋白R、REG、罗丹明绿、罗丹明异硫氰酸酯、罗丹明红、ROX、TAMRA、TET、TRIT(四甲基罗丹明异硫醇)、四甲基罗丹明和德克萨斯红中的任一种。
在一种或多种实施方式中，所述放射性同位素包括 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 64Cu、 67Cu、 67Ga、 68Ga、 86Y、 90Y、 89Zr、 94mTc、 94Tc、 99mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 55Co、 72As、 75Br、 76Br、 82mRb和 83Sr中的任一种。
在一种或多种实施方式中，所述酶包括辣根过氧化酶、碱性磷酸酶和葡萄糖氧化酶中的任一种。
在一种或多种实施方式中，所述荧光微球为：聚苯乙烯荧光微球，内部包裹有稀土荧光离子铕。
如在一种或多种实施方式中，本公开提供用于确定例如感染登革热的受试者中的NS1蛋白存在的试剂盒，所述试剂盒包含至少一种本公开提供的结合蛋白，相关的缓冲剂，用于使液体样品与所述结合蛋白反应所需的试剂，以及用于确定NS1蛋白和结合蛋白之间存在阳性或阴性结合反应的试剂。为了确定NS1蛋白的存在，所述试剂盒可以例如利用带有标记的结合蛋白作为抗体，其中所述标记可以是任何合适的标记，如胶体金标记。
本公开还提供如本文所述的结合蛋白，在检测登革热感染中的应用。
本公开还提供一种检测登革热感染方法，包括：
A)在足以发生结合反应的条件下，使来自受试者的样品与本文所述的结合蛋白接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物，
其中，所述免疫复合物的存在指示登革热感染的存在。
在一种或多种实施方式中，所述方法基于荧光免疫技术、化学发光免疫分析技术、免疫层析技术、放射免疫分析和/或酶联免疫技术。
在一种或多种实施方式中，所述方法基于酶联免疫测定。
在一种或多种实施方式中，所述方法基于胶体金免疫测定法。
在一种或多种实施方式中，所述样品选自全血，外周血，血清或血浆中的至少一种。
在一种或多种实施方式中，所述受试者为哺乳动物，例如为灵长类动物，例如为人类。
下文提供了一些实例用于示例性说明本公开，而不是限制本公开的范围。
实施例1
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。SMARTERTM RACE cDNA扩增试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen公司完成。分泌抗登革病毒NS1 7D9单克隆抗体的杂交瘤细胞株为申请人新筛选得到的杂交瘤细胞株。
1.1引物
扩增重链和轻链5’RACE引物：
SMARTER II A寡核苷酸：
5’-AAGCAGTGGTATCAACGCAGAGTACXXXXX-3’；
5’-RACE CDS引物(5’-CDS)：5’-(T) 25VN-3‘(N＝A,C,G,orT；V＝A,G,orC)；
通用引物A混合物(UPM)：
5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’；
巢式通用引物A(NUP)：5’-AAGCAGTGGTATCAACGCAGAGT-3‘；
mkR：5’-TTTTCCTTTTGAATTCCTAACACTCATTCCTGTTGAAGC-3’；
mHR：5’-ACACTCATTCCTGTTGAAGCTCTTGACAATG-3’。
1.2、抗体可变区基因克隆及测序
从分泌抗登革病毒NS1 7D9单克隆抗体的杂交瘤细胞株中提取中RNA，用SMARTERTM RACE cDNA扩增试剂盒及试剂盒中的SMARTER II A寡核苷酸和5’-CDS引物进行第一链cDNA合成，获得的第一链cDNA产物作为PCR扩增模板。轻链基因以通用引物A混合物(UPM)、巢式通用引物A(NUP)和mkR引物进行扩增，重链基因以通用引物A混合物(UPM)、巢式通用引物A(NUP)和mHR引物进行扩增。其中轻链的引物对扩增出0.8KB左右的目的条带，重链的引物对扩增出1.4KB左右的目的条带。用琼脂糖凝胶电泳纯化回收，产物用rTaq DNA聚合酶进行加A反应后插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取重链及轻链基因克隆各4个克隆送Invitrogen公司进行测序。
