WO2020043068A1 - 一种ns1蛋白的结合蛋白以及应用 - Google Patents

一种ns1蛋白的结合蛋白以及应用 Download PDF

Info

Publication number
WO2020043068A1
WO2020043068A1 PCT/CN2019/102631 CN2019102631W WO2020043068A1 WO 2020043068 A1 WO2020043068 A1 WO 2020043068A1 CN 2019102631 W CN2019102631 W CN 2019102631W WO 2020043068 A1 WO2020043068 A1 WO 2020043068A1
Authority
WO
WIPO (PCT)
Prior art keywords
cdr
binding protein
sequence
complementarity determining
antibody
Prior art date
Application number
PCT/CN2019/102631
Other languages
English (en)
French (fr)
Inventor
崔鹏
何志强
孟媛
钟冬梅
Original Assignee
东莞市朋志生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 东莞市朋志生物科技有限公司 filed Critical 东莞市朋志生物科技有限公司
Priority to KR1020217004457A priority Critical patent/KR20210031946A/ko
Priority to CA3109433A priority patent/CA3109433A1/en
Priority to US17/267,025 priority patent/US20210301000A1/en
Publication of WO2020043068A1 publication Critical patent/WO2020043068A1/zh

Links

Images

Classifications

    • 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
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • C07K2317/50—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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • 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 and application thereof.
  • 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 supportive therapy, 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. Virus isolation takes a long time and cannot achieve the purpose of rapid diagnosis, and conventional serological diagnosis is disturbed by the existence of extensive cross-reactions.
  • the existing monoclonal antibodies specific for NS1 protein have low activity and poor affinity and cannot be applied to the detection of NS1 protein. Therefore, there is a strong interest in the field for antibodies that effectively and specifically bind to NS1 protein and detect it. demand.
  • the present disclosure provides an isolated binding protein comprising an antigen-binding domain, wherein the antigen-binding domain comprises at least one complementarity determining region selected from the amino acid sequence described below, or has At least 80% sequence identity and KD ⁇ 6.55 ⁇ 10 -8 mol / L affinity to NS1 protein;
  • CDR-VH1 is G-Y-X1-F-T-X2-Y-W-I-G, where:
  • X1 is S or T and X2 is D or E;
  • CDR-VH2 is D-M-X1-P-G-D-X2-Y-I-N-Y-X3-E-K-F-K-G, where:
  • X1 is F or V
  • X2 is V, L or I
  • X3 is Q or N
  • CDR-VH3 is T-N-F-X1-T-X2-G-G-X3-D-Y, where:
  • X1 is I or L
  • X2 is L or V
  • X3 is V, L or I
  • CDR-VL1 is K-S-S-X1-S-L-L-X2-S-D-G-X3-T-Y-L-N, where:
  • X1 is Q or N
  • X2 is E or D
  • X3 is R or K
  • the CDR-VL2 is L-V-X1-K-X2-D-S, where:
  • X1 is S or T and X2 is V, I or L;
  • the complementarity determining region CDR-VL3 is W-X1-G-T-H-F-X2-H-T, where:
  • X1 is Q, Y or W, and X2 is A or P.
  • X1 is T
  • X1 is F
  • X2 is L
  • X2 is D
  • X1 is S
  • X2 is P.
  • X2 is D
  • X2 is E
  • X2 is V and X3 is Q;
  • X2 is V and X3 is N;
  • X2 is L and X3 is Q;
  • X2 is L and X3 is N;
  • X2 is I and X3 is Q;
  • X2 is I and X3 is N;
  • X1 is I and X3 is V;
  • X1 is I and X3 is L;
  • X1 is I and X3 is I;
  • X1 is L and X3 is V;
  • X1 is L and X3 is L;
  • X1 is L and X3 is I;
  • X1 is Q and X2 is R;
  • X1 is Q and X2 is K;
  • X1 is N and X2 is R;
  • X1 is N and X2 is K;
  • X2 is V
  • X2 is I
  • X2 is L
  • X1 is Q
  • X1 is Y
  • X1 is W in the CDR-VL3.
  • amino acids at the corresponding positions of the complementarity determining regions are 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 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 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 an isolated nucleic acid encoding the aforementioned binding protein.
  • the present disclosure also provides a vector including the above-mentioned nucleic acid.
  • the present disclosure also provides a host cell including the aforementioned nucleic acid or the aforementioned vector.
  • the present disclosure also provides a method for producing the above-mentioned binding protein, comprising the steps of: culturing the above-mentioned host cell in a medium, and recovering the produced binding protein from the medium or from the cultured host cell.
  • the present disclosure also provides an application of the above binding protein in the preparation of a product for detecting a dengue infection.
  • the present disclosure also provides a kit comprising one or more of the aforementioned binding protein, the aforementioned isolated nucleic acid, or the aforementioned vector.
  • the present disclosure also provides an application of the above binding protein in detecting dengue infection.
  • the present disclosure also provides a method for detecting a dengue infection, comprising: A) contacting a sample from a subject with the above-mentioned binding protein to perform a binding reaction under conditions sufficient to cause a binding reaction; and B) detecting a result of the binding reaction Immune complex; wherein the presence of the immune complex indicates the presence of a dengue infection.
  • the present invention provides an isolated binding protein including an antigen-binding domain that binds to the NS1 protein.
  • the binding protein can specifically recognize and bind to the NS1 protein, and has high sensitivity and specificity, thereby achieving detection of dengue virus.
  • FIG. 1 is an electrophoresis diagram of a monoclonal antibody against a binding protein of NS1 protein provided in Example 1 of the present disclosure.
  • amino acid refers to naturally-occurring or non-naturally-occurring fussy alpha-amino acids.
  • amino acid as used in this application 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
  • 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 substantially 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 antigenic compound binding fragments of these antibodies, including Fab, F (ab ') 2, Fd, Fv, scFv, bispecific antibodies, and minimal antibody recognition units, And single chain derivatives of these antibodies and fragments.
  • 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). As used herein, the term “antibody” is used interchangeably with “immunoglobulin”.
  • variable region refers to the amino-terminal domain of the heavy or light chain of an antibody.
  • the variable domain of a heavy chain may be referred to as "VH”.
  • variable domain of a light chain can be referred to as "VL”.
  • These domains are usually the most variable part of an antibody and contain an antigen binding site.
  • the light or heavy chain variable region consists of three hypervariable regions called “complementarity determining regions” or "CDRs" and a framework region (FR) separating them.
  • CDRs complementarity determining regions
  • FR framework region
