[go: up one dir, main page]

CN108191973B - High-affinity single-domain antibody targeting Ebola virus envelope protein and preparation method and application thereof - Google Patents

High-affinity single-domain antibody targeting Ebola virus envelope protein and preparation method and application thereof Download PDF

Info

Publication number
CN108191973B
CN108191973B CN201810183021.XA CN201810183021A CN108191973B CN 108191973 B CN108191973 B CN 108191973B CN 201810183021 A CN201810183021 A CN 201810183021A CN 108191973 B CN108191973 B CN 108191973B
Authority
CN
China
Prior art keywords
envelope protein
ebola virus
seq
ebov4h9
gly
Prior art date
Legal status (The legal status 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 status listed.)
Active
Application number
CN201810183021.XA
Other languages
Chinese (zh)
Other versions
CN108191973A (en
Inventor
龚睿
张怀东
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuhan Institute of Virology of CAS
Original Assignee
Wuhan Institute of Virology of CAS
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 Wuhan Institute of Virology of CAS filed Critical Wuhan Institute of Virology of CAS
Priority to CN201810183021.XA priority Critical patent/CN108191973B/en
Publication of CN108191973A publication Critical patent/CN108191973A/en
Application granted granted Critical
Publication of CN108191973B publication Critical patent/CN108191973B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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

Landscapes

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

Abstract

本发明公开了一种靶向埃博拉病毒囊膜蛋白的高亲和力单域抗体及应用。本发明从人IgG的VH结构域m0E1为骨架的多肽及蛋白质文库中，以埃博拉病毒囊膜蛋白GP为抗原，筛选得到一种靶向埃博拉病毒囊膜蛋白的高亲和力单域抗体，相对于全长单抗(分子量～150kD)，本发明的抗体分子量较小，具有更好的组织渗透性和结合存在位阻效应的抗原表位的能力，可在原核表达系统进行表达，生产成本低、周期短；其中由本发明筛选出的EBOV4H9(～14kD)，针对埃博拉病毒囊膜蛋白具有较高亲和力，应用前景好。The invention discloses a high-affinity single-domain antibody targeting Ebola virus envelope protein and its application. In the present invention, a high-affinity single-domain antibody targeting Ebola virus envelope protein is obtained by screening from the polypeptide and protein library with the VH structural domain m0E1 of human IgG as the skeleton and the Ebola virus envelope protein GP as the antigen. , Compared with the full-length monoclonal antibody (molecular weight ~ 150kD), the antibody of the present invention has a smaller molecular weight, better tissue permeability and the ability to bind antigenic epitopes with steric hindrance effect, and can be expressed in a prokaryotic expression system, producing The cost is low and the cycle is short; the EBOV4H9 (~14kD) screened by the present invention has a high affinity for the Ebola virus envelope protein and has a good application prospect.