1.3、抗登革病毒NS1 7D9抗体可变区基因的序列分析
将上述测序得到的基因序列放在IMGT抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中轻链扩增出的基因片段中，VL基因序列为339bp，属于VkII基因家族，其前方有57bp的前导肽序列；重链引物对扩增出的基因片段中，VH基因序列为364bp，属于VH1基因家族，其前方有57bp的前导肽序列。
1.4、重组抗体表达质粒的构建
pcDNA TM 3.4
Figure PCTCN2019102632-appb-000003
载体为构建的重组抗体真核表达载体，该表达载体已经引入HindIII、BamHI、EcoRI等多克隆酶切位点，并命名为pcDNA 3.4A表达载体，后续简称3.4A表达载体；根据上述pMD-18T中抗体基因测序结果，设计抗登革病毒NS1 7D9抗体的重链和轻链基因特异性引物，两端分别带有HindIII、EcoRI酶切位点和保护碱基，引物如下：
DN7D9-HF：5’-CCCAAGCTTATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATG-3’；
DN7D9-HR：5’-CACGAATTCTTACTAACACTCATTCCTGTTGAAGCTCTTGACAATG-3’；
DN7D9-LF：5’-CATGAAGCTTATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATG-3’；
DN7D9-LR：5’-CATGGAATTCTTACTAACACTCATTCCTGTTGAAGCTCTTGACA-3’。
通过PCR扩增方法扩出0.72KB的轻链基因片段和1.4KB的重链基因片段。重链和轻链基因片段分别采用HindIII/EcoRI双酶切，3.4A载体采用HindIII/EcoRI双酶切，将片段和载体纯化回收后重链基因和轻链基因分别连接3.4A表达载体中，分别得到重链和轻链的重组表达质粒。
实施例2
1、表达上清结合蛋白活性鉴定
质粒用超纯水稀释至400ng/ml，调节中国仓鼠卵巢(CHO)细胞1.43×10 7cells/mL于离心管中，100μL质粒与700μL细胞混合，转入电转杯，电转，转入10mL含CD CHO AGT培养基中，37℃摇床中培养(8％CO 2、震幅150)；每天取样检测细胞活率，当细胞活率低于50％，离心细胞培养上清，得到蛋白样品。
抗原DN-II-Ag#(菲鹏生物产品)用CB稀释1000倍，聚苯乙烯酶标块每孔加样100μL，4℃过夜；次日，洗涤液PBST清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，37℃，1h，拍干；加入稀释后的细胞上清，100μL/孔，37℃，30min(部分上清1h)；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100μL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50μL/孔)，加入显色液B液(50μL/孔)，10min；加入终止液，50μL/孔；酶标仪上450nm(参考630nm)处读OD值。
2、结合蛋白纯化
将上述样品用蛋白A亲和层析柱进行亲和纯化，纯化后得到500mg重组抗体，取4μg纯化的抗体进行还原性SDS-PAGE。在还原性SDS-PAGE后显示两条带，1条为28KD的轻链(序列如SEQ ID NO:11所示)，另一条为50KD的重链(序列如SEQ ID NO:12所示)。
3、抗体亲和力测定
利用AMC传感器，纯化出来的抗体用PBST稀释到10μg/ml，DN-II质控品重组蛋白(公司自产)用PBST进行梯度稀释：500nmol/ml、250nmol/ml、125nmol/ml、62.5nmol/ml、31.3nmol/ml、15.6nmol/ml、7.81nmol/ml、0nmol/ml；