  • backbone region or "FR” means the exclusion of an antibody variable domain from those regions defined as those of the CDR.
  • Each antibody variable domain backbone region can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs.
  • 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.
  • variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the terms “purified” or “isolated” associated with a polypeptide or nucleic acid refer to a polypeptide or nucleic acid that is not in its natural medium or form.
  • isolated includes a polypeptide or nucleic acid removed from its original environment, for example if it is naturally occurring.
  • an isolated polypeptide is typically free of at least some proteins or other cellular components that are typically associated with or generally mixed with or in solution.
  • An isolated polypeptide includes the naturally occurring polypeptide contained in a cell lysate, the polypeptide in a purified or partially purified form, a recombinant polypeptide, the polypeptide expressed or secreted by a cell, and in a heterologous host cell or culture Of the polypeptide.
  • the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).
  • the present disclosure provides an isolated binding protein comprising an antigen-binding domain, the antigen-binding domain comprising at least one complementarity determining region selected from the following amino acid sequence, or having a complementarity determining region having at least 80 % sequence identity with the NS1 protein and having a K D ⁇ 6.55 ⁇ 10 -8 mol / L affinity;
  • CDR-VH1 is G-Y-X1-F-T-X2-Y-W-I-G, where:
  • X1 is S or T and X2 is D or E;
  • CDR-VH2 is D-M-X1-P-G-D-X2-Y-I-N-Y-X3-E-K-F-K-G, where:
  • X1 is F or V
  • X2 is V, L or I
  • X3 is Q or N
  • CDR-VH3 is T-N-F-X1-T-X2-G-G-X3-D-Y, where:
  • X1 is I or L
  • X2 is L or V
  • X3 is V, L or I
  • CDR-VL1 is K-S-S-X1-S-L-L-X2-S-D-G-X3-T-Y-L-N, where:
  • X1 is Q or N
  • X2 is E or D
  • X3 is R or K
  • the CDR-VL2 is L-V-X1-K-X2-D-S, where:
  • X1 is S or T and X2 is V, I or L;
  • the complementarity determining region CDR-VL3 is W-X1-G-T-H-F-X2-H-T, where:
  • X1 is Q, Y or W, and X2 is A or P.
  • 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 an amino acid substitution.
  • a functional derivative retains a detectable binding protein activity, such as 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% of the complementarity determining region of the amino acid sequence described below, Or at least 75%, 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 and KD ⁇ 6.550 ⁇ 10 -8 mol / L with NS1 protein, for example, the KD value can be 4.587 ⁇ 10 -8 mol / L, 2.798 ⁇ 10 -8 mol / L, 1.282 ⁇ 10 -8 mol / L, 1.891 ⁇ 10 -9 mol / L, 4.601 ⁇ 10 -9 mol / L, 6.592 ⁇ 10 -9 mol / L, 8.002 ⁇ 10
  • X1 is T; in the complementarity determining region CDR-VH2, X1 is F; in the complementarity determining region CDR-VH3, X2 is L; the complementarity determining region CDR-VL1 In the above, X2 is D; in the CDR-VL2, X1 is S; in the CDR-VL3, X2 is P.
  • X2 is D.
  • X2 is E.
  • X2 is V and X3 is Q.
  • X2 is V and X3 is N.
  • X2 is L and X3 is Q.
  • X2 is L and X3 is N.
  • X2 is I and X3 is Q.
  • X2 is I and X3 is N.
  • X1 is I and X3 is V.
  • X1 is I and X3 is L.
  • X1 is I and X3 is I.
  • X1 is L and X3 is V.
  • X1 is L and X3 is L.
  • X1 is L and X3 is I.
  • X1 is Q and X2 is R.
  • X1 is Q and X2 is K.
  • X1 is N and X2 is R.
  • X1 is N and X2 is K.
  • X2 is V in the CDR-VL2.
  • X2 is I.
  • X2 is L.
  • X1 is Q.
  • X1 is Y in the CDR-VL3.
  • X1 is W 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; for example, the constant region sequence is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD constant Sequence of zones.
  • 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; for example, 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 an isolated nucleic acid that encodes the aforementioned binding protein.
  • nucleic acid includes variants (e.g., degenerate codon replacements) and complementary sequences that are conservatively substituted.
  • nucleic acid and polynucleotide are synonymous and encompass genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.
  • the present disclosure also provides a vector comprising the aforementioned nucleic acid.
  • 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 Chinese hamster egg cells (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 present disclosure also provides a method for producing the above-mentioned binding protein, including the following steps:
  • the above-mentioned host cells are cultured in a medium, and the produced binding protein is recovered from the medium or from the cultured host cells.
  • the method may be, for example, transfection of a host cell with a nucleic acid vector encoding at least a portion of a binding protein, and culturing the host cell under appropriate conditions such that the binding protein is expressed.
  • the host cell may also be transfected with one or more expression vectors, which may alone or in combination contain DNA encoding at least a portion of the binding protein.
  • Binding proteins can be separated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (such as ion exchange, gel filtration, affinity chromatography, etc.) and / or Electrophoresis.
  • Construction of appropriate vectors containing the coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques well known in the art.
  • the isolated plasmid, DNA sequence or synthetic oligonucleotide is cut, tailed and religated as required. Any method can be used to introduce mutations into the coding sequence to produce variants of the present disclosure, and these mutations can include deletions or insertions or substitutions and the like.
  • the present disclosure also provides antibodies that react with epitopes of the NS1 protein, including monoclonal and polyclonal antibodies.
  • the antibody may contain a complete binding protein, or a fragment or derivative thereof.
  • Preferred antibodies contain all or part of the binding protein.
  • the present disclosure also provides an application of the above binding protein in the preparation of a product for detecting a dengue infection.
  • 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.
  • the immunoassay of the present disclosure includes colloidal gold immunoassay, and also includes ELISA and other tests or methods using antigen-antibody reaction.
  • 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 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.
  • the present disclosure also provides a detection kit including the above-mentioned binding protein, nucleic acid, or vector.