Description

High-affinity single-domain antibody targeting Ebola virus envelope protein and preparation method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a high-affinity single-domain antibody of a targeting Ebola virus envelope protein and application thereof.
Background
Ebola virus (EBoV) belongs to filoviridae, has a length of 970 nm, is in the form of long filament, has single-strand negative strand RNA virus (RNA virus) with 18959 bases, and has a molecular weight of 4.17 × 106. The virus has an envelope, the diameter of virus particles is about 80nm, the size is 100nm multiplied by (300-1500) nm, the length of the virus with stronger infection capacity is about 665-805 nm generally, and the virus has a branch shape, a U shape, a 6 shape or a ring shape, and the branch shape is common. Has a capsule membrane, the surface has fiber protrusions with the length of 8-10 nm, and the pure virus particles are composed of a spiral ribose nucleocapsid complex and contain minus-strand linear RNA molecules and 4 virion structural proteins.
At present, clinically, no approved antibody medicines aiming at the Ebola virus exist. In the existing studies of ebola neutralizing antibodies, the use of the antibody drug ZMapp in emergency situations has achieved some exciting results, but its clinical efficacy also requires extensive validation efforts.
In the course of antibody studies, it was found that full-length antibodies have a large molecular weight (-150 kD), making them less tissue permeable and also difficult to bind to some sterically hindered key epitopes, thus affecting activity. One of the strategies to solve is to miniaturize the full-length antibody, thereby developing a series of antibody fragments with binding function. Among them, the VH domain of an antibody Fab fragment is called a single domain antibody (dAb). Currently VH-based dAb single domain antibodies have been used to develop candidate antibody-based drugs against viruses. Compared with full-length antibodies, the antibody has the advantages of tissue permeability and capability of entering into a steric hindrance effect site due to small molecular weight.
Disclosure of Invention
The invention aims to provide a high-affinity single-domain antibody targeting an Ebola virus envelope protein, a preparation method and application thereof.
In order to realize the aim, the invention provides a high-affinity single-domain antibody of a targeting Ebola virus envelope protein, which is obtained by taking a VH structural domain m0E1 of an IgG antigen binding region of a human antibody as a framework, constructing a polypeptide and protein library and then taking an Ebola virus envelope protein GP as an antigen for screening; the amino acid sequence of m0E1 is seq ID No. 15.
In the above scheme, the coding gene sequence of m0E1 is seq ID No. 16.
m0E1 is a mutant of the VH domain m0 of human antibody IgG, with a higher stability than m0, obtained by mutating the VSA of frame 2(FR2) upstream of CDR2 of m0 to IGE. In the experimental design, due to the diversity of primers, the obtained framework is different from m0, and the physicochemical stability, such as thermal stability, of the obtained antibody is increased by adopting m0E1 as the framework and constructing a polypeptide and protein library for screening.
In the above scheme, both polypeptide and protein libraries capable of realizing antibody screening can be used in the present invention, such as yeast surface display libraries, and in the prior art, phage surface display libraries are preferably used.
The invention also provides a high-affinity single-domain antibody of the targeted Ebola virus envelope protein, which is named as EBOV4H9, and complementary determinants of the antibody are CDR1, CDR2 and CDR3 respectively; the amino acid sequence of the CDR1 is Seq ID No.2, the amino acid sequence of the CDR2 is Seq ID No.4, and the amino acid sequence of the CDR3 is Seq ID No. 6.
Preferably, the EBOV4H9 has the gene sequence encoding CDR1 of Seq ID No.1, the gene sequence encoding CDR2 of Seq ID No.3 and the gene sequence encoding CDR3 of Seq ID No. 5.
Preferably, the full-length amino acid sequence of the EBOV4H9 is Seq ID No. 7.
Optionally, the full-length sequence of the EBOV4H9 gene is Seq ID No. 8.
Alternatively, base and amino acid mutations are made in regions of EBOV4H9 other than the CDR regions, and antibodies functionally similar to EBOV4H9 are obtained and fall within the scope of the present invention. Other regions act as framework linkages and folding functions relative to the core functional CDR regions of EBOV4H9, are simple mutations and have no substantial effect on EBOV4H9 function, and should be considered as the same as in the present invention.