运行流程：缓冲液1(PBST)中平衡60s，抗体溶液中固化抗体300s，缓冲液2(PBST)中孵育180s，抗原溶液中结合420s，缓冲液2中解离1200s，用10mM pH 1.69GLY溶液及缓冲液3进行传感器再生，输出数据。(KD表示平衡解离常数，用来衡量亲和力；kon表示结合速率；koff表示解离速率)，输出数据。
实施例3
实施例2得到的抗体(具有序列如SEQ ID NO:11以及12所示的轻链和重链)虽然具备结合NS1蛋白的能力，但亲和力和抗体活性均不够理想，因而申请人对该抗体的轻链CDR及重链CDR进行突变。
经分析，重链的互补决定区：
CDR-VH1为A-S-G-Y-T(X1)-F-T-G-F(X2)-Y-M-H；
CDR-VH2为R-V-N-P-Q(X1)-N-G-G-S(X2)-S-Y-N-Q-K-F-V(X3)-G；
CDR-VH3为A-Q(X1)-E-G-V(X2)-H-Y-D-R-A；
轻链的互补决定区：
CDR-VL1为Q-S-L-L-Y-S(X1)-S-N-N(X2)-K-N-S-L-A；
CDR-VL2为W-A-S-T-Q(X1)-E-S；
CDR-VL3为Q-Q-Y-F(X1)-T-Y-A(X2)-Y-T。
其中，X1、X2、X3均为突变位点。
在突变后应用实施例2提供的方法对抗体活性进行检测，部分结果如下：
表1 与抗体活性有关的突变位点
位点 CDR-VH1 X1 CDR-VH2 X2/X3 CDR-VL1 X2 CDR-VL3 X2
WT T S/V N A
突变1 S T/I Q P
突变2 S T/F H G
突变3 L K/H V Q
突变4 R E/A I V
突变5 C R/Q P T
表2 抗体活性分析数据
  WT 突变1 突变2 突变3 突变4 突变5
原倍 2.279 2.488 2.382 1.575 1.135 -
稀释3倍 2.228 2.376 2.253 0.924 0.422 -
稀释9倍 2.372 2.463 2.337 0.312 0.059 -
稀释27倍 2.304 2.547 2.452 0.011 - -
稀释81倍 2.150 2.486 2.389 - - -
稀释243倍 1.013 1.763 1.601 - - -
稀释729倍 0.438 0.787 0.702      
空白孔 0.127 0.169 0.092 - - -
“-”代表无活性。
从上表可知，突变1的活性效果最佳，因而以突变1作为骨架序列筛选效价较好的突变位点，部分结果如下。
表3 与抗体亲和力有关的突变位点
Figure PCTCN2019102632-appb-000004
Figure PCTCN2019102632-appb-000005
表4 亲和力分析数据
  KD(M) Kon(1/Ms) Koff(1/S)
突变1 3.77E-09 4.18E+04 1.58E-04
突变1-1 5.34E-10 4.45E+05 2.38E-04
突变1-2 7.05E-10 4.24E+05 2.99E-04
突变1-3 3.13E-10 4.81E+05 1.51E-04
突变1-4 2.58E-09 4.03E+04 1.04E-04
突变1-5 5.20E-10 4.06E+05 2.11E-04
突变1-6 6.65E-10 4.09E+05 2.72E-04
突变1-7 8.11E-10 4.82E+05 3.91E-04
突变1-8 4.92E-10 4.73E+05 2.33E-04
突变1-9 2.94E-09 4.58E+04 1.35E-04
突变1-10 4.02E-10 4.83E+05 1.94E-04
突变1-11 4.25E-10 4.14E+05 1.76E-04
突变1-12 2.32E-09 4.88E+04 1.13E-04
突变1-13 3.54E-09 4.01E+04 1.42E-04
突变1-14 2.44E-09 4.75E+04 1.16E-04
突变1-15 3.61E-09 4.35E+04 1.57E-04
突变1-16 3.40E-09 4.47E+04 1.52E-04
突变1-17 3.08E-09 4.13E+04 1.27E-04
突变1-18 3.70E-09 4.68E+04 1.73E-04
突变1-19 2.49E-09 4.38E+04 1.09E-04
