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

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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
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envelope protein
ebov4h9
ebola virus
gly
ser
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CN108191973A (en
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龚睿
张怀东
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Wuhan Institute of Virology of CAS
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Wuhan Institute of Virology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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

Abstract

The invention discloses a high-affinity single-domain antibody of a targeting Ebola virus envelope protein and application thereof. In the invention, a high-affinity single-domain antibody targeting the Ebola virus envelope protein is obtained by screening from a polypeptide and protein library taking a VH structure domain m0E1 of human IgG as a framework and taking the Ebola virus envelope protein GP as an antigen, and compared with a full-length monoclonal antibody (the molecular weight is 150kD), the antibody has smaller molecular weight, better tissue permeability and capability of combining with an epitope with steric hindrance 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.

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
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tca 363

Claims (5)

1. A high affinity single domain antibody targeting the Ebola virus envelope protein, characterized by: the high affinity single domain antibody is EBOV4H9, the complementarity determining clusters of which 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.
2. The high affinity single domain antibody targeting the ebola virus envelope protein of claim 1, wherein: the gene sequence of EBOV4H9 for coding CDR1 is Seq ID No.1, the gene sequence of EBOV4H9 for coding CDR2 is Seq ID No.3, and the gene sequence of EBOV4H9 for coding CDR3 is Seq ID No. 5.
3. The high affinity single domain antibody targeting the ebola virus envelope protein of claim 1, wherein: the full-length amino acid sequence of the EBOV4H9 is Seq ID No. 7.
4. The high affinity single domain antibody targeting the ebola virus envelope protein of claim 3, wherein: the full-length sequence of the EBOV4H9 gene is Seq ID No. 8.
5. The high affinity single domain antibody targeting the ebola virus envelope protein of claim 1, wherein: the regions of EBOV4H9 other than the CDR regions were subjected to base and amino acid mutation, and an antibody functionally similar to EBOV4H9 was obtained.
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