The invention also provides a preparation method of the high-affinity single-domain antibody of the targeted Ebola virus envelope protein, which comprises the following steps:
(1) constructing a eukaryotic expression vector containing an Ebola virus envelope protein GP gene sequence, expressing the virus envelope protein, and separating and purifying to obtain the envelope protein GP;
(2) screening by using purified envelope protein GP as an antigen from a phage surface display library with m0E1 as a framework, and obtaining a clone with high affinity and specific binding after several rounds; the amino acid sequence of m0E1 is seq ID No. 15;
(3) the clone is expressed and purified to obtain the high-affinity single-domain antibody of the target Ebola virus envelope protein.
The invention also discloses the application of the high-affinity single-domain antibody of the targeted Ebola virus envelope protein in the preparation of medicaments for treating Ebola virus infection;
and the high affinity single domain antibody of the targeted Ebola virus envelope protein, and the application thereof in preparing an Ebola virus detection reagent, such as a detection probe.
The application comprises the steps of preparing the high-affinity single-domain antibody into a protein containing the antibody, a derivative (such as coupling other molecules) based on the antibody and the like, such as a fusion antibody or a coupling antibody; the fusion antibody is obtained by fusing the antibody and peptide segments of a plurality of amino acids into protein, including but not limited to self and Fc segments of the antibody; the conjugated antibody includes but is not limited to the antibody of the present invention obtained by coupling with radioactive isotope and toxin.
The invention has the beneficial effects that: the provided single-domain antibody is an antibody for virus detection, diagnosis and treatment, has smaller molecular weight compared with a full-length monoclonal antibody (molecular weight is 150kD), has better tissue permeability and capability of combining an epitope with steric effect, can be expressed in a prokaryotic expression system, and has low production cost and short period; the EBOV4H9 (about 14kD) screened by the invention has higher affinity for Ebola virus envelope protein and good application prospect.
Drawings
FIG. 1 shows V of human IgGHSchematic structural diagram of (1).
FIG. 2 shows SDS-PAGE detection of proteins purified from the single domain antibody EBOV4H 9.
FIG. 3 is the binding of EBOV4H9 to Ebola virus envelope protein as determined by ELISA.
FIG. 4 is a graph of the binding of EBOV4H9, chimeraH9-1 and chimeraH9-2 (chimeras formed by replacing the backbone of EBOV4H9 with the other family VH backbones) to the Ebola virus envelope protein determined by ELISA.
Detailed Description
The invention is described in further detail below with reference to the figures and specific embodiments. The following examples are carried out on the premise of the technical scheme of the invention, and detailed embodiments and specific operation procedures are given, but the scope of the invention is not limited to the following examples.
Example 1: construction and screening of phage display libraries
V of human IgG selected in examplesHThe structural domain m0E1 is a framework, the amino acid sequence is seq ID No.15, and the coding gene sequence is seq ID No. 16. Three CDR region sequences of m0E 1: the gene sequence of the code CDR1 is Seq ID No.9, and the amino acid sequence is Seq ID No. 10; the gene sequence of the code CDR2 is Seq ID No.11, and the amino acid sequence is Seq ID No. 12; the gene sequence encoding CDR3 is Seq ID No.13, and the amino acid sequence is Seq ID No. 14.
Phage libraries (W Chen, et al, Methods Mol Biol,2009:81-99) were constructed according to the existing literature using m0E1 as a backbone and screened for eukaryotic expressed antigens. After the purified antigen is incubated overnight at 4 ℃ in a 96-well plate, panning is carried out in a phage library, specific phage are captured by the antigen, washing is carried out by PBS + 0.05% Tween-20, and an enriched clone is obtained after 4 rounds of screening, wherein the enriched clone is named as EBOV4H 9.