  • 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 NS1 protein in a subject infected with dengue fever, the kit comprising at least one binding protein provided by the present disclosure, an associated buffer Reagents, reagents required for reacting a liquid sample with the binding protein, and reagents 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 fluorescent antibody technology, radioimmunoassay, and / or enzymatic immunotechnology.
  • 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 11E2 monoclonal antibodies is a hybridoma cell line newly screened by the inventors' laboratory.
  • SMARTER II A oligonucleotide: 5’-AAGCAGTGGTATCAACGCAGAGTACXXXX-3 ’;
  • NUP Nested Universal Primer A
  • MIgG-CKR 5'-CTAACACTCATTCCTGTTGAAGC-3 ';
  • MIgG-CHR 5'-TCATTTACCCGGAGTCCGGGAGAAGCTC-3 '.
  • Strand cDNA synthesis, and the obtained first strand cDNA product is used as a template for PCR amplification.
  • the light chain genes were amplified by Universal Primer A Mix (UPM), Nested Universal Primer A (NUP), and MIgG-CKR primers, and the heavy chain genes were amplified by Universal Primer A Mix (UPM), Nested Universal Primer A (NUP), and MIgG-CHR primers were used for amplification.
  • 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 leader peptide sequence of 60 bp in front; among the gene fragments amplified by the heavy chain primer pair, the VH gene sequence is 354 bp, belonging to the VH1 gene family, with 57 bp in front Leader peptide sequence.
  • pcDNA TM 3.4 Vector is the constructed eukaryotic expression vector of the recombinant antibody.
  • the expression vector has been introduced into polyclonal restriction sites such as HindIII, BamHI, EcoRI, and named pcDNA 3.4A expression vector, which will be referred to as 3.4A expression vector for short; according to the above 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 11E2 antibody, with HindIII and EcoRI restriction sites and protective bases at both ends.
  • the primers are as follows:
  • DN11E2-HF 5’-CATAAGCTTATGAAATGGAGCTGGGTTATCCTCTTCTTCC-3 ’;
  • DN11E2-HR 5’-GACGAATTCTCATTTACCCGGAGTCCGGGAGAAGCTC-3 ’;
  • DN11E2-LF 5’-CCCAAGCTTATGGAGACAGACACACTCCTGCTATGG-3 ’;
  • DN11E2-LR 5'-CCCGAATTCCTAACACTCATTCCTGTTGAAGCTCTTGAC-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 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 to an electric rotor, and electrolyzed. Samples were counted on days 3, 5, and 7. On the seventh day, samples were collected and tested.
  • the coating solution diluted the corresponding antigen to the specified concentration, 100 ⁇ L per well at 4 ° C overnight; the next day, the washing solution was washed twice and patted dry; adding blocking 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 the washing solution 5 times, pat dry; add goat anti-mouse IgG-HRP, 100 ⁇ L per well, 37 °C, 30min; Wash the washing solution 5 times, pat dry; Add chromogenic solution A (50 ⁇ L / well), add chromogenic solution B (50 ⁇ L / well), 10min; add stop solution, 50 ⁇ L / well; microplate reader Read the OD value at 450nm (refer to 630nm). The standard concentration and OD value were used as a standard curve to calculate the antibody content in the cell supernatant.
  • Recombinant antibody expression plasmid is stably transfected, and stable cell lines are screened under pressure:
  • the plasmid was diluted to 400ng / ml with ultrapure water, and 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 to an electric rotor, electrotransformed, and counted the next day; 25 ⁇ mol / L MSX 96 wells Pressurize for about 25 days.
  • the cells were resuscitated, they were first cultured in a 125 ml shake flask with an inoculation volume of 30 ml and a 100% Dynamis medium. The cells were placed in a shaker at a speed of 120 r / min, a temperature of 37 ° C, and a carbon dioxide of 8%. Cultivate for 72 hours, inoculate the expanded culture at a density of 500,000 cells / ml, the volume of the expanded culture is calculated according to production requirements, and the culture medium is 100% Dynamis medium. After every 72h, the culture was expanded. When the amount of cells meets production needs, strictly control the seeding density to about 500,000 cells / ml for production.
  • Shake flask parameters rotation speed is 120r / min, temperature is 37 ° C, carbon dioxide is 8%.
  • Feed-feeding Start feeding every day when the flask is cultured to 72h.
  • HyCloneTM Cell BoostBoost Feed 7a feeds 3% of the initial culture volume daily.
  • Feed7b feeds one-thousandth of the initial culture volume daily.
  • Glucose was supplemented with 3g / L on the sixth day.
  • the samples were collected on the 13th day.
  • Affinity purification was performed using a proteinA affinity chromatography column. After purification, 500 mg of recombinant antibody was obtained, and 4 ⁇ g of the purified antibody was subjected to reducing SDS-PAGE.
  • the electrophoresis chart is shown in FIG. 1. Two bands are shown after reducing SDS-PAGE, one is a light chain of about 24KD (sequence is shown in SEQ ID NO: 11) and the other is a heavy chain of about 49KD (sequence is shown in SEQ ID NO: 12 ).
  • Example 1 Although the antibody obtained in Example 1 (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 inventors Light chain CDRs and heavy chain CDRs were subjected to mutation analysis and design.
  • CDR-VH1 is G-Y-S (X1) -F-T-D (X2) -Y-W-I-G;
  • CDR-VH2 is D-M-V (X1) -P-G-D-L (X2) -Y-I-N-Y-Q (X3) -E-K-F-K-G;
  • CDR-VH3 is T-N-F-I (X1) -T-V (X2) -G-G-L (X3) -D-Y;
  • CDR-VL1 is K-S-S-Q (X1) -S-L-L-E (X2) -S-D-G-R (X3) -T-Y-L-N;
  • CDR-VL2 is L-V-T (X1) -K-V (X2) -D-S;
  • CDR-VL3 is W-Q (X1) -G-T-H-F-A (X2) -H-T;
  • X1, X2, and X3 are all mutation sites.
  • Sheep anti-mouse IgG was diluted by the coating solution to 1 ⁇ g / ml for micro-well plate coating, 100 ⁇ L per well, 4 ° C overnight; the next day, the washing solution was washed twice, patted dry; and the blocking solution was added.
  • mutation 1 has the best activity effect, so mutation 1 is used as the backbone sequence to screen mutation sites with better titers (to ensure that the antibody activity obtained is similar to mutation 1 and antibody activity ⁇ 10%). as follows.
  • the purified antibody was diluted to 10ug / ml with PBST, and the recombinant protein of the DN quality control product (recombinant antigen produced by the company) was diluted with PBST gradient: 465.1nmol / ml, 232.6nmol / ml, 116.3nmol / ml, 58.1 nmol / ml, 29.1 nmol / ml, 14.5 nmol / ml, 7.27 nmol / ml, 0 nmol / ml.
  • the present invention provides an isolated binding protein including an antigen-binding domain that binds to the NS1 protein.
  • the binding protein can specifically recognize and bind to the NS1 protein, and has high sensitivity and specificity, thereby achieving detection of dengue virus.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Communicable Diseases (AREA)
  • Pulmonology (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种包含与NS1蛋白结合的抗原结合结构域的分离的结合蛋白，该结合蛋白能够识别并结合NS1蛋白，实现对登革热病毒的检测。