突变1-20 2.61E-09 4.67E+04 1.22E-04
突变1-21 2.41E-09 4.44E+04 1.07E-04
突变1-22 3.29E-09 4.96E+04 1.63E-04
突变1-23 3.71E-09 4.58E+04 1.70E-04
突变1-24 4.45E-10 4.18E+05 1.86E-04
突变1-25 2.81E-09 4.16E+04 1.17E-04
突变1-26 3.13E-09 4.35E+04 1.36E-04
突变1-27 3.11E-09 4.86E+04 1.51E-04
突变1-28 4.06E-10 4.56E+05 1.85E-04
突变1-29 3.56E-09 4.81E+04 1.71E-04
突变1-30 3.80E-09 4.18E+04 1.59E-04
突变1-31 2.31E-09 4.90E+04 1.13E-04
突变1-32 3.76E-09 4.12E+04 1.55E-04
突变1-33 3.07E-09 4.49E+04 1.38E-04
突变1-34 2.81E-09 4.77E+04 1.34E-04
突变1-35 2.24E-09 4.90E+04 1.10E-04
突变1-36 2.95E-09 4.58E+04 1.35E-04
突变1-37 3.61E-09 4.71E+04 1.70E-04
突变1-38 2.28E-09 4.95E+04 1.13E-04
突变1-39 3.10E-09 4.74E+04 1.47E-04
突变1-40 3.90E-09 4.21E+04 1.64E-04
以WT作为骨架序列重复上述实验，进行突变位点的亲和力验证，部分结果如下。
表5 以WT为骨架进行的突变
Figure PCTCN2019102632-appb-000006
Figure PCTCN2019102632-appb-000007
表6 亲和力分析数据
  KD(M) Kon(1/Ms) Koff(1/S)
WT 3.93E-08 4.12E+03 1.62E-04
WT 1-1 5.99E-08 4.86E+03 2.91E-04
WT 1-5 6.12E-08 4.77E+03 2.92E-04
WT 1-6 2.98E-08 4.92E+03 1.46E-04
WT 1-11 2.42E-08 4.67E+03 1.13E-04
从以上数据分析，在保证具有抗体活性的前提下，基于突变1的抗体整体亲和力高于WT。
实施例4
将以上自产抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对21天样品进行活性检测，结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降越势，说明自产抗体稳定好，下表为考核21天的酶免活性检测OD结果。
表7 抗体稳定性考核
样品浓度(ug/ml) 3 0.75 0
4℃，21天样品 1.518 0.665 0.019
-80℃，21天样品 1.647 0.599 0.026
37℃，21天样品 1.626 0.682 0.032
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性
本公开提供的包括与NS1蛋白结合的抗原结合结构域的分离的结合蛋白，包括特定的重链CDR和轻链CDR。该结合蛋白能够特异性地识别并结合NS1蛋白，具有较高的灵敏度和特异性，从而实现对登革热病毒的检测。并且，无需利用小鼠腹腔诱生杂交瘤细胞来生产该结合蛋白，生产难度小的同时，抗体功能更加稳定。

Claims (15)

  1. 一种包括与NS1蛋白结合的抗原结合结构域的分离的结合蛋白，其中所述抗原结合结构域包括选自下述氨基酸序列的至少一个互补决定区，
    或，与下述氨基酸序列的互补决定区具有至少80％的序列同一性且与NS1蛋白具有KD≤6.12×10-8mol/L的亲和力；
    互补决定区CDR-VH1为A-S-G-Y-X1-F-T-G-X2-Y-M-H，其中，
    X1是S或T，X2是F、Y或L；
    互补决定区CDR-VH2为R-V-N-P-X1-N-G-G-X2-S-Y-N-Q-K-F-X3-G，其中，
