CN111320688A - Flavivirus neutralizing antibody, preparation method and application thereof - Google Patents
Flavivirus neutralizing antibody, preparation method and application thereof Download PDFInfo
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- CN111320688A CN111320688A CN201811542435.3A CN201811542435A CN111320688A CN 111320688 A CN111320688 A CN 111320688A CN 201811542435 A CN201811542435 A CN 201811542435A CN 111320688 A CN111320688 A CN 111320688A
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- 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
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- A61P31/12—Antivirals
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- 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
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- 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
Abstract
The invention relates to a flavivirus neutralizing antibody, wherein the light chain antigen complementarity determining region of the antibody has an amino acid sequence shown in SEQ ID NO. 1-3; the heavy chain antigen complementarity determining region of the antibody has an amino acid sequence shown in SEQ ID NO. 4-6. The antibody can be combined with E protein on the surfaces of particles of dengue virus, West Nile virus, Zika virus and yellow fever virus; moreover, the antibody of the invention can well neutralize different viruses of flaviviruses, including dengue virus (DENV), West Nile Virus (WNV), Zika virus (ZIKV) and Yellow Fever Virus (YFV) of 4 serotypes, which is a broad-spectrum neutralizing antibody; the antibody can be used as a diagnostic antibody and a therapeutic antibody, and has important economic and social significance.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a flavivirus neutralizing antibody, and a preparation method and application thereof.
Background
Flaviviruses (flaviviruses) are single-stranded positive-strand RNA viruses with a membrane that is composed of multiple members of the Flavivirus. Members of this genus mainly include Yellow Fever Virus (YFV), dengue fever (Denguevirus, DENV), West Nile virus (West Nile virus, WNV), ZIKA virus (ZIKA virus, ZIKV), Tick-borne encephalitis (TBEV), and Japanese Encephalitis Virus (JEV).
Dengue virus (DENV), composed of four serotypes, dengue 1-4 types (DENV1-DENV4), primarily by mosquito transmission, DENV-induced diseases ranging from mild dengue fever to severe dengue hemorrhagic fever or dengue shock syndrome, inside the dengue virion there is displayed an 11kb single-stranded positive sense RNA gene surrounded by a capsid protein, this RNA being first translated into a polyprotein and subsequently cleaved under the action of viral and host proteases into three structural proteins (C, prM/M, E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS 5). the inner nucleocapsid of the flavivirus particle is surrounded by a bilayer lipid membrane, outside the bilayer lipid membrane there are 180 envelope proteins (velonope, E) and membrane proteins (Memerane, M) E and M proteins arranged into an icosahedral symmetric unit E protein, the three envelope proteins and M proteins are arranged into an icosahedral symmetric unit E protein, the three regions are composed of a surface domain of a DIII, a fusion domain of a DIII, a C, DII domain, a fusion domain, a D23, a C domain, a D23, a D domain, a C domain, a D domain.
Three regions of the extracellular domain of flavivirus E protein can be targeted by neutralizing antibodies. Flavivirus neutralizing antibodies can be classified into 4 major classes based on the known structure of antibody-antigen E protein complexes and antibody-virion complexes. The first is an antibody to DII region Fusion Loop Epitope (FLE), which is generally recognized by the FLE antibody as a conserved fusion loop region, so this type of antibody generally has cross-protection, e.g., the murine monoclonal antibody 2a10G6 is capable of broadly neutralizing dengue 1-4, WNVE, YFV, and ZIKA. Another FLE antibody was E53, and the resolved complex structure of E53 with WNVE E protein showed that it bound to the fusion loop region at an oblique angle. Both these FLE antibodies specifically bind to the bc loop.
The second type of antibody is a DI region specific neutralizing antibody, including crystal structures of 5H2 and 1 F4.5H2 with flavivirus E protein showing that the epitope of 5H2 is located at F0G0 of the DI β fold edge on the DI-DII junction region, including the F0G0H0 chain, the loop connected between them, and downstream of the I0 chain its neutralizing mechanism is hypothesized to block the binding of E protein to the receptor surface because of binding at the DI-DI junction region, while 1F4 binds to DENV1 virus particles rather than E protein, and electron microscopy shows that the antibody neutralization may be due to steric hindrance caused by binding blocking the binding of the N67 sugar to the host receptor DC-539n, and locks the DI-DI hinge region preventing the conformational changes in E protein required for membrane fusion to occur.