Description

一种NS1蛋白的结合蛋白以及应用
相关申请的交叉引用
本申请要求于2018年08月28日提交中国国家知识产权局的申请号为201810999045.2、名称为“一种NS1蛋白的结合蛋白以及应用”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蛋白的结合蛋白以及应用。
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％，经过有效的支持疗法，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。
现有的NS1蛋白的特异性单克隆抗体活性低、亲和力差，无法很好地应用于NS1蛋白的检测中，因此本领域对于有效且特异性结合NS1蛋白并对其进行检测的抗体存在着强烈需求。
发明内容
本公开提供了一种包含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≤6.55×10 -8mol/L的亲和力；
互补决定区CDR-VH1为G-Y-X1-F-T-X2-Y-W-I-G，其中，
X1是S或T，X2是D或E；
互补决定区CDR-VH2为D-M-X1-P-G-D-X2-Y-I-N-Y-X3-E-K-F-K-G，其中，
X1是F或V，X2是V、L或I，X3是Q或N；
互补决定区CDR-VH3为T-N-F-X1-T-X2-G-G-X3-D-Y，其中，
X1是I或L，X2是L或V，X3是V、L或I；
互补决定区CDR-VL1为K-S-S-X1-S-L-L-X2-S-D-G-X3-T-Y-L-N，其中，
X1是Q或N，X2是E或D，X3是R或K；
互补决定区CDR-VL2为L-V-X1-K-X2-D-S，其中，
X1是S或T，X2是V、I或L；
互补决定区CDR-VL3为W-X1-G-T-H-F-X2-H-T，其中，
X1是Q、Y或W，X2是A或P。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X1是T；
所述互补决定区CDR-VH2中，X1是F；
所述互补决定区CDR-VH3中，X2是L；
所述互补决定区CDR-VL1中，X2是D；
所述互补决定区CDR-VL2中，X1是S；
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D；
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是E；
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是V，X3是Q；
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是V，X3是N；
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是L，X3是Q；
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是L，X3是N；
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是I，X3是Q；
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是I，X3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I，X3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I，X3是L；
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是I，X3是I；
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是L，X3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L，X3是L；
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是L，X3是I；
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是Q，X2是R；
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是Q，X2是K；
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是N，X2是R；
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是N，X2是K；
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是V；
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是I；
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是L；
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是Q；
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W。
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表，
Figure PCTCN2019102631-appb-000001
Figure PCTCN2019102631-appb-000002
在一种或多种实施方式中，所述结合蛋白中包括至少3个CDR；或者，所述结合蛋白包括至少6个CDR。
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；
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分离的核酸，该核酸编码上述结合蛋白。
本公开还提供了一种载体，该载体包括上述的核酸。
本公开还提供了一种宿主细胞，该宿主细胞包括上述核酸或上述载体。
本公开还提供了一种生产上述结合蛋白的方法，包括如下步骤：在培养基中培养上述宿主细胞，从培养基中或从所培养的宿主细胞中回收产生的结合蛋白。
本公开还提供了上述结合蛋白在制备用于检测登革热感染的产品中的应用。
本公开还提供了一种试剂盒，该试剂盒包括上述结合蛋白、上述分离的核酸或上述载体中的一种或多种。
本公开还提供了上述结合蛋白在检测登革热感染中的应用。
本公开还提供了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上述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。
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，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。
附图说明
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一种或多种实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本公开实施例1提供的抗NS1蛋白的结合蛋白的单克隆抗体电泳图。
具体实施方式
本公开可通过后续对于本公开一些实施方式描述以及其中所包括的实施例的详细内容而更容易被了解。
在进一步叙述本公开之前，应明了本公开不会被局限于所述具体实施方式中，因为这些实施方式必然是多样的。亦应明了本说明书中所使用的用语仅是为了阐述具体实施方式，而非作为限制，因为本公开的范围将会被仅仅界定在所附的权利要求中。
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。在本申请中，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梭基α-氨基酸。术语“氨基酸”用在本申请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大然结合的组分。
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。如本文所用，术语“抗体”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)。如本文所用，术语“抗体”可与“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。重链的可变结构域可以被称为“VH”。轻链的可变结构域可以被称为“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，FR)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，“骨架区”或“FR”意味着抗体可变结构域的排除被定义为CDR的那些区域之外的区域。每个抗体可变结构域骨架区可以被进一步细分成被CDR分隔开的毗邻区域(FR1、FR2、FR3和FR4)。
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。
通常情况下，重链和轻链的可变区VL/VH可由以下编号的CDR与FR按如下组合排列连接获得：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
当在本文中使用时，与多肽或核酸相关联的术语“纯化的”或“分离的”是指多肽或核酸不是处于其天然介质中或天然形式下。因此，术语“分离的”包括从其原始环境，例如如果它是天然存在的，从天然环境取出的多肽或核酸。例如，分离的多肽通常不含通常与其结合 或通常与其混合或在溶液中的至少某些蛋白质或其他细胞组分。分离的多肽包括细胞裂解物中包含的天然生产的所述多肽，纯化或部分纯化形式的所述多肽，重组多肽，被细胞表达或分泌的所述多肽，以及在异源宿主细胞或培养物中的所述多肽。与核酸相关联，术语分离的或纯化的指示例如所述核酸不在其天然的基因组背景中(例如在载体中，作为表达盒，连接到启动子，或人工引入到异源宿主细胞中)。
本公开提供的一种包含抗原结合结构域的分离的结合蛋白，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K D≤6.55×10 -8mol/L的亲和力；
互补决定区CDR-VH1为G-Y-X1-F-T-X2-Y-W-I-G，其中，
X1是S或T，X2是D或E；
互补决定区CDR-VH2为D-M-X1-P-G-D-X2-Y-I-N-Y-X3-E-K-F-K-G，其中，
X1是F或V，X2是V、L或I，X3是Q或N；
互补决定区CDR-VH3为T-N-F-X1-T-X2-G-G-X3-D-Y，其中，
X1是I或L，X2是L或V，X3是V、L或I；
互补决定区CDR-VL1为K-S-S-X1-S-L-L-X2-S-D-G-X3-T-Y-L-N，其中，
X1是Q或N，X2是E或D，X3是R或K；
互补决定区CDR-VL2为L-V-X1-K-X2-D-S，其中，
X1是S或T，X2是V、I或L；
互补决定区CDR-VL3为W-X1-G-T-H-F-X2-H-T，其中，
X1是Q、Y或W，X2是A或P。
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，例如为能结合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。