    X1是Q、D或N，X2是S或T，X3是V、I或F；
    互补决定区CDR-VH3为A-X1-E-G-X2-H-Y-D-R-A，其中，
    X1是Q、N或R，X2是V、F或Y；
    互补决定区CDR-VL1为Q-S-L-L-Y-X1-S-N-X2-K-N-S-L-A，其中，
    X1是S或T，X2是N、Q或H；
    互补决定区CDR-VL2为W-A-S-T-X1-E-S，其中，
    X1是Q、R或N；
    互补决定区CDR-VL3为Q-Q-Y-X1-T-Y-X2-Y-T，其中，
    X1是F、Y或V，X2是A、P或G。
  2. 根据权利要求1所述的结合蛋白，其中，
    所述互补决定区CDR-VH1中，X1是S；
    所述互补决定区CDR-VH2中，X2是T，X3是I；
    所述互补决定区CDR-VL1中，X2是Q；
    所述互补决定区CDR-VL3中，X2是P。
  3. 根据权利要求2所述的结合蛋白，其中所述互补决定区对应位点的氨基酸如下，
    Figure PCTCN2019102632-appb-100001
    Figure PCTCN2019102632-appb-100002
  4. 根据权利要求1-3任一项所述的结合蛋白，其中，所述结合蛋白中包括至少3个CDRs；或者，所述结合蛋白包括至少6个CDRs；
    优选的，所述结合蛋白为纳米抗体、F(ab’)2、Fab’、Fab、Fv、scFv、双特异抗体和抗体最小识别单位中的一种；
    优选的，所述结合蛋白包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
  5. 根据权利要求1-4任一项所述的结合蛋白，其中，所述结合蛋白还包含抗体恒定区序列；
    优选的，所述恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列；
    优选的，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人；
    优选的，所述恒定区来源于小鼠；
    轻链恒定区序列如SEQ ID NO:9所示；
    重链恒定区序列如SEQ ID NO:10所示。
  6. 一种核酸，其中，所述核酸编码权利要求1-5任一项所述的结合蛋白。
  7. 一种载体，其中，所述载体包括权利要求6所述的核酸。
  8. 一种宿主细胞，其中，所述宿主细胞包括权利要求6所述的核酸或权利要求7所述的载体。
  9. 一种试剂盒，其中，所述试剂盒包括权利要求1-5任一项所述的结合蛋白、权利要求6所述的核酸或权利要求7所述的载体中的一种或多种。
    优选地，所述试剂盒还包括用于标记所述结合蛋白的标记。
  10. 一种生产权利要求1-5任一项所述结合蛋白的方法，其中，包括制备权利要求6所述的核酸或权利要求7所述的载体的步骤；
    优选包括如下步骤：
    在培养基中培养权利要求8所述的宿主细胞，从培养基中或从所培养的宿主细胞中回收如此产生的结合蛋白。
  11. 如权利要求1-5任一项所述的结合蛋白在制备用于检测登革热感染的产品中的应用。
  12. 如权利要求1-5任一项所述的结合蛋白，在检测登革热感染中的应用。
  13. 一种检测登革热感染方法，包括：
    A)在足以发生结合反应的条件下，使来自受试者的样品与权利要求1-5中任一项所述的结合蛋白接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物，
    其中，所述免疫复合物的存在指示登革热感染的存在。
  14. 根据权利要求13所述的方法，所述方法基于荧光免疫技术、化学发光免疫分析技术、免疫层析技术、放射免疫分析和/或酶联免疫技术。
  15. 根据权利要求13或14所述的方法，其中所述受试者为哺乳动物，优选地为灵长类动物，更优选地为人类。
PCT/CN2019/102632 2018-08-28 2019-08-26 Ns1蛋白的结合蛋白 WO2020043069A1 (zh)

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