The third type of antibody is the DIII region where the binding is of the E protein to the cell receptor. Such as 4E11, 2H12, 1A1D-2 are all antibodies with DENV cross protection effect. Where 4E11 shared an overlapping epitope with 1A1D-2, but 1A1D-2 was unable to neutralize DENV4 because it exhibited a lower charge complementation with DENV4 DIII. Two additional antibodies, E111 and E104 dengue-specific, neutralized DENV1 and DENV2, respectively.
The fourth type of antibody is an antibody that binds to the E protein dimer (EDE), which, as is well known, blocks the pre-fusion structure by binding to the E protein dimer, preventing the conformational changes necessary for membrane fusion to occur. Four crystal structures showed that the EDE antibodies can be further divided into EDE1(C8, C10) and EDE2(a11, B7). The EDE1 antibody binds directly to the N67 glycosylation site, whereas the EDE2 antibody requires binding of both the N67 and N153 glycosylation sites. Notably, these four anti-dengue EDE antibodies were also found to be able to neutralize ZIKA in recent studies.
CN 106589116A discloses a flavivirus human monoclonal antibody and application thereof, and finds that three new antibodies can be combined with ZIKV-E protein, and the combination sites are determined, and all have strong Zika virus E protein combination capacity. But it is of great significance to continuously search for a flavivirus neutralizing antibody which has broad spectrum, high affinity and good specificity.
Disclosure of Invention
Aiming at the defects and practical requirements of the prior art, the invention provides a flavivirus neutralizing antibody, a preparation method and application thereof, wherein the flavivirus neutralizing antibody provides a new treatment method for the prevention and treatment of flavivirus, provides a new choice for the diagnosis of flavivirus infection and the detection of flavivirus surface antigen E protein, and has important economic and social meanings.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a flavivirus neutralizing antibody, wherein the light chain antigen complementarity determining region of said antibody has an amino acid sequence as shown in SEQ ID No. 1-3; the heavy chain antigen complementarity determining region of the antibody has an amino acid sequence shown in SEQ ID NO. 4-6.
The amino acid sequence of the light chain antigen complementarity determining region is as follows:
CDR1(SEQ ID NO.1)：SSNIGAGYD；
CDR2(SEQ ID NO.2)：GNN；
CDR3(SEQ ID NO.3)：QSYDSSLSGGV；
the amino acid sequence of the heavy chain antigen complementarity determining region is as follows:
CDR1(SEQ ID NO.4)：GFTFSSQV；
CDR2(SEQ ID NO.5)：IHTGGSST；
CDR3(SEQ ID NO.6)：AKGSAYGDYVEY.
in the invention, the inventor finds that the antibody or the antigen binding fragment with the light chain and heavy chain antigenic determinant regions can well neutralize the flavivirus and can be combined with the antigen E protein of the flavivirus through screening, thereby inhibiting the replication of the virus and having the treatment effect on the flavivirus.
According to the invention, the light chain variable region of the antibody has an amino acid sequence shown as SEQ ID NO.7, and the heavy chain variable region of the antibody has an amino acid sequence shown as SEQ ID NO. 8.
The amino acid sequence of the variable region of the light chain of the antibody (SEQ ID NO.7) is as follows:
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQPPGTAPKLLIYGNNNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGGV；
the amino acid sequence of the heavy chain variable region (SEQ ID NO.8) of the antibody is as follows:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSQVMSWVRQAPGRGLEWVSVIHTGGSSTYYADSVKGRFTISRDNSKNTVFLHMNSLRVEDTALYYCAKGSAYGDYVEY.
according to the invention, the antibody is a humanized monoclonal antibody.
According to the invention, the C-terminus of the light chain of the flavivirus neutralizing antibody carries 4-8 HIS tags, which may be, for example, 4, 5, 6, 7 or 8, preferably 6 HIS tags.
In a second aspect, the present invention provides a DNA fragment encoding a flavivirus neutralizing antibody as described in the first aspect.