在一种或多种实施方式中，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≤6.550×10 -8mol/L，例如，KD值可以为4.587×10 -8mol/L、2.798×10 -8mol/L、1.282×10 -8mol/L、1.891×10 -9mol/L、4.601×10 -9mol/L、6.592×10 -9mol/L、8.002×10 -9mol/L、9.614×10 -9mol/L、2.324×10 -10mol/L、3.540×10 -10mol/L、6.121×10 -10mol/L、9.876×10 -10mol/L，或者2.324×10 -10mol/L≤KD≤6.550×10 -8mol/L，或者2.324×10 -10mol/L≤KD≤9.614× 10 -9mol/L，或KD≤1.03×10 -10mol/L、≤1.15×10 -10mol/L、≤1.21×10 -10mol/L、≤0.32×10 -9mol/L、≤0.46×10 -9mol/L、≤0.70×10 -9mol/L、≤0.80×10 -9mol/L、≤0.90×10 -9mol/L、≤1.07×10 -9mol/L或≤1.14×10 -9mol/L。其中，亲和力按照本公开说明书中的方法测定。
在一种或多种实施方式中，
所述互补决定区CDR-VH1中，X1是T；所述互补决定区CDR-VH2中，X1是F；所述互补决定区CDR-VH3中，X2是L；所述互补决定区CDR-VL1中，X2是D；所述互补决定区CDR-VL2中，X1是S；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D。
在一种或多种实施方式中，所述互补决定区CDR-VH1中，X2是E。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是V，X3是Q。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是V，X3是N。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是L，X3是Q。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是L，X3是N。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是I，X3是Q。
在一种或多种实施方式中，所述互补决定区CDR-VH2中，X2是I，X3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I，X3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I，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是I，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是L，X3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L，X3是L。
在一种或多种实施方式中，所述互补决定区CDR-VH3中，X1是L，X3是I。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是Q，X2是R。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是Q，X2是K。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是N，X2是R。
在一种或多种实施方式中，所述互补决定区CDR-VL1中，X1是N，X2是K。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是V。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是I。
在一种或多种实施方式中，所述互补决定区CDR-VL2中，X2是L。
在一种或多种实施方式中，所述互补决定区CDR-VL3中，X1是Q。
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W。
在一种或多种实施方式中，所述结合蛋白中包括至少3个CDR；或者，所述结合蛋白包括至少6个CDR。
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；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。
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；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分离的核酸，所述核酸编码上述的结合蛋白。
在本文中，核酸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。
本公开还提供了一种载体，所述载体包含上述的核酸。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，例如中国仓鼠卵细胞(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。
本公开还提供了一种生产上述结合蛋白的方法，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产生的结合蛋白。
所述方法可以是例如，用编码至少一部分结合蛋白的核酸载体转染宿主细胞，在合适的条件下培养该宿主细胞使其表达该结合蛋白。宿主细胞也可以用一个或多个表达载体转染，该表达载体可以单独或结合地包含编码至少一部分结合蛋白的DNA。利用常规的纯化蛋白质和肽的技术可从培养基或细胞裂解物中分离结合蛋白，所述技术包括硫酸铵沉淀，层析(如离子交换，凝胶过滤，亲合层析等)和/或电泳。
构建合适的含有目的编码和调控序列的载体可以使用本领域公知的标准连接和限制技术进行。将分离的质粒、DNA序列或合成的寡核苷酸按需要的形式切割、加尾和再连接。可以用任何方法向编码序列中引入突变以产生本公开的变体，这些突变可以包含缺失或插入或置换等。
本公开也提供抗体，能与NS1蛋白的表位发生反应，包含单克隆的和多克隆的抗体。该抗体可以含有完整的结合蛋白，或其片段或衍生物。优选的抗体含有全部或部分的结合蛋白。
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。
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。
本公开的免疫测定包括胶体金免疫测定，还包括ELISA以及其它利用抗原抗体反应的试验或方法。
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。
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，本公开还提供了包括上述的结合蛋白、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；
在此实施方式中，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；
在此实施方式中，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。
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，所述方法基于荧光抗体技术、放射免疫分析和/或酶免疫技术。
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 11E2单克隆抗体的杂交瘤细胞株为本发明人实验室新筛选的杂交瘤细胞株。
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’；
MIgG-CKR：5’-CTAACACTCATTCCTGTTGAAGC-3’；
MIgG-CHR：5’-TCATTTACCCGGAGTCCGGGAGAAGCTC-3’。
2、抗体可变区基因克隆及测序
从分泌抗登革病毒NS1 11E2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MIgG-CKR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MIgG-CHR引物进行扩增。其中轻链的引物对扩增出0.72KB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。
3、抗登革病毒NS1 11E2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60bp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354bp，属于VH1基因家族，其前方有57bp的前导肽序列。
4、重组抗体表达质粒的构建
pcDNA TM 3.4
Figure PCTCN2019102631-appb-000003
vector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 11E2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：
DN11E2-HF：5’-CATAAGCTTATGAAATGGAGCTGGGTTATCCTCTTCTTCC-3’；
DN11E2-HR：5’-GACGAATTCTCATTTACCCGGAGTCCGGGAGAAGCTC-3’；
DN11E2-LF：5’-CCCAAGCTTATGGAGACAGACACACTCCTGCTATGG-3’；
DN11E2-LR：5’-CCCGAATTCCTAACACTCATTCCTGTTGAAGCTCTTGAC-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。
5、筛选稳定细胞株
5.1质粒用超纯水稀释至400ng/ml，调节CHO细胞1.43×10 7细胞/ml于离心管中，100μl质粒与700μl细胞混合，转入电转杯，电转，第3、5、7天取样计数，第7天收样检测。
包被液稀释相应抗原到指定浓度，每孔100μL，4℃过夜；次日，洗涤液清洗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。以标准品浓度和OD值作标准曲线，计算细胞上清中抗体含量。
5.2重组抗体表达质粒线性化
准备下述试剂：Buffer 50μl、DNA 100μg/管、PuvⅠ酶10μl、无菌水补至500μl，37℃水浴酶切过夜；先用等体积酚/氯仿/异戊醇(下层)25:24:1，再用氯仿(水相)依次进行抽提；0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀，70％乙醇漂洗沉淀，去除有机溶剂，待乙醇挥发完全用适量的灭菌水进行复融，最后进行浓度的测定。
重组抗体表达质粒稳定转染，加压筛选稳定细胞株：