Sequencing is carried out to obtain the complementarity determining clusters of EBOV4H9, namely CDR1, CDR2 and CDR 3. The gene sequence of the code CDR1 is Seq ID No.1, and the amino acid sequence is Seq ID No. 2; the gene sequence of the coded CDR2 is Seq ID No.3, and the amino acid sequence is Seq ID No. 4; the gene sequence of the code CDR3 is Seq ID No.5, and the amino acid sequence is Seq ID No. 6; the full-length sequence of the EBOV4H9 gene is Seq ID No.7, and the full-length sequence of the amino acid is Seq ID No. 8.
Example 2: expression purification of EBOV4H9
EBOV4H9 was expressed and purified according to the literature (Gong R, et al, Methods Mol biol., 2012). An EBOV4H9 prokaryotic expression vector is constructed and transformed into E.coli HB 2151. Then inoculated into SB medium (1L medium containing 30g tryptone, 20g yeast extract and 10g MOPS, pH adjusted to 7.0 with NaOH) containing 100. mu.g/ml ampicillin until OD is reached600When reaching 0.7-1.0, IPTG is added to the final concentration of 200 mug/ml, and the induction expression is carried out for 14-16 h under the conditions of 37 ℃ and 220 rpm. Centrifuging at 4 deg.C and 6000rpm for 15min to collect thallus, discarding culture medium, resuspending the precipitate in Buffer A (50mM Tris-HCl, 450mM NaCl, pH 8.0), and passing through polymyxin B (pol)ymyxin B) treatment for 1 hour, the supernatant was collected by centrifugation. The purified product was purified by Ni-NTA filler and the purity was verified by SDS-PAGE, as shown in FIG. 2, lane M is the molecular weight standard and protein H9 is the target protein eluted by imidazole concentration gradient. Then ultrafiltering and concentrating with ultrafiltration centrifuge tube with molecular weight cutoff of 3 kD. The resulting EBOV4H9 contained a 6 XHis tag and a FLAG tag at the C-terminus.
Experimental example 1: ELISA determination of binding of EBOV4H9 to envelope protein
The binding capacity of EBOV4H9 was identified by ELISA, with a library backbone m0 control.
The envelope protein (2. mu.g/mL) was coated on ELISA plates, incubated overnight at 4 ℃ and blocked with PBS + 3% mil k at 37 ℃ for 1 h. Serial dilutions of EBOV4H9 were added, incubated for 2 hours at 37 ℃ and washed four times with PBST (PBS + 0.05% Tween 20), followed by horseradish peroxidase (HRP) -labeled murine anti-FLAG monoclonal antibody incubated for 1 hour at 37 ℃, washed four times with PBST, and additional ABTS was added for detection. Backbone m0 served as a negative control. The results are shown in FIG. 3, EC associated with EBOV4H9 and Ebola virus envelope protein50At 0.8nM, the affinity is higher, whereas m0 is unable to bind to envelope protein.
Example 2: ELISA assays for binding of EBOV4H9, chimeraH9-1, chimeraH9-2, m0, m0E1 and envelope proteins
The envelope protein (2. mu.g/mL) was coated on ELISA plates, incubated overnight at 4 ℃ and blocked with PBS + 3% mil k at 37 ℃ for 1 h. Adding serial diluted EBOV4H9, chimeraH9-1, chimeraH9-2, m0 and m0E1(chimeraH9-1 amino acid sequence seq ID No.17, gene sequence seq ID No. 18; chimeraH9-2 amino acid sequence seq ID No.19, gene sequence seq ID No. 20; amino acid and gene sequence of m0 are disclosed in the prior art), incubating for 2 hours at 37 deg.C, washing with PBST (PBS + 0.05% Tween 20) four times, washing with PBS 1 time, adding horseradish peroxidase (HRP) -labeled mouse anti-FLAG monoclonal antibody, incubating for 1 hour at 37 deg.C, washing with PBST four times, washing with PBS once, adding ABTS to OD405And (6) detecting. Scaffolds m0 and m0E1 served as negative controls.
The results are shown in FIG. 4, (■) is the curve for EBOV4H9 binding to antigenic proteins, (. tangle-solidup.) is the curve for chimera H9-1 binding to antigenic proteins, (. diamond-solid.) is the curve for chimera H9-2 binding to antigenic proteins, and scaffolds m0(□) and m0E1(●) are negative controls.
EC of EBOV4H9 binding to Ebola virus envelope protein50At 0.8nM, chimeraH9-1 and chimeraH9-2 bound weakly to the Ebola virus envelope protein, and m0 and m0E1 did not bind to the envelope protein.
Sequence listing
<110> Wuhan Virus institute of Chinese academy of sciences
<120> high-affinity single-domain antibody of targeted Ebola virus envelope protein, and preparation method and application thereof
<130> 2018
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> EBOV4H9 CDR1
<400> 1
Gly Gly Ser Ile Ser Thr Ala Gly Tyr Gly
1 5 10
<210> 2
<211> 30
<212> DNA
<213> EBOV4H9 CDR1
<400> 2
ggtggtagca taagcactgc tggttatggt 30
<210> 3
<211> 7
<212> PRT
<213> EBOV4H9 CDR2
<400> 3
Ile Asn His Ser Gly Ser Thr
1 5
<210> 4
<211> 21
<212> DNA
<213> EBOV4H9 CDR2
<400> 4
atcaatcata gtggaagcac c 21