According to the invention, the light chain of the antibody has the nucleotide sequence shown in SEQ ID NO.9, and the heavy chain of the antibody has the nucleotide sequence shown in SEQ ID NO. 10.
The nucleotide sequence of the variable region of the light chain of the antibody (SEQ ID NO.9) is as follows:
Cagtctgtgctgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcaccgggagcagctccaacatcggggcaggttatgatgtacactggtatcagcagcctccaggaacagcccccaaactcctcatctatggtaacaacaatcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcctatgacagcagcctgagtgggggggtgt；
the nucleotide sequence of the heavy chain variable region (SEQ ID NO.10) of the antibody is as follows:
Gaggtgcagctgttggagtcggggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacgtttagcagccaagtcatgagctgggtccgccaggctccagggagggggctggagtgggtctcagttattcataccggtggaagtagtacatattatgctgactccgtgaagggccggttcaccatctccagagataattccaagaacacggtatttctgcatatgaacagcctgagagtcgaggacacggccctgtattactgtgcgaagggatcagcctacggtgactacgtggagtact.
in a third aspect, the present invention provides an expression vector comprising at least one copy of a DNA fragment according to the second aspect.
In a fourth aspect, the present invention provides a host cell comprising an expression vector according to the third aspect.
In a fifth aspect, the present invention provides a method for preparing a flavivirus neutralizing antibody according to the first aspect, comprising the steps of:
(1) PBMC in the peripheral blood of an infected person is separated, RNA is extracted, and cDNA is reversely transcribed;
(2) amplifying the sequences of the highly variable regions of the heavy chain and the light chain, selecting and synthesizing according to the abundance of the CDR;
(3) the synthesized antibody fragment was constructed into an expression vector.
According to the invention, the vector in step (3) is a mammalian expression vector, preferably a pCAGGS mammalian expression vector.
In a sixth aspect, the present invention provides the use of a flavivirus neutralizing antibody according to the first aspect, or a DNA fragment of a flavivirus neutralizing antibody according to the second aspect, or an expression vector according to the third aspect, or a host cell according to the fourth aspect, in the manufacture of a medicament and/or agent for inhibiting influenza virus.
According to the invention, the flavivirus comprises any one of, or a combination of at least two of, a dengue virus (DENV) serotype, a West Nile Virus (WNV) serotype, a ZIKV serotype, or a Yellow Fever Virus (YFV) serotype.
In a seventh aspect, the present invention provides the use of a flavivirus neutralizing antibody as described in the first aspect for the preparation of a medicament and/or agent having affinity for the E protein antigen of a flavivirus and/or neutralizing activity against a flavivirus.
In the present invention, the antibody binds to the fusion loop region of the E protein or its peripheral region, thereby inhibiting the fusion of the virus with the cell membrane and thus inhibiting the replication of the virus.
Preferably, the flavivirus comprises any one of, or a combination of at least two of, a dengue virus (DENV) serotype, a West Nile Virus (WNV) serotype, a ZIKV serotype, or a Yellow Fever Virus (YFV) serotype.
Compared with the prior art, the invention has the following beneficial effects:
(1) the antibody can well neutralize flavivirus, can be combined with antigen E proteins of dengue virus, yellow fever virus, Zika virus and West Nile virus in the flavivirus genus, and inhibits the fusion of the virus and cell membrane, thereby inhibiting the replication of the virus in vivo;
(2) the antibody of the invention can bind to flavivirus with an affinity of 0.13nM to 0.85nM and a neutralizing capacity of 0.04 to 1.31 mug/ml for neutralizing flavivirus;
(3) the acquisition of the antibody provides a new candidate for the prevention and treatment of the flavivirus, and has important economic and social significance.
Drawings
FIG. 1 is a graph showing the results of purification of the antibody prepared according to the present invention by HiloadTM 16/600 Superdex 200pg molecular sieve chromatography;
FIG. 2 is a graph showing the results of neutralizing dengue virus 1-4 and West Nile Virus in the flavivirus genus by the antibody of the present invention, wherein DENV1 is dengue virus 1, DENV2 is dengue virus 2, DENV3 is dengue virus 3, DENV4 is dengue virus 4, and WNV is West Nile Virus;
FIG. 3 is a graph showing the results of competitive binding of an antibody of the present invention to an epitope with an antibody to a known epitope, wherein TIB12 TIB12+ mab11 indicates that TIB12 is the primary antibody and TIB12+ mab11 is the competing antibody; buffer buffer + mab11 indicates buffer as the primary antibody and buffer + mab11 as the competing antibody; mab11 mab11+ TIB12 indicates mab11 as the primary antibody and mab11+ TIB12 as the competing antibody; buffer buffer + mab11 indicates buffer as the primary antibody and buffer + mab11 as the competing antibody.