质粒用超纯水稀释至400ng/ml，调节CHO细胞1.43×10 7细胞/ml于离心管中，100μl质粒与700μl细胞混合，转入电转杯，电转，次日计数；25μmol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔,并记录汇合度；取培养上清，送样检测；挑选抗体浓度、相对浓度高的细胞株转24孔，3天左右转6孔；3天后保种批培，调整细胞密度0.5×10 6细胞/ml，2.2ml进行批培养，细胞密度0.3×10 6细胞/ml，2ml进行保种；7天6孔批培上清送样检测，挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。
6、生产重组抗体
6.1细胞扩培
细胞复苏之后先在125ml规格的摇瓶中培养，接种体积为30ml，培养基为100％Dynamis培养基，放置于转速120r/min，温度为37℃，二氧化碳为8％的摇床中。培养72h，以50万细胞/ml接种密度接种扩培，扩培体积根据生产需求进行计算，培养基为100％Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时，严格控制接种密度为50万细胞/ml左右进行生产。
6.2摇瓶生产及纯化
摇瓶参数：转速120r/min，温度为37℃，二氧化碳为8％。流加补料：在摇瓶中培养至72h时开始每天补料，HyCloneTM Cell BoostTM Feed 7a每天流加初始培养体积的3％，Feed7b每天流加量为初始培养体积的千分之一，一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第13天收样。用proteinA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，电泳图如图1所示。在还原性SDS-PAGE后显示两条带，1条为约24KD的轻链(序列如SEQ ID NO:11所示)，另一条为约49KD的重链(序列如SEQ ID NO:12所示)。
实施例2
实施例1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，因而发明人对该抗体的轻链CDR及重链CDR进行突变分析和设计。
经分析，重链的互补决定区(WT)：
CDR-VH1为G-Y-S(X1)-F-T-D(X2)-Y-W-I-G；
CDR-VH2为D-M-V(X1)-P-G-D-L(X2)-Y-I-N-Y-Q(X3)-E-K-F-K-G；
CDR-VH3为T-N-F-I(X1)-T-V(X2)-G-G-L(X3)-D-Y；
轻链的互补决定区：
CDR-VL1为K-S-S-Q(X1)-S-L-L-E(X2)-S-D-G-R(X3)-T-Y-L-N；
CDR-VL2为L-V-T(X1)-K-V(X2)-D-S；
CDR-VL3为W-Q(X1)-G-T-H-F-A(X2)-H-T；
其中，X1、X2、X3均为突变位点。
表1 与抗体活性有关的突变位点
Figure PCTCN2019102631-appb-000004
Figure PCTCN2019102631-appb-000005
在突变后对抗体活性进行检测，包被液稀释羊抗鼠IgG 1μg/ml进行微孔板包被，每孔100μL，4℃过夜；次日，洗涤液清洗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DN单克隆抗体，100μL/孔，37℃，60min；甩掉板内液体，拍干，加入20％鼠阴性血封闭，每孔120μl，37℃，1h；甩掉板内液体，拍干，加入稀释10倍的(DN-(I+II+III+IV)-NS)抗原(公司自产昆虫表达)，每孔100μL，37℃，40min；洗涤液清洗5次，拍干；加入标记HRP的另一株DN单克隆抗体(1:4K)，每孔100μL，37℃，30min；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。部分结果如下：
表2 抗体活性分析数据
抗体浓度(ng/ml) WT 突变1 突变2 突变3 突变4 突变5
1000 2.316 2.403 2.075 0.847 0.514 -
333.333 2.376 2.413 2.153 0.347 0.061 -
111.111 2.198 2.331 1.998 0.056 - -
37.037 1.655 1.806 1.414 - - -
12.346 0.838 0.970 0.645 - - -
4.115 0.448 0.453 0.234 - - -
1.372 0.297 0.315 0.147 - - -
0 0.163 0.191 0.078 - - -
“-”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(保证筛选得到的抗体活性与突变1相近，抗体活性±10％)，部分结果如下。
表3 与抗体亲和力有关的突变位点
Figure PCTCN2019102631-appb-000006
Figure PCTCN2019102631-appb-000007
亲和力分析
利用AMC传感器，纯化出来的抗体用PBST稀释到10ug/ml，DN质控品重组蛋白(公司自产重组抗原)用PBST进行梯度稀释：465.1nmol/ml、232.6nmol/ml、116.3nmol/ml、58.1nmol/ml、29.1nmol/ml、14.5nmol/ml、7.27nmol/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 1.69 GLY溶液 及缓冲液3进行传感器再生，输出数据。KD表示平衡解亲常数即亲和力；Kon表示结合速率；Koff表示解离速率。
表4 亲和力分析数据
位点 KD(M) Kon(1/Ms) Koff(1/S)
突变1 1.554E-09 7.927E+04 1.232E-04
突变1-1 7.726E-09 6.584E+04 5.087E-04
突变1-2 8.838E-09 7.619E+04 6.734E-04
突变1-3 9.719E-09 6.198E+04 6.024E-04
突变1-4 9.488E-09 4.774E+04 4.530E-04
突变1-5 4.871E-09 5.717E+04 2.785E-04
突变1-6 8.176E-10 4.942E+04 4.041E-05
突变1-7 9.672E-09 8.666E+04 8.382E-04
突变1-8 5031E-09 7333E+04 3689E-04
突变1-9 7.232E-09 3.765E+04 2.723E-04
突变1-10 4.352E-10 2.835E+04 1.234E-05
突变1-11 2.821E-09 3.485E+04 9.831E-05
突变1-12 5.356E-09 5.656E+04 3.029E-04
突变1-13 6.239E-09 5.164E+04 3.222E-04
突变1-14 3.546E-09 4.241E+04 1.504E-04
突变1-15 8.619E-09 3.378E+04 2.912E-04
突变1-16 3.249E-09 4.492E+04 1.465E-04
突变1-17 4.087E-09 5.330E+04 2.178E-04
突变1-18 6.896E-09 4.570E+04 3.151E-04
突变1-19 4.716E-09 5.917E+04 2.790E-04
突变1-20 1.891E-09 5.020E+04 9.490E-05
突变1-21 4.601E-09 8.734E+04 4.018E-04
突变1-22 9.876E-10 2.400E+04 2.370E-05
突变1-23 2.779E-09 4.824E+04 1.341E-04
突变1-24 3.540E-10 2.165E+04 7.665E-06
突变1-25 6.039E-09 4.514E+04 2.726E-04
突变1-26 7.467E-09 5.287E+04 3.948E-04
突变1-27 2.305E-09 6.827E+04 1.574E-04
突变1-28 6.592E-09 5.118E+04 3.374E-04
突变1-29 7.943E-09 2.325E+04 1.847E-04
突变1-30 5.021E-09 2.269E+04 1.139E-04
突变1-31 8.220E-09 5.848E+04 4.807E-04
突变1-32 8.655E-09 3.594E+04 3.111E-04
突变1-33 9.614E-09 6.228E+04 5.988E-04
突变1-34 4.940E-10 5.470E+04 2.702E-05
突变1-35 4.004E-09 4.753E+04 1.903E-04
突变1-36 6.466E-09 6.416E+04 4.149E-04
突变1-37 8.896E-09 8.636E+04 7.683E-04
突变1-38 3.472E-09 7.387E+04 2.565E-04
突变1-39 2.324E-10 5.196E+04 1.208E-05
突变1-40 4.051E-09 4.230E+04 1.714E-04
突变1-41 3.875E-10 3.022E+04 1.171E-05
突变1-42 1.703E-09 8.932E+04 1.521E-04
突变1-43 5.851E-09 2.254E+04 1.319E-04
突变1-44 4.263E-09 5.708E+04 2.433E-04
突变1-45 6.592E-09 6.527E+04 4.302E-04
突变1-46 8.002E-09 5.991E+04 4.794E-04
突变1-47 6.121E-10 2.749E+04 1.683E-05
突变1-48 5.609E-09 4.111E+04 2.306E-04
突变1-49 7.953E-09 6.449E+04 5.129E-04
突变1-50 6.051E-09 7.696E+04 4.657E-04
突变1-51 2.687E-09 4.289E+04 1.152E-04
突变1-52 1.554E-09 7.927E+04 1.232E-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 PCTCN2019102631-appb-000008
表6 亲和力分析数据
位点 KD(M) Kon(1/Ms) Koff(1/S)
WT 5.884E-08 6.771E+03 3.984E-04
WT 1-1 1.282E-08 4.568E+03 5.856E-05
WT 1-4 2.798E-08 5.079E+03 1.421E-04
WT 1-7 6.550E-08 3.528E+03 2.311E-04
WT 1-20 4.587E-08 3.121E+03 1.432E-04
WT 1-30 2.041E-08 7.284E+03 1.487E-04
从表5和表6分析，在保证具有抗体活性的前提下，基于突变1作为框架的抗体亲和力整体高于WT。
实施例3
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 2.432 2.043 0.075
-80℃，21天样品 2.546 2.054 0.057
37℃，21天样品 2.499 2.033 0.089
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，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 (16)