<210> 5
<211> 24
<212> PRT
<213> EBOV4H9 CDR3
<400> 5
Ala Arg Gly Ser Met Leu Pro Asn Tyr Gly Leu Asp Val Trp Gly Gln
1 5 10 15
Gly Thr Thr Val Thr Val Ser Ser
20
<210> 6
<211> 72
<212> DNA
<213> EBOV4H9 CDR3
<400> 6
gcgagaggct ctatgcttcc taactacggt ctagacgtct ggggccaagg gaccacggtc 60
accgtctcct ca 72
<210> 7
<211> 121
<212> PRT
<213> EBOV4H9
<400> 7
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Ser Ile Ser Thr Ala
20 25 30
Gly Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu
35 40 45
Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
65 70 75 80
Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr
85 90 95
Cys Ala Arg Gly Ser Met Leu Pro Asn Tyr Gly Leu Asp Val Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 8
<211> 363
<212> DNA
<213> EBOV4H9
<400> 8
caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60
tcctgtgcag cctctggtgg tagcataagc actgctggtt atggtatgag ctgggtccgc 120
caggctccag ggaaggccct ggagtggatt ggggaaatca atcatagtgg aagcaccaac 180
tacaacccat ccctcaagag tcgagtcacc atctccagag acaattccaa gaacacgctg 240
tatctgcaaa tgaacaccct gagagccgag gacacggctt tgtattactg tgcgagaggc 300
tctatgcttc ctaactacgg tctagacgtc tggggccaag ggaccacggt caccgtctcc 360
tca 363
<210> 9
<211> 8
<212> PRT
<213> m0E1 CDR1
<400> 9
Gly Phe Ser Phe Ser Thr Tyr Glu
1 5
<210> 10
<211> 24
<212> DNA
<213> m0E1 CDR1
<400> 10
ggattcagct tcagtactta tgaa 24
<210> 11
<211> 8
<212> PRT
<213> m0E1 CDR2
<400> 11
Ile Ser Gly Ser Gly Gly Asn Ser
1 5
<210> 12
<211> 24
<212> DNA
<213> m0E1 CDR2
<400> 12
attagtggta gtggtggtaa ctca 24
<210> 13
<211> 18
<212> PRT
<213> m0E1 CDR3
<400> 13
Ala Lys Gly Pro Pro Val Trp Ser Gly Tyr Tyr Phe Ala Asp Gly Phe
1 5 10 15
Asp Ile
<210> 14
<211> 54
<212> DNA
<213> m0E1 CDR3
<400> 14
gcgaaaggcc ccccggtttg gagtggttat tatttcgctg atggttttga tatc 54
<210> 15
<211> 125
<212> PRT
<213> m0E1
<400> 15
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Thr Tyr
20 25 30
Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Ser Gly Ser Gly Gly Asn Ser Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Gly Pro Pro Val Trp Ser Gly Tyr Tyr Phe Ala Asp Gly Phe
100 105 110
Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 16
<211> 375
<212> DNA
<213> m0E1
<400> 16
caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60
tcctgtgcag cctctggatt cagcttcagt acttatgaaa tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg gattggggaa attagtggta gtggtggtaa ctcaaactac 180
aacccatccc tcaagagtcg agtcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acaccctgag agccgaggac acggccgtat attactgtgc gaaaggcccc 300
ccggtttgga gtggttatta tttcgctgat ggttttgata tctggggcca agggacaatg 360
gtcaccgtct cttca 375
<210> 17
<211> 121
<212> PRT
<213> chimeraH9-1
<400> 17
Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Gly Ser Ile Ser Thr Ala
20 25 30
Gly Tyr Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45
Trp Leu Ala Leu Ile Asn His Ser Gly Ser Thr Arg Tyr Ser Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val
65 70 75 80
Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Gly Ser Met Leu Pro Asn Tyr Gly Leu Asp Val Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 18
<211> 363
<212> DNA
<213> chimeraH9-1
<400> 18
cagatcacct tgaaggagtc tggtcctacg ctggtgaaac ccacacagac cctcacgctg 60
acctgcacct tctctggtgg tagcataagc actgctggtt atggtgtggg ctggatccgt 120
cagcccccag gaaaggccct ggagtggctt gcactcatca atcatagtgg aagcacccgc 180
tacagcccat ctctgaagag caggctcacc atcaccaagg acacctccaa aaaccaggtg 240
gtccttacaa tgaccaacat ggaccctgtg gacacagcca catattactg tgcgagaggc 300
tctatgcttc ctaactacgg tctagacgtc tggggccaag ggaccacggt caccgtctcc 360
tca 363
<210> 19
<211> 121
<212> PRT
<213> chimeraH9-2
<400> 19
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Gly Ser Ile Ser Thr Ala
20 25 30
Gly Tyr Gly Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu
35 40 45
Trp Met Gly Ile Ile Asn His Ser Gly Ser Thr Arg Tyr Ser Pro Ser
50 55 60
Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala
65 70 75 80
Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr
85 90 95
Cys Ala Arg Gly Ser Met Leu Pro Asn Tyr Gly Leu Asp Val Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 20
<211> 363
<212> DNA
<213> chimeraH9-2
<400> 20
gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60
tcctgtaagg gttctggtgg tagcataagc actgctggtt atggtatcgg ctgggtgcgc 120
cagatgcccg ggaaaggcct ggagtggatg gggatcatca atcatagtgg aagcaccaga 180
tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 240
tacctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgagaggc 300
tctatgcttc ctaactacgg tctagacgtc tggggccaag ggaccacggt caccgtctcc 360
tca 363