FIG. 4 is a graph showing the results of the infection-enhancing effect of the antibody of the present invention on dengue virus types 1-4, in which DENV1 is dengue virus 1, DENV2 is dengue virus 2, DENV3 is dengue virus 3, and DENV4 is dengue virus 4.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.
The instrument comprises the following steps:
octet RED96 instrument (ForteBio)
The instrument is mainly characterized in that a biological molecule compatible layer is arranged on the bottom surface of a biological chip made of optical fibers and used for fixing a stationary phase of sample molecules to be analyzed to form a biological film layer; when visible light with a certain wavelength range is incident on the biological film layer, the reflection and refraction phenomena of the light can occur, the incident light can be divided into two parts on the surface of the biological film layer, one part of the incident light forms reflected light, and when the incident light is emitted in the same way on a second interface of the biological film layer, a beam of reflected light is formed; when the light beam is vertically incident, the two parts of reflected light are overlapped to form interference waves which can be detected by a spectrometer; when there is an interaction between molecules, the interference curve formed by the reflected light of the two parts shifts to a direction of increasing wavelength, and the interference curve changes when the molecules are combined or dissociated.
EXAMPLE 1 preparation and purification of antibodies
This example provides a method for preparing a flavivirus neutralizing antibody, comprising the steps of:
(1) PBMC in the peripheral blood of an infected person is separated, RNA is extracted, and cDNA is reversely transcribed;
(2) amplifying the sequences of the highly variable regions of the heavy chain and the light chain, sequencing the amplified target fragment by using Miseq 2X300bp, and analyzing the sequencing result;
(3) selecting high-frequency variable region sequences of infected patients by taking CDR abundance as a main parameter, then selecting heavy chain variable region (VH) and light chain variable region (VL) sequences of CDR1, CDR2 and CDR3 which appear at high frequency, adding respective constant regions and Fc required by a full antibody for whole gene synthesis;
the nucleotide sequence of the variable region of the light chain of the antibody (SEQ ID NO.9) is as follows:
Cagtctgtgctgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcaccgggagcagctccaacatcggggcaggttatgatgtacactggtatcagcagcctccaggaacagcccccaaactcctcatctatggtaacaacaatcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcctatgacagcagcctgagtgggggggtgt；
the nucleotide sequence of the heavy chain variable region (SEQ ID NO.10) of the antibody is as follows:
Gaggtgcagctgttggagtcggggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacgtttagcagccaagtcatgagctgggtccgccaggctccagggagggggctggagtgggtctcagttattcataccggtggaagtagtacatattatgctgactccgtgaagggccggttcaccatctccagagataattccaagaacacggtatttctgcatatgaacagcctgagagtcgaggacacggccctgtattactgtgcgaagggatcagcctacggtgactacgtggagtact；
(4) adding secretion signal peptides of corresponding heavy chains and light chains at the N end in an IgG form, and constructing an antibody fragment into a pCAGGS mammalian expression vector;
(5) co-transfecting 293T cells with a PEI transfection reagent by using a pCAGGS expression vector inserted with heavy chain and light chain sequences for mass expression and purifying;
the purification method of the antibody comprises the following steps:
(1') the supernatant was harvested 96h after transfection, centrifuged at 6000rpm for 30min, filtered through a 0.22. mu.M filter and bound to HisTrap overnight by a peristaltic pumpTMHP 5mL pre-column (Fab format antibody purification) or Protein a pre-column (IgG format antibody purification);
(2') eluting the target protein on AKTA machine under the following conditions: buffer 1(10mM Tris, 40mM NaCl elution of hetero-proteins), buffer 2(10mM Tris, 40mM NaCl, 300mM imidazole) and buffer 1 elute the Protein of interest using a 100% B pump on AKTA machine for 30min (20 mM phosphoric acid (pH 7.0) for hetero-proteins if the column is prepacked with Protein A, 0.1M glycine (pH 3.0) for IgG proteins bound to Protein A);
(3') concentrating the eluted protein, exchanging buffer solution (PBS), and subjecting to HiloadTM16/600SuperdexTMThe 200pg molecular sieve was further purified, the result after purification is shown in FIG. 1, and the purified antibody is named TIB 12.
As can be seen from FIG. 1, a single IgG form of the desired antibody can be obtained by purification.
Example 2 antigen expression purification
The preparation method of the flavivirus E protein antigen comprises the following steps:
(1) expression of Inclusion bodies: constructing genes of surface antigen E proteins of different viruses of flaviviridae, such as dengue virus, west nile virus, Zika virus and yellow fever virus, on a PET21a vector, then transforming BL21 competent cells, selecting a monoclonal grown on an LB solid culture medium to be 10ml of a liquid LB (ampicillin resistance) culture medium, and shaking the bacteria at 200rpm and 37 ℃ overnight; the next day 10ml of the shaken bacteria were transferred to 1L of LB (ampicillin resistant) medium, shaken at 200rpm and 37 ℃ for 4h, followed by addition of 1M IPTG1ml to a final concentration of 1mM, continued shaking at 200rpm and 37 ℃ for 4h, followed by centrifugation at 6000rpm for 10min to harvest the bacteria;
(2) purification of inclusion bodies: the above centrifugally collected pellet of the thallus is resuspended with 20ml PBS and then disrupted using a high pressure disrupter, then centrifuged at 10000rpm for 30min and then the supernatant is discarded, the bacterial debris on the bottom inclusion body pellet is stripped off, then centrifuged with 20ml washing buffer (2% Triton X-100, 50mM Tris pH8.0, 30mM NaCl, 10mM EDTA, 10mM DTT) and suspended at 10000rpm for 20min and then washed once with washing buffer to strip off the bacterial debris as much as possible, then resuspended with resuspension buffer (50mM Tris pH8.0, 100mM NaCl, 10mM EDTA, 10mM DTT), 10000rpm, centrifuged for 20min and then the supernatant is discarded, after the pellet of the inclusion body is weighed, 1ml lysis buffer (6M guanidine hydrochloride, 10% glycerol, 100mM NaCl, 50mM Tris pH8.0, 10mM EDTA, 10mM DTT) is added per 30mg and stirred for solubilization at 4 ℃ and then 10000rpm, centrifuging for 30min to remove impurities, collecting supernatant as treated inclusion body, and standing at-80 deg.C;
(3) renaturation and purification of soluble E protein: dropping 12ml of inclusion body into 2L of renaturation solution (100mM Tris pH8.0, 400mM L-Arg HCl, 2mM EDTA, 5mM GSH, 1mM GSSG) through a needle syringe, stirring at low speed for 8h, dropping 12ml of inclusion body again, concentrating to about 40ml through a concentration cup after overnight, changing the solution with 20mM Tris pH8.0, 150mM NaCl and 10% glycerol for 20 times, concentrating to 2ml in volume and HiloadTM200 molecular sieve purified protein.
Example 3 antigen antibody affinity assay
This example provides a method for testing the affinity of antigen and antibody, specifically using an Octet RED96 instrument, PBST buffer solution for the whole experimental procedure, and a detection temperature of 30 ℃.
The Protein A probe was first bound to 25nM of the antibody prepared in example 1 for 60s, followed by PBST wash for 10s, and the probe was subsequently placed in gradient diluted dengue E Protein antigen (200nM starting, diluted 7 gradients) for 300s binding and then dissociated for 500s in PBST buffer, and the affinity was calculated using OCTET's analysis software, the results are shown in Table 1.
TABLE 1 affinity assay of TIB12 antibody with different E proteins
As can be seen from Table 1, the antibody TIB12 almost agreed with the binding dissociation pattern of dengue virus, Zika virus, West Nile virus and yellow fever virus in the flavivirus genus, and all were slow binding and dissociation patterns, with the affinity ranging from 0.13nM to 0.85 nM.