  1. 一种包含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K D≤6.55×10 -8mol/L的亲和力；
    互补决定区CDR-VH1为G-Y-X1-F-T-X2-Y-W-I-G，其中，
    X1是S或T，X2是D或E；
    互补决定区CDR-VH2为D-M-X1-P-G-D-X2-Y-I-N-Y-X3-E-K-F-K-G，其中，
    X1是F或V，X2是V、L或I，X3是Q或N；
    互补决定区CDR-VH3为T-N-F-X1-T-X2-G-G-X3-D-Y，其中，
    X1是I或L，X2是L或V，X3是V、L或I；
    互补决定区CDR-VL1为K-S-S-X1-S-L-L-X2-S-D-G-X3-T-Y-L-N，其中，
    X1是Q或N，X2是E或D，X3是R或K；
    互补决定区CDR-VL2为L-V-X1-K-X2-D-S，其中，
    X1是S或T，X2是V、I或L；
    互补决定区CDR-VL3为W-X1-G-T-H-F-X2-H-T，其中，
    X1是Q、Y或W，X2是A或P；
    优选地，所述互补决定区CDR-VH1中，X1是T；
    所述互补决定区CDR-VH2中，X1是F；
    所述互补决定区CDR-VH3中，X2是L；
    所述互补决定区CDR-VL1中，X2是D；
    所述互补决定区CDR-VL2中，X1是S；
    所述互补决定区CDR-VL3中，X2是P；
    优选地，所述互补决定区CDR-VH1中，X2是D；
    优选地，所述互补决定区CDR-VH1中，X2是E；
    优选地，所述互补决定区CDR-VH2中，X2是V，X3是Q；
    优选地，所述互补决定区CDR-VH2中，X2是V，X3是N；
    优选地，所述互补决定区CDR-VH2中，X2是L，X3是Q；
    优选地，所述互补决定区CDR-VH2中，X2是L，X3是N；
    优选地，所述互补决定区CDR-VH2中，X2是I，X3是Q；
    优选地，所述互补决定区CDR-VH2中，X2是I，X3是N；
    优选地，所述互补决定区CDR-VH3中，X1是I，X3是V；
    优选地，所述互补决定区CDR-VH3中，X1是I，X3是L；
    优选地，所述互补决定区CDR-VH3中，X1是I，X3是I；
    优选地，所述互补决定区CDR-VH3中，X1是L，X3是V；
    优选地，所述互补决定区CDR-VH3中，X1是L，X3是L；
    优选地，所述互补决定区CDR-VH3中，X1是L，X3是I；
    优选地，所述互补决定区CDR-VL1中，X1是Q，X2是R；
    优选地，所述互补决定区CDR-VL1中，X1是Q，X2是K；
    优选地，所述互补决定区CDR-VL1中，X1是N，X2是R；
    优选地，所述互补决定区CDR-VL1中，X1是N，X2是K；
    优选地，所述互补决定区CDR-VL2中，X2是V；
    优选地，所述互补决定区CDR-VL2中，X2是I；
    优选地，所述互补决定区CDR-VL2中，X2是L；
    优选地，所述互补决定区CDR-VL3中，X1是Q；
    优选地，所述互补决定区CDR-VL3中，X1是Y；
    优选地，所述互补决定区CDR-VL3中，X1是W。
  2. 根据权利要求1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表，
    Figure PCTCN2019102631-appb-100001
    Figure PCTCN2019102631-appb-100002
  3. 根据权利要求1或2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。
  4. 根据权利要求1至3中任一项所述的结合蛋白，其中，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；
    优选地，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中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；
    优选地，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；
    优选地，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；
    优选地，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. 一种分离的核酸，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，其包含权利要求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. 一种生产权利要求1-5中任一项所述的结合蛋白的方法，其中，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产生的结合蛋白。
  10. 权利要求1-5中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。
  11. 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所述的分离的核酸或权利要求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。
  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，其中，所述方法基于荧光免疫技术、化学发光免疫技术、胶体金免疫测定、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，所述样品选自全血，外周血，血清或血浆中的至少一种。
  16. 根据权利要求13-15中任一项所述的方法，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/CN2019/102631 2018-08-28 2019-08-26 一种ns1蛋白的结合蛋白以及应用 WO2020043068A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
KR1020217004457A KR20210031946A (ko) 2018-08-28 2019-08-26 Ns1 단백질의 결합 단백질 및 응용
CA3109433A CA3109433A1 (en) 2018-08-28 2019-08-26 Ns1-binding protein and uses thereof
US17/267,025 US20210301000A1 (en) 2018-08-28 2019-08-26 NS1-Binding Protein and Uses Thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810999045.2 2018-08-28
CN201810999045.2A CN109053882B (zh) 2018-08-28 2018-08-28 一种ns1蛋白的结合蛋白以及应用

Publications (1)

Publication Number Publication Date
WO2020043068A1 true WO2020043068A1 (zh) 2020-03-05

Family

ID=64758644

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/102631 WO2020043068A1 (zh) 2018-08-28 2019-08-26 一种ns1蛋白的结合蛋白以及应用

Country Status (5)

Country Link
US (1) US20210301000A1 (zh)
KR (1) KR20210031946A (zh)
CN (1) CN109053882B (zh)
CA (1) CA3109433A1 (zh)
WO (1) WO2020043068A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109053882B (zh) * 2018-08-28 2019-08-23 东莞市朋志生物科技有限公司 一种ns1蛋白的结合蛋白以及应用
CN115785262B (zh) * 2021-09-10 2023-09-22 东莞市朋志生物科技有限公司 一种抗登革ns1蛋白的抗体及其应用
CN115785274B (zh) * 2021-09-10 2023-10-31 东莞市朋志生物科技有限公司 一种抗胃蛋白酶原i的抗体及其应用
CN116082499B (zh) * 2021-11-08 2023-10-27 东莞市朋志生物科技有限公司 一种抗登革ns1蛋白的抗体及其应用

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1360678A (zh) * 1999-06-09 2002-07-24 巴斯德研究所 用ns1糖蛋白对黄病毒进行早期检测
CN102143979A (zh) * 2008-08-04 2011-08-03 拜耳医药保健有限公司 针对组织因子途径抑制剂(tfpi)的单克隆抗体
TW201533059A (zh) * 2014-02-27 2015-09-01 Univ Nat Cheng Kung 抗登革病毒非結構性蛋白１之單株抗體及其用途
CN106279410A (zh) * 2016-09-29 2017-01-04 华南理工大学 一种二型登革热病毒ns1蛋白多价纳米抗体及制备方法
CN107406503A (zh) * 2014-11-18 2017-11-28 詹森药业有限公司 Cd47抗体、方法和用途
WO2017223286A1 (en) * 2016-06-23 2017-12-28 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Dengue virus non-structural protein 1 specific binding polypeptides and methods of using the same
WO2018109170A2 (en) * 2016-12-16 2018-06-21 Singapore Health Services Pte Ltd Il-11ra antibodies
CN109053882A (zh) * 2018-08-28 2018-12-21 东莞市朋志生物科技有限公司 一种ns1蛋白的结合蛋白以及应用
WO2019152742A1 (en) * 2018-02-01 2019-08-08 Pfizer Inc. Chimeric antigen receptors targeting cd70

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1360678A (zh) * 1999-06-09 2002-07-24 巴斯德研究所 用ns1糖蛋白对黄病毒进行早期检测
CN102143979A (zh) * 2008-08-04 2011-08-03 拜耳医药保健有限公司 针对组织因子途径抑制剂(tfpi)的单克隆抗体
TW201533059A (zh) * 2014-02-27 2015-09-01 Univ Nat Cheng Kung 抗登革病毒非結構性蛋白１之單株抗體及其用途
CN107406503A (zh) * 2014-11-18 2017-11-28 詹森药业有限公司 Cd47抗体、方法和用途
WO2017223286A1 (en) * 2016-06-23 2017-12-28 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Dengue virus non-structural protein 1 specific binding polypeptides and methods of using the same
CN106279410A (zh) * 2016-09-29 2017-01-04 华南理工大学 一种二型登革热病毒ns1蛋白多价纳米抗体及制备方法
WO2018109170A2 (en) * 2016-12-16 2018-06-21 Singapore Health Services Pte Ltd Il-11ra antibodies
WO2019152742A1 (en) * 2018-02-01 2019-08-08 Pfizer Inc. Chimeric antigen receptors targeting cd70
CN109053882A (zh) * 2018-08-28 2018-12-21 东莞市朋志生物科技有限公司 一种ns1蛋白的结合蛋白以及应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GAO, HONGLI ET AL.: "The Preparation and Preliminary Application of the Group-specific Monoclonal Antibodies against the Nonstructural Glycoprotein 1 of Dengue Virus", JOURNAL OF TROPICAL MEDICINE, vol. 11, no. 6, 28 June 2011 (2011-06-28), pages 620 - 646, ISSN: 1672-3619 *
LEBANI, K.: "Isolation of serotype-specific antibodies against dengue virus non-structural protein 1 using phage display and application in a multi-plexed serotyping assay", PLOS ONE, vol. 12, no. 7, 6 July 2017 (2017-07-06), pages e0180669, XP055696960, ISSN: 1932-6203 *
LI, DUO: "Production of Universal Monoclonal Antibodies for Dengue Serotype I-IV Virus Nonstructural Protein NS1, and Establishment of NS1 Antigen Capture ELISA", CHINESE MASTER'S DISSERTATIONS FULL-TEXT DATABASE, MEDICAL AND HEALTH SCIENCES, 15 January 2016 (2016-01-15), pages 1674 - 0246 *
ROCHA, L.B.: "Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies", ANTIBODIES (BASEL, 15 October 2017 (2017-10-15), pages 4, XP055696961, ISSN: 2073-4468 *

Also Published As

Publication number Publication date
KR20210031946A (ko) 2021-03-23
CN109053882B (zh) 2019-08-23
CN109053882A (zh) 2018-12-21
US20210301000A1 (en) 2021-09-30
CA3109433A1 (en) 2020-03-05

Similar Documents

Publication Publication Date Title
WO2020043068A1 (zh) 一种ns1蛋白的结合蛋白以及应用
CA3200150A1 (en) Antibody against novel coronavirus, and kit for novel coronavirus detection
WO2020043067A1 (zh) Ns1蛋白的结合蛋白
WO2020043069A1 (zh) Ns1蛋白的结合蛋白
WO2020043066A1 (zh) Ns1蛋白的结合蛋白
CN111217912B (zh) 一种抗pgⅱ的抗体及其应用
JP7307158B2 (ja) 抗ヒト心筋型トロポニンｉ抗体及びその応用
CN111349168B (zh) 一种抗人ckmb的抗体及其应用
US20240117012A1 (en) Anti-sars-cov-2 antibody
CN112745390B (zh) 一种包含NT-proBNP抗原结合结构域的结合蛋白
CN111217913B (zh) 一种抗pg ii的抗体及其应用
KR102671155B1 (ko) Ns1 단백질의 결합 단백질
WO2020073833A1 (zh) 一种抗人心肌肌钙蛋白i的抗体及其应用
CN112707964B (zh) 一种抗n末端脑钠肽前体的重组抗体
CN111018991B (zh) 一种抗ca50的抗体及其应用
CN111018979B (zh) 一种抗人心肌肌钙蛋白i的抗体及其应用
JP2012157353A (ja) 抗トリガーファクターモノクローナル抗体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19855062

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3109433

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 20217004457

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19855062

Country of ref document: EP

Kind code of ref document: A1