Claims (5)

1.一种靶向埃博拉病毒囊膜蛋白的高亲和力单域抗体，其特征在于：所述高亲和力单域抗体为EBOV4H9，它的互补决定簇分别为CDR1、CDR2和CDR3；所述CDR1的氨基酸序列为SeqID No.2，所述CDR2的氨基酸序列为seq ID No.4，所述CDR3的氨基酸序列为Seq ID No.6。1. a high-affinity single-domain antibody targeting Ebola virus envelope protein, characterized in that: the high-affinity single-domain antibody is EBOV4H9, and its complementary determinants are respectively CDR1, CDR2 and CDR3; the CDR1 The amino acid sequence of the CDR2 is Seq ID No.2, the amino acid sequence of the CDR2 is seq ID No.4, and the amino acid sequence of the CDR3 is Seq ID No.6. 2.根据权利要求1所述靶向埃博拉病毒囊膜蛋白的高亲和力单域抗体，其特征在于：所述EBOV4H9的编码CDR1的基因序列为seq ID No.1，编码CDR2的基因序列为Seq ID No.3，编码CDR3的基因序列为Seq ID No.5。2. the high-affinity single domain antibody targeting Ebola virus envelope protein according to claim 1, is characterized in that: the gene sequence of coding CDR1 of described EBOV4H9 is seq ID No.1, and the gene sequence of coding CDR2 is Seq ID No.3, the gene sequence encoding CDR3 is Seq ID No.5. 3.根据权利要求1所述靶向埃博拉病毒囊膜蛋白的高亲和力单域抗体，其特征在于：所述EBOV4H9的氨基酸全长序列为Seq ID No.7。3. The high-affinity single-domain antibody targeting Ebola virus envelope protein according to claim 1, wherein the amino acid full-length sequence of the EBOV4H9 is Seq ID No.7. 4.根据权利要求3所述靶向埃博拉病毒囊膜蛋白的高亲和力单域抗体，其特征在于：所述EBOV4H9的基因全长序列为Seq ID No.8。4 . The high-affinity single-domain antibody targeting Ebola virus envelope protein according to claim 3 , wherein the full-length gene sequence of the EBOV4H9 is Seq ID No.8. 5 . 5.根据权利要求1所述靶向埃博拉病毒囊膜蛋白的高亲和力单域抗体，其特征在于：对EBOV4H9除CDR区以外的区域进行碱基和氨基酸的突变，且获得与EBOV4H9功能类似的抗体。5. the high-affinity single domain antibody targeting Ebola virus envelope protein according to claim 1, is characterized in that: carry out the mutation of base and amino acid to EBOV4H9 except CDR region, and obtain the function similar to EBOV4H9 of antibodies.
CN201810183021.XA 2018-03-06 2018-03-06 High-affinity single-domain antibody targeting Ebola virus envelope protein and preparation method and application thereof Active CN108191973B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810183021.XA CN108191973B (en) 2018-03-06 2018-03-06 High-affinity single-domain antibody targeting Ebola virus envelope protein and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810183021.XA CN108191973B (en) 2018-03-06 2018-03-06 High-affinity single-domain antibody targeting Ebola virus envelope protein and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN108191973A CN108191973A (en) 2018-06-22
CN108191973B true CN108191973B (en) 2021-04-27