Example 4 neutralization assay of antibodies
This example provides a method for antibody neutralization assay, which comprises the following steps:
(1) preparing a 24-well plate Vero cell with 90% confluence, preparing an antibody and virus mixed solution, diluting the antibody from a final concentration of 400 mu g/ml by a 3-fold ratio (DMEM containing 1% FBS is used as a diluent), adding a dengue virus with the same volume and quantitative (FACS detection positive rate is 8% -20%), uniformly mixing the dengue virus and the antibody, and then, acting for 1h at 37 ℃;
(2) the antibody-virus mixture was added to Vero cells in 24-well plates washed twice with PBS in an amount of 300. mu.l per well, 2 wells were set for each antibody dilution, and the 24-well plates to which the antibody-virus mixture was added were placed at 37 ℃ with 5% CO2Culturing in an incubator for 1h, shaking every 15min, directly adding 700 μ l DMEM medium containing 10% FBS into each hole, and performing flow detection after 48 h;
(3) flow detection: the cells in the 24-well plate were washed once with PBS and the supernatant was discarded, 150. mu.l of pancreatin (0.25% final concentration) was added thereto at 37 ℃ with 5% CO2The incubator was digested for 3min, and then 100. mu.l of PBS containing 10% FBS was added to stop the digestion; then transferring the cell suspension in the 24-well plate into a round-bottom 96-well plate, and horizontally centrifuging at the temperature of 4 ℃ for 10min at 500 g; sucking and discarding the supernatant, washing the supernatant once with PBS (containing 1% FBS), adding 100 μ l of fixing solution (BD Biosciences), placing the supernatant on ice away from light for 20min, then adding 100 μ l of washing buffer solution (BD Biosciences), centrifuging the supernatant, adding 50 μ l of primary antibody (antibody Z6 capable of binding dengue E protein and 2 μ g/ml final concentration) on ice, incubating the mixture for 30min, then adding 150 μ l of washing buffer solution, sucking and discarding the supernatant after centrifuging, adding 50 μ l of goat anti-human secondary antibody (diluted by 1:200 with the washing buffer solution), then adding 150 μ l of washing buffer solution, washing the supernatant once with PBS after centrifuging, adding 180 μ l of PBS, blowing and mixing the mixture evenly, transferring the mixture to a flow tube of Facs, and detecting the mixture on a machine;
the obtained results were analyzed in flow. jo, and then IC50 was calculated by the Nonlinear regression method of Graphpad software, and the results are shown in fig. 2 and table 2.
TABLE 2 neutralization assay of TIB12 antibody with different viruses
As can be seen from FIG. 2 and Table 2, TIB12 was prepared to neutralize dengue virus types 1-4, West Nile virus and yellow fever virus in the flavivirus genus at a neutralization level of between 0.04 and 1.31. mu.g/ml.
Example 5 Competition test of antibodies with known epitopes
The present embodiment provides a competitive assay for detecting an antibody and an antibody with a known epitope, which uses an OctetRED96 instrument, and specifically includes the following steps:
the Ni-NTA biosensor was immobilized on the chip surface by placing it in 200nM DV2E protein for 300s, then contacted with the first antibody (200nM) for about 900s until saturation of binding was reached, followed by direct contact of the biosensor with the second antibody for 300s, where the first antibody was present in the second antibody (final concentration of both antibodies is 200nM), and the real-time binding reaction was monitored by Octet RED96 instrument;
two experiments were performed, the first antibody in the first experiment being TIB12 antibody prepared in example 1 of the present application and the second antibody being mAb11 antibody; in the second experiment the primary antibody was mAb11 antibody and the secondary antibody was TIB12 antibody prepared in example 1 of the present application; the change of the binding curve was compared to determine whether the antigen-binding epitopes of the two antibodies were identical, and the results are shown in fig. 3.
From fig. 3, it can be seen that the antibody TIB12 antibody of the present application has a competitive binding relationship with mAb11 antibody, indicating that the antibody TIB12 of the present application binds to the fusion loop or peripheral region of the E protein.
Example 6 infection-enhancing Effect (ADE) of antibodies against dengue Virus
This example provides a test of the infection-enhancing effect of an antibody against dengue virus, as follows:
(1) by 5 timesDiluting the antibody and dengue virus (MOI 0.2-0.5) with different phenotypes at a multiple ratio, adding 96U plate at a volume of 100. mu.l per well, incubating at 37 deg.C for 1h (dilution is RPMI1640), adding RPMI1640 to the mixture, washing K562 cells (5 × 104cells per well), setting 2 multiple wells per antibody dilution, placing 96 well plate with antibody-virus-cell mixture at 37 deg.C, 5% CO2Culturing for 2h in an incubator;
(2) the cells were washed once with RPMI1640, resuspended in 100. mu.l of 2% FBS-containing RPMI1640, and subjected to flow detection 48 hours later, in the same manner as in example 4, and the results are shown in FIG. 4;
as can be seen from fig. 4, the TIB12 antibody of the present application had a weaker ADE effect on DENV1 and DENV2, and a stronger effect on the ADE of DENV3 and DENV 4.
In conclusion, the antibody of the invention can well neutralize dengue virus 1-4 and west nile virus in flavivirus. Can inhibit the virus from generating membrane fusion, thereby inhibiting the virus from replicating in vivo, and the antibody has important economic and social significance.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> flavivirus neutralizing antibody, preparation method and application thereof
<130>2018
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<170>PatentIn version 3.3
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Claims (10)
1. A flavivirus neutralizing antibody, wherein the light chain antigen complementarity determining region of said antibody has an amino acid sequence as shown in SEQ id nos. 1-3; the heavy chain antigen complementarity determining region of the antibody has an amino acid sequence shown in SEQ ID NO. 4-6.
2. The antibody of claim 1, wherein the light chain variable region of said antibody has the amino acid sequence shown in SEQ id No. 7;
preferably, the heavy chain variable region of the antibody has an amino acid sequence shown as SEQ ID No. 8.
3. The antibody of claim 1 or 2, wherein the antibody is a humanized monoclonal antibody;
preferably, the C-terminus of the light chain of the antibody carries 4-8 HIS tags, preferably 6 HIS tags.
4. A DNA fragment encoding the flavivirus neutralizing antibody of claim 1.
5. An expression vector comprising at least one copy of the DNA fragment of claim 4.
6. A host cell comprising the expression vector of claim 5.
7. A method of producing the flavivirus neutralizing antibody of claim 1, comprising the steps of:
(1) PBMC in the peripheral blood of an infected person is separated, RNA is extracted, and cDNA is reversely transcribed;
(2) amplifying the sequences of the highly variable regions of the heavy chain and the light chain, selecting and synthesizing according to the abundance of the CDR;
(3) the synthesized antibody fragment was constructed into an expression vector.
8. The method according to claim 7, wherein the vector of step (3) is a mammalian expression vector, preferably a pCAGGS mammalian expression vector.
9. Use of the flavivirus neutralizing antibody of any one of claims 1-3, the DNA fragment of the flavivirus neutralizing antibody of claim 4, the expression vector of claim 5, or the host cell of claim 6 for the preparation of a medicament and/or agent for the prevention and treatment of flavivirus;
preferably, the flavivirus comprises any one of, or a combination of at least two of, a dengue virus serotype, a west nile virus serotype, a zika virus serotype, or a yellow fever virus serotype.
10. Use of a flavivirus neutralizing antibody according to any of claims 1-3 for the preparation of a medicament and/or agent having affinity for the E protein antigen of a flavivirus and/or neutralizing activity against a flavivirus;
preferably, the flavivirus comprises any one of, or a combination of at least two of, a dengue virus serotype, a west nile virus serotype, a zika virus serotype, or a yellow fever virus serotype.
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| CN116655781A (en) * | 2023-04-21 | 2023-08-29 | 中国人民解放军军事科学院军事医学研究院 | Antibody FY1 for broad-spectrum recognition of flavivirus E protein |
| CN116514963B (en) * | 2023-04-21 | 2023-09-26 | 中国人民解放军军事科学院军事医学研究院 | Antibody FY2 for broad-spectrum recognition of flavivirus E protein and application thereof |
| CN116655781B (en) * | 2023-04-21 | 2023-10-27 | 中国人民解放军军事科学院军事医学研究院 | Antibody FY1 for broad-spectrum recognition of flavivirus E protein |
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