Family

ID=62594608

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810183021.XA Active CN108191973B (en) 2018-03-06 2018-03-06 High-affinity single-domain antibody targeting Ebola virus envelope protein and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN108191973B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111929439B (en) * 2020-10-10 2021-01-05 中国农业科学院蜜蜂研究所 A rapid diagnosis of bee filovirus test strip and its application

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101861165A (en) * 2007-10-12 2010-10-13 麻省理工学院 Vaccine Nanotechnology
CN103596985A (en) * 2011-04-19 2014-02-19 美国政府(由卫生和人类服务部的部长所代表) Human monoclonal antibodies specific for glypican-3 and use thereof
CN103619875A (en) * 2011-01-28 2014-03-05 加拿大国家研究委员会 Engineering of immunoglobulin domains
CN106188286A (en) * 2016-07-21 2016-12-07 中国科学院武汉病毒研究所 A kind of nano antibody neutralizing Ebola virus

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101861165A (en) * 2007-10-12 2010-10-13 麻省理工学院 Vaccine Nanotechnology
CN103619875A (en) * 2011-01-28 2014-03-05 加拿大国家研究委员会 Engineering of immunoglobulin domains
CN103596985A (en) * 2011-04-19 2014-02-19 美国政府(由卫生和人类服务部的部长所代表) Human monoclonal antibodies specific for glypican-3 and use thereof
CN105968209A (en) * 2011-04-19 2016-09-28 美国政府(由卫生和人类服务部的部长所代表) Human monoclonal antibodies specific for glypican-3 and use thereof
CN106188286A (en) * 2016-07-21 2016-12-07 中国科学院武汉病毒研究所 A kind of nano antibody neutralizing Ebola virus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Selection, characterization, and thermal stabilization of llama single domain antibodies towards Ebola virus glycoprotein";Jinny L. Liu et al.;《Microb Cell Fact》;20171212;第16卷(第223期);摘要，第2页右栏第2段-第10页右栏第2段 *

Also Published As

Publication number Publication date
CN108191973A (en) 2018-06-22

Similar Documents

Publication Publication Date Title
CN111320694B (en) Nano antibody GN2 composed of variable region of heavy chain antibody and preparation method and application thereof
JP4992068B2 (en) Ultra high affinity neutralizing antibody
CN111303279A (en) Single-domain antibody for novel coronavirus and application thereof
CN111647076A (en) Neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 and application thereof
CN113563463B (en) A kind of neutralizing nanobody against novel coronavirus SARS-CoV-2 and its application
CN111592595A (en) Neutralizing antibody against novel coronavirus SARS-Cov-2 and application thereof
CN113045647B (en) Neutralizing antibody of novel coronavirus SARS-CoV-2 and application thereof
JP2011521662A5 (en)
JP7171737B2 (en) Nanobodies capable of binding to SFTSV and uses thereof
CN102906111A (en) Human antibody that specifically binds to the surface antigen of hepatitis B virus
CN108484771A (en) EpCAM single domain antibody G7
CN106188286A (en) A kind of nano antibody neutralizing Ebola virus
CN114656554B (en) Nano antibody of chikungunya virus E2 protein and application thereof
CN114478757B (en) Nano antibody targeting new coronavirus, and preparation method and application thereof
CN114805560B (en) Construction body of nano antibody R14 and application thereof
CN112028997A (en) Anti-CEACAM5 Nanobody
CN110862455A (en) Polypeptides that can bind to CD47 and their applications
CN117050165A (en) Antibody targeting monkey poxvirus, antigen binding fragment thereof and application thereof
CN114805563B (en) Camel-derived high-affinity nanobodies against SARS-CoV-2 α, γ, δ, and ο mutants
EP4286409A1 (en) Antigen-binding protein targeting staphylococcus aureus ?-hemolysis and application thereof
CN108191973B (en) High-affinity single-domain antibody targeting Ebola virus envelope protein and preparation method and application thereof
CN116284377A (en) Anti-human angiopoietin 3 nanobody and application thereof
CN108530538A (en) Epcam single domain antibodies e6
WO2023040834A1 (en) Alpaca-derived nanobody and use thereof
CN116120438B (en) Nanobody of targeting novel coronavirus RBD structural domain and derivative protein thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant