CN117659177B - Antibodies against monkey poxvirus or antigen binding fragments thereof and uses thereof - Google Patents

Antibodies against monkey poxvirus or antigen binding fragments thereof and uses thereof Download PDF

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CN117659177B
CN117659177B CN202410129976.2A CN202410129976A CN117659177B CN 117659177 B CN117659177 B CN 117659177B CN 202410129976 A CN202410129976 A CN 202410129976A CN 117659177 B CN117659177 B CN 117659177B
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CN117659177A (en
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程功
曲园园
肖望成
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Tsinghua University
Shenzhen Bay Laboratory
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Shenzhen Bay Laboratory
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Abstract

The invention discloses an antibody against a monkey pox virus or an antigen binding fragment thereof and application thereof, and relates to the technical field of antibodies. The invention specifically discloses a plurality of antibodies (A012, A046, A051, A073, A094, A100, A124, A129 and A138) with good specificity and affinity to M1R protein, and the antibodies have good neutralization activity to orthopoxviruses such as monkey pox viruses and vaccinia viruses, can be used for detecting M1R antigen proteins, MPXV and VACV, can be used for developing medicaments for treating orthopoxvirus infection such as smallpox viruses, vaccinia viruses, monkey pox viruses and vaccinia viruses, and have good application prospects.

Description

Antibodies against monkey poxvirus or antigen binding fragments thereof and uses thereof
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-monkey poxvirus antibody or an antigen binding fragment thereof and application thereof.
Background
Monkey pox is a viral zoonotic disease caused by the monkey pox virus. The monkey poxvirus (Monkeypox virus, MPXV) belongs to the family Poxviridae, the genus orthopoxvirus, and also belongs to the genus Smallpox, vaccinia virus, and the like. Serological studies have shown that historical smallpox vaccine (MVA-BN vaccine) induces low levels of monkey pox virus neutralizing antibodies and does not provide complete protection for high risk populations. There is no specific vaccine or drug against MPXV infection. Therefore, it is important to develop an antibody that can help prevent infection by the monkey poxvirus.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides an anti-poxvirus antibody or an antigen binding fragment thereof, which has good affinity to M1R protein and good neutralizing activity to both monkey poxvirus and vaccinia virus.
The invention also provides a recombinant protein.
The invention also provides biological materials related to the antibodies or antigen binding fragments thereof, or recombinant proteins.
The invention also provides a conjugate.
The invention also provides the use of the above antibodies or antigen binding fragments thereof, the above recombinant proteins, the above biological materials or the above conjugates.
The invention also provides a product comprising the above antibody or antigen binding fragment thereof, the above recombinant protein, the above biological material or the above conjugate.
The invention also provides a preparation method of the antibody or the antigen binding fragment thereof or the recombinant protein.
An anti-poxvirus antibody or antigen-binding fragment thereof according to the embodiments of the first aspect of the invention, selected from any one of a 1) to a 9); and has heavy and light chain variable regions containing complementarity determining regions CDR1, CDR2, CDR3, respectively;
a1 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26 th to 33 th positions, 51 th to 58 th positions and 97 th to 110 th positions in SEQ ID No. 1; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 32 th positions, 50 th to 52 th positions and 89 th to 97 th positions in SEQ ID No. 2;
a2 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26 th to 33 th, 51 th to 60 th and 99 th to 115 th positions in SEQ ID No. 5; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 32 th, 50 th to 52 th and 89 th to 97 th positions in SEQ ID No. 6;
a3 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26 th to 33 th positions, 51 th to 58 th positions and 97 th to 109 th positions in SEQ ID No. 9; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 26 th to 33 th, 50 th to 52 th and 89 th to 97 th positions in SEQ ID No. 10;
a4 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26 th to 33 th, 51 th to 58 th and 97 th to 111 th positions in SEQ ID No. 13; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 32 th positions, 50 th to 52 th positions and 89 th to 96 th positions in SEQ ID No. 14;
a5 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26 th to 33 th positions, 51 th to 58 th positions and 97 th to 103 th positions in SEQ ID No. 17; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 32 th positions, 50 th to 52 th positions and 89 th to 97 th positions in SEQ ID No. 18;
a6 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26 th to 33 th positions, 51 th to 58 th positions and 97 th to 112 th positions in SEQ ID No. 21; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 32 th positions, 50 th to 52 th positions and 89 th to 97 th positions in SEQ ID No. 22;
a7 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26 th to 33 th positions, 51 th to 59 th positions and 97 th to 112 th positions in SEQ ID No. 25; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 32 th positions, 50 th to 52 th positions and 89 th to 97 th positions in SEQ ID No. 26;
a8 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26 th to 33 th positions, 51 th to 58 th positions and 97 th to 112 th positions in SEQ ID No. 29; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 32 th, 50 th to 52 th and 89 th to 97 th positions in SEQ ID No. 30;
a9 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26 th to 33 th, 51 th to 58 th and 97 th to 109 th positions in SEQ ID No. 33; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 32 th, 50 th to 52 th and 89 th to 97 th positions in SEQ ID No. 34.
The antibody or antigen binding fragment thereof according to the embodiments of the present invention has at least the following beneficial effects:
The antibody or antigen binding fragment thereof has good affinity to M1R protein, has good neutralizing activity to trans-complement defective monkey pox virus and vaccinia virus (VACV), can more effectively neutralize orthopoxviruses such as MPXV and VACV when used in combination with other MPXV surface protein specific antibodies, and is expected to replace Vaccinia Immunoglobulin (VIG) in future MPXV treatment. The antibody or the antigen binding fragment thereof can be used for detecting M1R antigen protein, MPXV and VACV, and can also be used for developing medicines for treating orthopoxvirus infection such as smallpox virus, vaccinia virus and the like.
According to some embodiments of the invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof of a 1) comprises:
a111 Amino acid sequence shown as SEQ ID No. 1; or (b)
A112 Amino acid sequence with the same function as the protein shown in SEQ ID No.1 by substituting and/or deleting and/or adding one or more amino acids in SEQ ID No. 1; or (b)
A113 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 1 and the same function as the protein shown in SEQ ID No. 1;
a1 The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
a121 Amino acid sequence shown as SEQ ID No. 2; or (b)
A122 Amino acid sequence with the same function as the protein shown in SEQ ID No.2 by substituting and/or deleting and/or adding one or more amino acids in SEQ ID No. 2; or (b)
A123 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 2 and the same function as the protein shown in SEQ ID No. 2;
a2 The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises:
a211 Amino acid sequence shown as SEQ ID No. 5; or (b)
A212 Amino acid sequence with the same function as the protein shown in SEQ ID No. 5 by substituting and/or deleting and/or adding one or more amino acids in SEQ ID No. 5; or (b)
A213 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 5 and the same function as the protein shown in SEQ ID No. 5;
a2 The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
a221 Amino acid sequence shown as SEQ ID No. 6; or (b)
A222 Amino acid sequence with the same function as the protein shown in SEQ ID No. 6 by substituting and/or deleting and/or adding one or more amino acids in SEQ ID No. 6; or (b)
A223 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 6 and the same function as the protein shown in SEQ ID No. 6;
a3 The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises:
a311 Amino acid sequence shown as SEQ ID No. 9; or (b)
A312 Amino acid sequence of SEQ ID No. 9 with the same function as the protein shown in SEQ ID No. 9 through substitution and/or deletion and/or addition of one or several amino acids; or (b)
A313 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 9 and the same function as the protein shown in SEQ ID No. 9;
a3 The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
a321 Amino acid sequence shown as SEQ ID No. 10; or (b)
A322 Amino acid sequence of SEQ ID No. 10 with one or several amino acids to replace and/or delete and/or add and with the same function as the protein shown in SEQ ID No. 10; or (b)
A323 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 10 and the same function as the protein shown in SEQ ID No. 10;
a4 The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises:
a411 An amino acid sequence shown as SEQ ID No. 13; or (b)
A412 Amino acid sequence of SEQ ID No. 13 with the same function as the protein shown in SEQ ID No. 13 through substitution and/or deletion and/or addition of one or several amino acids; or (b)
A413 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 13 and the same function as the protein shown in SEQ ID No. 13;
a4 The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
a421 Amino acid sequence shown as SEQ ID No. 14; or (b)
A422 Amino acid sequence of SEQ ID No. 14 with the same function as the protein shown in SEQ ID No. 14 through substitution and/or deletion and/or addition of one or several amino acids; or (b)
A423 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 14 and the same function as the protein shown in SEQ ID No. 14;
a5 The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises:
a511 Amino acid sequence shown as SEQ ID No. 17; or (b)
A512 Amino acid sequence of SEQ ID No. 17 with the same function as the protein shown in SEQ ID No. 17 through substitution and/or deletion and/or addition of one or several amino acids; or (b)
A513 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 17 and the same function as the protein shown in SEQ ID No. 17;
a5 The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
a521 Amino acid sequence shown as SEQ ID No. 18; or (b)
A522 Amino acid sequence of SEQ ID No. 18 with the same function as the protein shown in SEQ ID No. 18 through substitution and/or deletion and/or addition of one or several amino acids; or (b)
A523 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 18 and the same function as the protein shown in SEQ ID No. 18;
a6 The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises:
a611 Amino acid sequence shown as SEQ ID No. 21; or (b)
A612 Amino acid sequence of SEQ ID No. 21 with one or several amino acids to replace and/or delete and/or add and with the same function as the protein shown in SEQ ID No. 21; or (b)
A613 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 21 and the same function as the protein shown in SEQ ID No. 21;
a6 The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
a621 Amino acid sequence shown as SEQ ID No. 22; or (b)
A622 Amino acid sequence of SEQ ID No. 22 with the same function as the protein shown in SEQ ID No. 22 through substitution and/or deletion and/or addition of one or several amino acids; or (b)
A623 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 22 and the same function as the protein shown in SEQ ID No. 22;
a7 The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises:
a711 An amino acid sequence as shown in SEQ ID No. 25; or (b)
A712 Amino acid sequence of SEQ ID No. 25 with the same function as the protein shown in SEQ ID No. 25 through substitution and/or deletion and/or addition of one or several amino acids; or (b)
A713 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 25 and the same function as the protein shown in SEQ ID No. 25;
a7 The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
a721 An amino acid sequence as shown in SEQ ID No. 26; or (b)
A722 Amino acid sequence of SEQ ID No. 26 with one or several amino acids to replace and/or delete and/or add and with the same function as the protein shown in SEQ ID No. 26; or (b)
A723 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 26 and the same function as the protein shown in SEQ ID No. 26;
a8 The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises:
a811 Amino acid sequence shown as SEQ ID No. 29; or (b)
A812 Amino acid sequence of SEQ ID No. 29 with one or several amino acid substitutions and/or deletions and/or additions and with the same function as the protein shown in SEQ ID No. 29; or (b)
A813 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 29 and the same function as the protein shown in SEQ ID No. 29;
a8 The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
a821 An amino acid sequence shown as SEQ ID No. 30; or (b)
A822 Amino acid sequence of SEQ ID No. 30 with the same function as the protein shown in SEQ ID No. 30 through substitution and/or deletion and/or addition of one or several amino acids; or (b)
A823 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 30 and the same function as the protein shown in SEQ ID No. 30;
a9 The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises:
a911 Amino acid sequence shown as SEQ ID No. 33; or (b)
A912 Amino acid sequence of SEQ ID No. 33 with one or several amino acids to replace and/or delete and/or add and with the same function as the protein shown in SEQ ID No. 33; or (b)
A913 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 33 and the same function as the protein shown in SEQ ID No. 33;
a9 The amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
a921 An amino acid sequence as shown in SEQ ID No. 34; or (b)
A922 Amino acid sequence of SEQ ID No. 34 with one or several amino acids to replace and/or delete and/or add and with the same function as the protein shown in SEQ ID No. 34; or (b)
A923 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 34 and the same function as the protein shown in SEQ ID No. 34.
According to some embodiments of the invention, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region;
the amino acid sequence of the heavy chain constant region comprises:
b11 37 of the amino acid sequence shown in SEQ ID No; or (b)
B12 37 by substitution and/or deletion and/or addition of one or more amino acids, and has the same function as the protein shown in SEQ ID No. 37; or (b)
B13 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 37 and the same function as the protein shown in SEQ ID No. 37;
the amino acid sequence of the light chain constant region comprises:
b21 Amino acid sequence shown as SEQ ID No. 38; or (b)
B22 Amino acid sequence of SEQ ID No. 38 with one or several amino acid substitutions and/or deletions and/or additions and with the same function as the protein shown in SEQ ID No. 38; or (b)
B23 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 38 and the same function as the protein shown in SEQ ID No. 38.
Herein, the term "homology" refers to the similarity between amino acid sequences or nucleotide sequences. Homology can be assessed by computer software.
Herein, the at least 80% homology may be at least 80%, 81%, 82%, 83%, 84%, 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% homology.
According to some embodiments of the invention, the antibody or antigen binding fragment thereof is selected from any one of Fab, fab ', F (ab') 2, fv, scFv, diabody, fusion antibody, and intact antibody.
According to a second aspect of the present invention, there is provided a recombinant protein comprising a tag and an antibody or antigen-binding fragment thereof as described above. The tag may be attached to the N-terminus or/and the C-terminus of the antibody or antigen binding fragment thereof. The tag may be one that facilitates solubilization and/or purification of the antibody or antigen binding fragment thereof.
According to some embodiments of the invention, the tag comprises at least one of a His tag, a GGGS sequence, a FLAG tag, a strep tag, a nus tag, a maltose binding protein, a GST tag. The recombinant proteins of the invention may comprise one or more tags; the plurality of tags may comprise a combination of a plurality of identical tags or a combination of a plurality of different tags.
A biological material associated with the above antibody or antigen-binding fragment thereof, or the above recombinant protein according to an embodiment of the third aspect of the present invention, which is any one of c 1) to c 5);
c1 A nucleic acid molecule encoding the above antibody or antigen-binding fragment thereof, or the above recombinant protein;
c2 An expression cassette comprising the nucleic acid molecule as described in c 1);
c3 A recombinant vector comprising the nucleic acid molecule as set forth in c 1) or the expression cassette as set forth in c 2);
c4 A recombinant microorganism comprising the nucleic acid molecule as described in c 1) or the expression cassette as described in c 2) or the recombinant vector as described in c 3);
c5 A transgenic cell line comprising the nucleic acid molecule as described in c 1) or the expression cassette as described in c 2) or the recombinant vector as described in c 3).
According to some embodiments of the invention, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
According to some embodiments of the invention, the nucleic acid molecule is a DNA molecule encoding the above antibody or antigen binding fragment thereof, or the above recombinant protein.
According to some embodiments of the invention, the DNA molecule is any one of d 1) to d 10) and comprises a nucleotide sequence encoding the heavy chain variable region and a nucleotide sequence encoding the light chain variable region;
d1 A nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 3; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 4;
d2 A nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 7; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 8;
d3 A nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 11; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 12;
d4 A nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 15; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 16;
d5 A nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 19; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 20;
d6 A nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 23; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 24;
d7 A nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 27; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 28;
d8 A nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 31; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 32;
d9 A nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 35; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 36;
d10 A DNA molecule having 80%, 85% or more than 90% homology to the nucleotide sequence defined in any of d 1) to d 9) and encoding said antibody or antigen binding fragment thereof;
d11 A DNA molecule which hybridizes under stringent conditions to a nucleotide sequence as defined in any one of d 1) to d 10) and which encodes said antibody or antigen binding fragment thereof.
The nucleotide sequence may be mutated by a person skilled in the art using known methods (e.g., directed evolution and point mutation), and those artificially modified nucleotide sequences having a certain homology with the antibody or antigen-binding fragment thereof of the present invention are derived from and are equivalent to the nucleotide sequence of the present invention as long as the antibody or antigen-binding fragment or variant thereof is encoded and has the same function as the antibody or antigen-binding fragment thereof.
According to some embodiments of the invention, the expression cassette refers to DNA capable of expressing the antibody or antigen-binding fragment thereof in a host cell, which DNA may include not only a promoter that initiates transcription of a DNA molecule encoding the antibody or antigen-binding fragment thereof, but also a terminator that terminates transcription of a DNA molecule encoding the antibody or antigen-binding fragment thereof. Further, the expression cassette may also include a replication initiation site, a transcription initiation sequence, an enhancer sequence, a selection element, or a reporter gene.
According to some embodiments of the invention, the recombinant vector may be a plasmid, phage, lentivirus, adenovirus, adeno-associated virus. The recombinant vector is used for realizing the replication, integration, amplification and/or expression of exogenous target genes in receptor cells; it may be a cloning vector or an expression vector. For example: c3 In particular, the recombinant vector may be a recombinant plasmid obtained by inserting the nucleic acid molecule into a CMV vector.
According to some embodiments of the invention, the recombinant microorganism may be a bacterium (e.g., E.coli or B.subtilis, etc.) or a fungus (e.g., yeast or Aspergillus, etc.). For example: c4 The recombinant microorganism may specifically be E.coli DN5α.
According to some embodiments of the invention, the transgenic cell line may be an insect cell (e.g., S2 drosophila cell or Sf9 cell, etc.), an animal cell line (e.g., 293T cell, 293F cell, CHO cell, COS cell, NSO cell, heLa cell, BHK cell or HEK293 cell, etc.), or a plant cell (e.g., arabidopsis thaliana or tobacco, etc.). The transgenic cell lines may all be non-propagating material.
A conjugate according to an embodiment of the fourth aspect of the invention comprises: the above antibody or antigen-binding fragment thereof, or the above recombinant protein;
and a coupling moiety comprising at least one of a detectable label, a drug, a toxin, a radionuclide, an enzyme.
According to some embodiments of the invention, the detectable label is selected from at least one of a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance.
Use of the above antibody or antigen binding fragment thereof, the above recombinant protein, the above biological material or the above conjugate according to an embodiment of the fifth aspect of the invention in any one of e 1) to e 5);
e1 Preparing a medicament for preventing and/or treating poxviruses;
e2 Non-diagnostic treatment destination to detect poxviruses;
e3 Preparing a kit for detecting poxviruses;
e4 Non-diagnostic treatment destination to detect the monkey poxvirus M1R protein;
e5 Preparing a kit for detecting the M1R protein of the monkey poxvirus;
The poxvirus comprises at least one of a monkey poxvirus and a vaccinia virus.
A product according to an embodiment of the sixth aspect of the invention, comprising at least one of f 1) to f 4);
f1 The above antibody or antigen-binding fragment thereof;
f2 A recombinant protein as described above;
f3 A) the above biological material;
f4 A conjugate as described above;
the product is at least one selected from the group consisting of a drug, a reagent, a test plate, a chip, a test paper and a kit.
According to some embodiments of the invention, the product has at least one of g 1) to g 3);
g1 Preventing and/or treating diseases associated with poxvirus infection;
g2 Detecting the presence or amount of poxvirus;
the poxvirus comprises at least one of a monkey poxvirus and a vaccinia virus;
g3 Detecting the presence or amount of the M1R protein of the monkey poxvirus.
According to some embodiments of the invention, the product is a medicament; the product also comprises pharmaceutically acceptable auxiliary materials. The dosage is nontoxic to recipients.
According to some embodiments of the invention, the adjuvant comprises at least one of a solvent, a dispersant, a diluent, a filler, a wetting agent, a binder, a disintegrant, a lubricant, a preservative, a suspending agent, an emulsifier, an excipient, a stabilizer, a buffer, an isotonic agent, a flavoring agent, a carrier. The adjuvant is used for promoting the absorption of the antibody or antigen binding fragment thereof to exert a prophylactic and/or therapeutic effect.
A method of preparing an antibody or antigen-binding fragment thereof according to an embodiment of the seventh aspect of the present invention comprises the steps of:
Obtained by culturing the recombinant microorganism or transgenic cell line described above.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 shows the electrophoresis results of recombinant M1R protein in Reduced and Non-Reduced gels (Non-Reduced); wherein M is Marker, and sample 1 is M1R-His protein;
FIG. 2 is a graph showing the results of activity detection of recombinant M1R-His protein and M1R-His-Biotin protein;
FIG. 3 shows the Pool ELISA assay results of eluted phage libraries obtained from three rounds of screening; wherein, the A graph is the experimental board result, and the B graph is the negative coating board result;
FIG. 4 shows the results of sequence analysis of the screened monkey pox specific antibodies; wherein, the A diagram is the statistics of the germ line gene ratio of the heavy chain variable region, the B diagram is the statistics of the germ line gene ratio of the light chain variable region, and the C diagram is the statistics of the length of the heavy chain CDR3 and the length of the light chain CDR 3;
FIG. 5 shows the results of detection of binding activity of 37 antibodies to M1R antigen;
FIG. 6 shows the results of the neutralization activity of 36 antibodies;
FIG. 7 shows the binding and dissociation curves of anti-monkey poxvirus monoclonal antibodies to M1R protein at different concentrations;
FIG. 8 shows the results of a neutralization assay of live, trans-complement defective monkey pox virus and vaccinia virus with anti-monkey pox virus monoclonal antibodies.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
In the description of the present invention, the terms "comprises" and "comprising," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
In the description of the present invention, "antibody" refers to any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that is capable of binding to a particular antigen. Intact antibodies are typically composed of two pairs of polypeptide chains, each pair comprising one light (L) chain and one heavy (H) chain, the light chains being classified as Kappa (Kappa) or Lambda (Lambda) types, whereas antibodies can be defined as IgM, igD, igG, igA and IgE, depending on the type of heavy chain, mu, delta, gamma, alpha and epsilon, respectively. The heavy chain comprises a heavy chain constant region (CH) and a heavy chain variable region (VH). The CH of different types of heavy chains contains 3 to 4 unequal domains (CH 1, CH2, CH3 and CH 4). The light chain comprises a light chain constant region (CL) and a light chain variable region (VL). Each of VH and VL comprises highly variable Complementarity Determining Regions (CDRs) and Framework Regions (FR) interspersed therewith, each VH and VL comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in that order from amino-terminus to carboxy-terminus.
In the description of the present invention, an "antigen binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, and specifically includes, but is not limited to, at least one of a Fab fragment, a Fab 'fragment, a F (ab') 2 fragment, an Fv fragment, an sc-Fv, and a diabody.
In the description of the invention, a "Fab fragment" comprises a light chain and a heavy chain CH1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.
In the description of the invention, a "Fab' fragment" contains a light chain and a heavy chain portion or fragment containing the VH domain and CH1 domain and the region between the CH1 and CH2 domains.
In the description of the invention, a "F (ab') 2 fragment" contains two light chains and two heavy chains, the heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. The F (ab ') 2 fragment thus consists of two Fab' fragments, which are linked together by a disulfide bond between the two heavy chains.
In the description of the invention, an "Fv fragment" comprises variable regions from the heavy and light chains, but lacks constant regions.
In the description of the invention, "single chain Fv" or "scFv" refers to an antibody fragment comprising the VH domain and the VL domain of an antibody, wherein these domains are present as a single polypeptide chain. Generally, fv polypeptides also comprise a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding.
In the description of the invention, "diabody" refers to a small antibody fragment having two antigen-binding sites, said fragment comprising in the same polypeptide chain a heavy chain variable domain (VH) and a light chain variable domain (VL) (VH-VL or VL-VH) linked thereto. By using a linker that is too short to allow pairing between two domains on the same strand, each domain is forced to pair with the complementary domain of the other strand, thereby creating two antigen binding sites.
Unless otherwise specified, the term "room temperature" in the present application means (25.+ -. 5). Degree.C.
"Amp" in the present application means Ampicillin (AMPICILLIN) unless otherwise specified; "Tet" means tetracycline (TETRACYCLINE); "Kana" refers to Kanamycin (KANAMYCIN).
Unless otherwise specified, "amino acid" in the present application refers to a basic unit constituting a protein, and imparts a specific molecular structural form to the protein, thereby giving the protein its molecule its biochemical activity. For example, "amino acids" as used herein include the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V) and tyrosine (Tyr or Y).
Unless otherwise specified, the amino acid sequences in the application are all in the order from N end to C end; the nucleotide sequences are all in order from the 5 'end to the 3' end.
The specific conditions are not noted in the examples below, and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the examples below, some of the experimental materials and reagents are described below:
Trans-complement defective monkey poxvirus system: the Shenzhen Bay laboratory Liu Jianying teacher gives away, and the reference patent is "Trans-complementary defective monkey pox Virus and its use" (application number: 2023113941163).
Vero E6 complementation cell line for stable expression of the monkey poxvirus g9r+a1r gene: the Shenzhen Bay laboratory Liu Jianying teacher gives away, and the reference patent is "Trans-complementary defective monkey pox Virus and its use" (application number: 2023113941163).
Recombinant monkey poxvirus surface protein M1R: reference is made to the VACV L1R protein expression literature (virology 2005 Oct 10;341 (1): 59-71) for self-made, M1R protein having the amino acid sequence MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAG.
Expression vectors CMV for heavy and light chains: the laboratory was given by university of Qinghua Zhang Linqi, described in Nature 2020 Aug; 584 (7819): 115-119.
M1R protein positive antibodies: anti-Monkeypox virus/MPXV M1R Anti-body, available from general Biotechnology Co., ltd., product number: SAA0283.
Negative irrelevant antibody (8 AH8 AL): reference (Proc NATL ACAD SCI U S A2006 Feb 7;103 (6): 1882-7.) is self-made.
Secondary antibody ：Goat-Anti-mouse-IgG-Fc-HRP(abcam; ab97265),Goat Anti-Human IgG-Fc-HRP (Jackson, 109-001-008),Mouse-Anti-M13Bacteriophage Antibody（HRP）.
Octet®Ni-NTA Biosensors (SARTORIUS，18-5102)。
PBST buffer: 1 XPBS with 0.1% Tween 20.
293T cells are highly trans-derived cell lines formed by transferring the SV 40T-antigen gene into HEK293 cells.
293F cells are a class of wild-type HEK293 cell lines capable of high protein expression in serum-free.
Example 1 (preparation of anti-monkey poxvirus monoclonal antibody)
1. Expression of recombinant monkey poxvirus surface protein M1R-His.
(1) 293F cells in the logarithmic growth phase with the activity rate higher than 90% were inoculated into fresh medium at a density of 1.5X10 6/mL, placed in a thermostatic shaker, and cultured at 37℃under a rotation speed of 5% CO 2 and 150 rpm for one day, and then the cell density and the activity rate (the activity rate is higher than 90%) were sampled and counted to adjust the cell density to 2X 10 6/mL, thereby obtaining 293F cell suspension.
(2) Extracting by using an endotoxin-free plasmid large extraction kit (Tiangen, DP 117-T) to obtain an M1R protein expression vector which is successfully constructed, regulating the concentration of the M1R protein expression vector to 80 mug/mL by using a 150 mM NaCl solution, uniformly mixing, and standing at room temperature for about 5 min; diluting the 120 mu L Sinofection transfection reagent with 150 mM NaCl solution to a total volume of 0.75 and mL, uniformly mixing, and standing at room temperature for about 5 min; mixing the diluted M1R protein expression carrier solution with Sinofection transfection reagent in equal volume, and standing at room temperature for about 10 min to obtain a transfection solution.
(3) 1.5 ML transfection solution was added dropwise to 200 mL of the 293F cell suspension, the flask was gently shaken while dropwise, the flask was returned to a constant temperature shaker after shaking, and the culture was continued, 2% (V/V) SMS 293-SUPI feed solution (Sino Biological, M293-SUPI) was added at 24h, 72h, 120 h post-transfection in this order, and the cell culture supernatant was collected after 5 days of transfection.
(4) Purifying the cell culture supernatant to obtain recombinant M1R-His protein; recombinant M1R-His protein was run in protein gel.
The results are shown in FIG. 1.
The recombinant M1R-His protein is about 25 KD protein in the reducing gel and non-reducing gel. This indicates that the M1R-His protein was successfully prepared and that the recombinant M1R-His protein was monomeric, consistent with literature.
2. Activity identification of recombinant monkey poxvirus surface protein M1R-His and biotinylated protein M1R-His-Biotin.
The activity of commercial M1R positive Antibody (Anti-Monkeypox virus/MPXV M1R Anti-body) was evaluated by detecting the binding activity of the recombinant M1R-His protein obtained in step 1 and its biotinylated protein M1R-His-Biotin. The method comprises the following steps:
(1) Coating: diluting M1R-His and M1R-His-Biotin to a concentration of 2 mug/mL by using PBS to obtain coating liquid; the coating solution was added to a 96-well ELISA plate at 30 μl/well and coated overnight at 4deg.C.
(2) Closing: the liquid in the 96-well ELISA plate was discarded, and a blocking solution (PBST buffer containing 5% (V/V) skimmed milk) was added to each well at 100. Mu.L/well, and blocked at room temperature of 2 h.
(3) Washing: the liquid in the 96-well ELISA plate was discarded, and the 96-well ELISA plate was washed 3 times with PBST buffer.
(4) Adding a sample: serial 10-fold dilutions of 10 μg/mL commercial M1R positive antibody solution were performed, each sample was added to each well separately at 100 μl/well, and incubated at room temperature for 1 h; other irrelevant antibody (8 AH8 AL) was treated as negative control and PBS as blank.
(5) Washing: the liquid in the 96-well ELISA plate was discarded, and the 96-well ELISA plate was washed 3 times with PBST buffer.
(6) Adding a secondary antibody: goat-Anti-mouse-IgG-Fc-HRP antibody was diluted with 1% BSA (w/v) in PBST buffer at 1:8000 dilution, and adding diluted secondary antibody solution to each well at 30 [ mu ] L/well, and incubating at room temperature for 1 h.
(7) Washing: the liquid in the 96-well ELISA plate was discarded, and the 96-well ELISA plate was washed 3 times with PBST buffer.
(8) Color development, termination and reading: after 50 [ mu ] L of color development solution is added to each well and incubated for 15 min ℃ at room temperature and in a dark place, 25 [ mu ] L of termination solution is added to each well, absorbance at 450 nm and 630 nm (respectively marked as OD450 and OD 630) is read by a microplate reader, and binding activities of commercial M1R positive antibodies to M1R-His and M1R-His-Biotin are analyzed.
And (3) carrying out regression curves on the concentration of the commercial M1R positive antibody and the corresponding OD450 to obtain EC50 values of the commercial M1R positive antibody on the M1R-His and the M1R-His-Biotin.
The results are shown in FIG. 2.
The Anti-Monkeypox virus/MPXV M1R Anti-body has affinity activity to M1R-His and M1R-His-Biotin, and the EC50 value is 0.025 mug/mL and 0.024 mug/mL respectively. This suggests that M1R-His and M1R-His-Biotin can be used for subsequent antibody screening.
3. Screening for monkey pox specific antibodies.
Phage libraries constructed using healthy human PBMCs were used to screen for Fab antibody sequences that specifically bind to M1R protein.
(1) Repeating the following steps 1) to 4) three times to perform three rounds of enrichment screening. The phage sub-library obtained in each round was partially used for the next round of screening and partially frozen for subsequent experiments.
1) First round antibody screening.
Antigen (M1R-His and M1R-His-Biotin) was coated on immune tubes (50. Mu.g/tube, coating solution was CBS, pH 9.6,2 mL/tube), slowly rotated overnight at 4℃while parallel coating with 50. Mu.g of 5V/V% skim milk was used as a control. The liquid in the immune tube was discarded, and the immune tube was washed 3 times with 2mL PBS at room temperature, 5min revolutions each. 2mL blocking solution (PBST buffer with 3% BSA (w/v)) was added and the mixture was spun at room temperature to block 2h. The blocking solution was discarded and the immune tube was washed 3 times with 1mL PBS room temperature revolutions for 5min each. 2mL PBS and phage library were added to the washed immune tubes and incubated at room temperature with 1 h. The liquid in the immune tube was discarded, and the immune tube was washed with 2mL PBST buffer at room temperature for 20 times, each rotation of 5: 5min. Adding 1mL of 0.25 mg/ML TRYPSIN solution into the cleaned immune tube, rotating and eluting at room temperature for 30 min hours, adding 10 mu L of 10% AEBSF to stop eluting, and transferring the solution in the immune tube into a new 1.5 mL centrifuge tube to obtain the phage eluent for the first round of screening.
2) The first round of screening phage eluate was tested for titer.
Inoculating the XL1-Blue strain into a2 XYT culture medium containing 10 mug/mL Tet of 5mL, culturing overnight at 37 ℃, transferring 250 mug bacterial liquid into a2 XYT culture medium containing 10 mug/mL Tet of 5mL, and culturing at 250rpm until the OD600 is 0.5-0.55 to obtain the XL1-Blue bacterial liquid. After 12 gradient 10 times dilution is continuously carried out on the first round of screening phage eluent, 90 mu L XL1-Blue bacterial liquid is respectively added into the 10 mu L diluted first round of screening phage eluent, the mixture is uniformly mixed, after 30 min is incubated at 37 ℃,5 mu L bacterial liquid is dripped into a2 XYT solid culture medium (Amp), the mixture is inverted at 37 ℃ for overnight culture, the number of single colonies corresponding to the dilution degree of single colonies can be obviously distinguished on the 2 XYT solid culture medium, and the titer of a phage library is calculated.
Phage library titers (T, pfu/mL) are the number of phagemids per milliliter of phage solution and are calculated as follows:
T=N×D×400；
Wherein D is dilution factor, N is the number of single colony on 2 XYT solid culture medium of corresponding dilution factor.
3) Amplification of the first round of phage eluate.
Inoculating the XL1-Blue strain into a 2 XYT culture medium containing 10 mug/mL Tet of 5mL, culturing overnight at 37 ℃, transferring 250 mug bacterial liquid into a 2 XYT culture medium containing 10 mug/mL Tet of 5mL, and culturing at 250rpm until the OD600 is 0.5-0.55 to obtain the XL1-Blue bacterial liquid. After 500 mu L of first-round phage eluent is added into XL1-Blue bacterial liquid and culture is continued for 30min under the conditions of 37 ℃ and 220 rpm, the whole bacterial liquid is uniformly coated on a 2% agarose plate (containing 100 mu g/mL Amp and 2% (W/V) glucose) and is cultured overnight at 37 ℃. 6mL of 2 XYT liquid culture medium (containing 10 mug/mL Tet) is added to the surface of a 2% agarose plate, colonies are scraped and collected, OD600 value is detected, and glycerin with the final concentration of 20% (V/V) is added to obtain a first round of bacterial library.
Transferring the first round of bacterial library into 100 mL of 2 XYT liquid culture medium (containing 10 mug/mL Tet and 100 mug/mL Amp), culturing under the conditions of initial OD600 of about 0.1, 37 ℃ and 250 rpm until OD600 is 0.5-0.55, and adding auxiliary phage M13K07 according to a formula (I) to enable the number of bacteria to be: phage number = 1:20, continuing to culture at 37 ℃ and 220 rpm under the condition of 30 min; kana (final concentration 50. Mu.g/mL) and IPTG (final concentration 0.2. Mu.M) were then added and incubated overnight at 37℃under 250 rpm.
(Formula I);
Wherein v is the volume of helper phage added, T helper-phage is the helper phage titer, and OD600 is the OD600 value of the bacterial liquid.
4) First round phage purification.
Centrifuging the bacterial liquid 4000 rpm obtained by the overnight culture in the step 3) at the temperature of 4 ℃ for 10 min, and taking a supernatant; adding 1/4 volume of pre-cooled 20% PEG/2.5M NaCl solution at 4deg.C into the supernatant, standing on ice for 30 min, centrifuging at 4000 rpm and 4deg.C for 20 min, discarding supernatant, and inverting 2 min on paper; re-suspending the pellet with 1mL PBS, centrifuging at 12000 rpm and 4deg.C for 20 and min, and collecting supernatant; adding 1/4 volume of 20% PEG/2.5M NaCl solution precooled at 4deg.C into the supernatant, standing on ice for 10 min after fully mixing, centrifuging at 12000 rpm and 4deg.C for 10 min, and discarding the supernatant; the pellet was resuspended in 1mL PBS, centrifuged at 12000 rpm at 4℃for 2 min, and the supernatant was taken to obtain the phage sub-library for the first round of screening for the next round of screening.
The three rounds of screening enrichment results are shown in table 1.
TABLE 1
The M1R-His-Biotin screening group showed significant enrichment in the third round.
(2) Pool ELISA detection.
Diluting the M1R-His protein to 2 mug/mL by using PBS to obtain coating liquid, adding the coating liquid into a 96-well ELISA plate according to 30 mug/well, and coating overnight at 4 ℃ to obtain an experimental plate; at the same time, PBS was used as a negative coating plate. The coating solution was discarded, and a blocking solution (PBST buffer containing 5% (V/V) skimmed milk) was added to each well at 100. Mu.L/well, and blocked at room temperature of 2 h. The blocking solution was discarded and the 96-well ELISA plate was washed 3 times with PBST buffer. The eluted Phage sub-library (Phage pool) obtained by three rounds of screening in step (1) was serially diluted 10-fold, and incubated with other irrelevant antigen Phage libraries (negative control) or helper Phage as control groups at room temperature of 1h. The samples were discarded and the 96-well ELISA plates were washed 3 times with PBST buffer. The secondary antibody (Mouse-Anti-M13 Bacteriophage Antibody (HRP) antibody) was diluted with 1% BSA (w/v) in PBST buffer at 1:8000 dilution, and adding diluted secondary antibody solution to each well at 30 [ mu ] L/well, and incubating at room temperature for 1h. The diluted secondary antibody solution was discarded and the 96-well ELISA plate was washed 3 times with PBST buffer. 50 mu L of color development liquid is added to each hole, after 15 min mu L of stop liquid is added to each hole after incubation at room temperature and in a dark place, OD450 is read by using an enzyme label instrument.
The results are shown in FIG. 3.
At the level of Phage, pool 1012-34, 1014-32, 1014-33, 1014-34 are enriched with the antigen M1R-His and M1R-His-Biotin. The monoclonal was selected from among the four screening Pool for the next screening.
(3) Monoclonal ELISA detection.
1) Inoculating the XL1-Blue strain into a2 XYT culture medium containing 10 mug/mL Tet of 5 mL, culturing overnight at 37 ℃, transferring 250 mug bacterial liquid into a2 XYT culture medium containing 10 mug/mL Tet of 5 mL, and culturing at 250rpm until the OD600 is 0.5-0.55 to obtain the XL1-Blue bacterial liquid. After phage eluents corresponding to screening numbers 1012-34, 1014-32, 1014-33 and 1014-34 (see Table 1) are continuously diluted 10 times with 12 gradients, 90 [ mu ] L XL1-Blue bacterial liquid is respectively added into the phage eluents diluted 10 [ mu ] L, the phage eluents are uniformly mixed, after the phage eluents are cultured at 37 ℃ and 220 rpm for 30 min, the bacterial liquid is uniformly coated on a solid culture medium plate containing 100 [ mu ] g/mL Amp, and the phage eluents are inverted at 37 ℃ and cultured overnight. Randomly picking monoclonal colonies on a flat plate into a sterile 96-well cell culture plate, adding 200 mu L of 2 XYT culture medium (containing 100 mu g/mL Amp and 10 mu g/mL Tet) into each well, and culturing overnight at 37 ℃ under 220 rpm conditions; transferring 2 mu L of bacterial liquid into 200 mu L of 2 XYT liquid culture medium, and standing and culturing for 3-5 h at 37 ℃; helper phage M13K07 was added according to formula (I) to give the bacterial count: phage number = 1:20, incubating at 37 ℃ for 30 min; kana (final concentration of 50. Mu.g/mL) and IPTG (final concentration of 0.2. Mu.M) were then added and incubated at 30℃overnight. 4000 Centrifugation at 10 min rpm and taking the supernatant.
2) The recombinant M1R protein was diluted to 2. Mu.g/mL with PBS to give a coating solution, which was added to a 96-well ELISA plate at 30. Mu.L/well, coated overnight at 4℃and simultaneously coated with PBS as a negative coating plate. The coating solution was discarded, and a blocking solution (PBST buffer containing 5% (V/V) skimmed milk) was added to each well at 100. Mu.L/well, and blocked at room temperature of 1 h. The blocking solution was discarded and the 96-well ELISA plate was washed 3 times with PBST buffer. Adding 30 mu L of 3% BSA and 80 mu L of the supernatant obtained in the step 1) into each hole, and incubating at room temperature for 1 h; commercial M1R antibody (5 mug/mL) was used as a positive control, and irrelevant murine antibody was used as a negative control. The samples were discarded and the 96-well ELISA plates were washed 3 times with PBST buffer. The Mouse-Anti-M13Bacteriophage Antibody (HRP) antibody was diluted with 1% BSA (w/v) in PBST buffer at 1:8000 dilution, adding diluted secondary antibody solution into each experimental hole according to 30 mu L/hole, and incubating at room temperature for 1 h; 30 μl of Anti-mouse-Fc-HRP diluted 1:8000 was added to each well of the positive and negative controls. The diluted secondary antibody solution was discarded and the 96-well ELISA plate was washed 3 times with PBST buffer. 50 mu L of developing solution is added to each hole, after 15min mu L of stopping solution is added to each hole after incubation at room temperature and in a dark place, an OD450 value is read by using an enzyme-labeling instrument.
(4) Positive clone sequencing.
The monoclonal with OD450 value 2.1 times greater than that of negative control OD450 was selected as positive monoclonal according to the monoclonal ELISA detection data. Inoculating positive monoclonal to a 2 XYT culture medium (containing 100 mug/mL Amp and 10 mug/mL Tet), culturing at 37 ℃ at 250 rpm until OD600 is 0.8-1.0, and taking a proper amount of bacterial liquid for sequencing.
Sequence alignment analysis was performed on the sequencing results using GENtle software and the antibody sequences were translated into amino acid sequences using GENtle software.
The screening results are shown in Table 2. The results of the antibody sequence analysis are shown in FIG. 4.
TABLE 2
Note that: positive rate = M1R-His positive clones/number of selected clones x 100%. unique sequences are not repeated in the variable region of the antibody obtained after sequencing.
540 Clones were selected for preliminary screening, 235 positive clones binding to the antigen M1R-His were obtained, 168 positive clones with OD450 value of 0.4 or more were sent and detected, and after sequence analysis, 37 unique clones were finally obtained.
37 Pairs of antibody sequences were obtained, the heavy chain variable regions were derived from 13 different germline genes, IGHV3-23 was at most 32%; analysis of the length of the CDR3 loop of the complementarity determining region shows that the heavy chain variable region has a length of 5aa to 19aa. The light chains are all kappa chains, and are mainly IGKV1-12 and IGLV 1-39; analysis of the length of the CDR3 loop of the complementarity determining region shows that the light chain variable region has 8aa to 9aa.
4. And constructing a human IgG antibody expression vector.
The human IgG antibody expression vector may be obtained by the following method, or may be obtained by artificial synthesis.
(1) Antibody sequence amplification.
And (3) designing a specific primer according to the sequencing result of the step (3), and performing PCR amplification by taking the expression plasmid of the positive monoclonal Fab antibody as a template. The PCR amplified products were subjected to agarose gel electrophoresis and gel recovered using a common agarose gel DNA recovery kit (Tiangen, DP 209).
The PCR reaction system (total volume: 20. Mu.L) was as follows:
PrimerSTAR Max Premix (2×)10 µL；
5' primer (10. Mu.M) 0.6. Mu.L;
3' primer (10. Mu.M) 0.6. Mu.L;
5 mu L of a template (0.2 mu g/mL);
and 3.8 mu L of sterilized distilled water.
The PCR reaction conditions were: pre-denaturation at 98 ℃ 2 min;98 ℃ for 10 s,58 ℃ for 5 s,72 ℃ for 10 s,35 cycles; final extension at 72℃5 min.
(2) And (3) respectively carrying out double enzyme digestion on cytomegalovirus expression vectors CMV of the heavy chain and the light chain by using restriction enzymes to obtain enzyme digestion products. The digested product was subjected to agarose gel electrophoresis, and gel recovery was performed using a general agarose gel DNA recovery kit (Tiangen, DP 209).
The double enzyme digestion reaction system (total volume: 30 [ mu ] L) of the heavy chain expression vector is as follows: CMV-H (1 [ mu ] g) 1 [ mu ] L;10 XNE Buffer 3 [ mu ] L; age I-HF 1 [ mu ] L; sal I-HF 1 [ mu ] L; deionized water 24 μl.
The double enzyme cleavage reaction system (total volume: 30 μl) of the light chain (κ) expression vector is: CMV-kappa (1 [ mu ] g) 1 [ mu ] L;10 XNE Buffer 3 [ mu ] L; age I-HF 1 [ mu ] L; bsiW I-HF 1 [ mu ] L; deionized water 24 μl.
The double enzyme digestion reaction system (total volume: 30 [ mu ] L) of the light chain (lambda) expression vector is as follows: CMV-lambda (1 [ mu ] g) 1 [ mu ] L;10 XNE Buffer 3 [ mu ] L; age I-HF 1 [ mu ] L; xho I-HF 1 [ mu ] L; deionized water 24 μl.
Reaction conditions: reaction at 37℃was carried out at 20 min.
(3) And (3) carrying out homologous recombination on the PCR product and the enzyme digestion product obtained by recycling the glue in the steps (1) and (2) to obtain a connection product.
The reaction system (total volume: 10 μl) of the CMV-H ligation product was: homologous recombination reagent (2×) (vazyme, C115) 5 μl; CMV-H cleavage product (about 50 ng) 1 μl; 1 [ mu ] L of a heavy chain gene fragment (about 10 ng); nuclease-free water 3 μl.
The reaction system (total volume: 10 μl) of the CMV- κ ligation product was: homologous recombination reagent (2×) 5 μl; CMV-kappa cleavage product (about 50 ng) 1. Mu.L; 1 [ mu ] L of a kappa chain gene fragment (about 10 ng); nuclease-free water 3 μl.
The reaction system (total volume: 10 μl) of the CMV- λ ligation product was: homologous recombination reagent (2×) 5 μl; CMV-lambda cleavage product (about 50 ng) 1 μl; lambda chain gene fragment (about 10 ng) 1 μl; nuclease-free water 3 μl.
Reaction conditions: reaction at 50℃was carried out at 20 min.
(4) Transforming the CMV-H ligation product obtained in the step (3) and the CMV-kappa ligation product into competent cells DH5 alpha respectively; and positive clones that were successfully transformed were identified by resistance screening and sequencing.
Example 2 (preparation of anti-monkey poxvirus monoclonal antibody)
1. Expression of 37 anti-monkey poxvirus monoclonal antibodies.
The successfully paired antibody heavy and light chain gene expression vectors were transiently transfected into 293T cells. The method comprises the following steps:
293T cells were inoculated into 12-well plates at 5X10 5 cells/well and cultured to a cell confluence of 70% -80%. Dissolving successfully paired 1 mu g of antibody heavy chain expression vector and 1 mu g of light chain gene expression vector in a 60 mu L Opti-MEM culture medium, diluting 4 mu L Lipofectamine ® 2000,2000, 2000 Reagent with the 60 mu L Opti-MEM culture medium, mixing in an equal volume to obtain a mixed solution, and incubating at room temperature for 5 min. 120 mu L of the mixed solution is gently added into 293T cells, after culturing at 37 ℃ with 5% CO 2 and 48 h, the mixture is centrifuged at 12000 and rpm for 2 min, and cell culture supernatant and cell sediment are collected. After washing the cell pellet 2 times with 1 mL precooled PBS, 100. Mu.L of cell lysate (Promega, E1531) was added to the cell pellet, ice-bath was performed for 10 min, centrifugation at 12000 rpm was performed for 5 min, and the cell lysate supernatant was harvested.
2. Antibody binding activity was detected as a primary screen for 37 anti-monkey pox virus monoclonal antibodies. The method comprises the following steps:
Diluting the recombinant M1R protein to 2 mug/mL by using PBS (phosphate buffer solution), obtaining coating liquid, adding the coating liquid into a 96-well ELISA plate according to 30 mug/well, and coating overnight at 4 ℃ to obtain an experimental plate; at the same time, PBS was used as a negative coating plate. The coating solution was discarded, and a blocking solution (PBST buffer containing 5% (V/V) skimmed milk) was added to each well at 100. Mu.L/well, and blocked at room temperature of 2 h. The blocking solution was discarded and the 96-well ELISA plate was washed 3 times with PBST buffer. The protein concentration in the cell culture supernatants was measured, and the cell lysis supernatants were serially diluted 10-fold with 8 gradients, respectively, incubated at room temperature for 1 h, treated with commercial M1R antibody as positive control, with irrelevant antibody (8 AH8 AL) as negative control, and PBS as BC (1% PBSM) group. The samples were discarded and the 96-well ELISA plates were washed 3 times with PBST buffer. The secondary antibody (Goat Anti-Human IgG-Fc-HRP antibody) was diluted 1 in 1% BSA (w/v) in PBST buffer: 8000 diluting, and adding the diluted secondary antibody solution into the sample, the negative control and the blank control respectively according to 30 mu L/hole; 30 μl of Anti-mouse-Fc-HRP diluted 1:8000 was added to each well of the positive control and incubated at room temperature for 50 min. The diluted secondary antibody solution was discarded and the 96-well ELISA plate was washed 3 times with PBST buffer. After 50 mu L of developing solution is added to each hole and incubated for 15 min mu L of stopping solution is added to each hole at room temperature and in a dark place, an OD450 value is read by an enzyme-labeling instrument, and a sample OD450 value is 2.1 times greater than a negative control OD450 value to be used as positive judgment.
The results are shown in FIG. 5. The EC50 statistics are shown in table 3.
TABLE 3 Table 3
Of the 37 antibodies obtained by the screening, 34 antibodies had binding activity to the M1R protein.
3. And (3) carrying out neutralization activity preliminary screening detection on 34 antibodies obtained through the combined activity preliminary screening. The method comprises the following steps:
Vero E6 complementation cells stably expressing the monkey poxvirus G9R+A1L gene were plated in 24 well plates. The concentration of the cell lysis antibody was determined, the antibody was adjusted to 10. Mu.g/mL, and the antibody was mixed with an equal volume of DMEM medium (concentration: 200 PUF/mL) containing the trans-complementary defective monkey pox virus, and incubated at 37℃for 1h to obtain a mixture. Adding 100 mu L of the mixture into a Vero E6 anaplerotic cell plate paved with a single layer of G9R+A1L gene for stable expression, and taking the mixture as an experimental group; simultaneously setting a virus control hole added with the same metering virus, only adding a cell control hole of a DMEM culture medium, and culturing at a constant temperature of 1h under the conditions of 5% CO 2 and 37 ℃; the plate was discarded, 1mL agar-cover (1.6 wt% agar (2 XDMEM+pancreatin mixture) =1:1 (V/V)) was added, and incubated in an incubator at 37℃for 5 days. Adding 2 mL of 4% paraformaldehyde fixing solution into each well, and fixing overnight; discarding paraformaldehyde fixing solution, lightly washing with water, picking out agar cover into disinfectant containing available chlorine to kill virus in agar, lightly washing with water to remove residual 4% paraformaldehyde fixing solution, and spin-drying; crystal violet was added for 5min, washed clean with tap water, air-dried, photographed on an LED lamp box, and plaques were counted, and the inhibition rate (%) =1- (average PFU number of experimental group/average PFU number of virus control wells) ×100%) was calculated. An antibody is considered to have neutralizing activity when the inhibition ratio of the antibody is more than 60% at 10. Mu.g/mL.
The results are shown in FIG. 6.
27 Of the 34 binding antibodies had neutralizing activity. In combination with EC50 values, a012, a046, a051, a073, a094, a100, a124, a129, a138 were selected for subsequent study.
4. Preparation of anti-monkey poxvirus monoclonal antibodies. The method comprises the following steps:
(1) 293F cells in the logarithmic growth phase with the activity rate higher than 90% were inoculated into fresh medium at a density of 1.5X10 6/mL, placed in a thermostatic shaker, and cultured at 37℃under a rotation speed of 5% CO 2 and 150 rpm for one day, and then the cell density and the activity rate (the activity rate is higher than 90%) were sampled and counted to adjust the cell density to 2X 10 6/mL, thereby obtaining 293F cell suspension.
(2) Extracting by using an endotoxin-free plasmid large extraction kit (Tiangen, DP 117-T) to obtain an anti-monkey pox virus monoclonal antibody A138 expression vector (the mole ratio of the heavy chain to the light chain is 1:1), regulating the concentration of the expression vector to 80 mug/mL by using a 150 mM NaCl solution, uniformly mixing, and standing at room temperature for about 5 min; diluting the 120 mu L Sinofection transfection reagent with 150 mM NaCl solution to a total volume of 0.75 and mL, uniformly mixing, and standing at room temperature for about 5 min; mixing the diluted solution containing the A138 expression vector with the diluted Sinofection transfection reagent in equal volume, and standing at room temperature for about 10min to obtain a transfection solution.
(3) The 1.5 mL transfection solution was added dropwise to the 293F cell suspension, the flask was gently shaken while dropwise, and after shaking, the flask was returned to a constant temperature shaker for continuous culture, 2% (V/V) SMS 293-SUPI feed (Sino Biological, M293-SUPI) was added sequentially at 24h, 72 h, 120 h post-transfection, and the cell culture supernatant was collected after 5 days of transfection.
(4) After filtration through a 0.45 μm filter, the cell culture supernatant was mixed with rProtein G Beads (Solarbio, R8300) and incubated 2: 2h on a shaker with slow shaking. Loading a proper amount of rProtein G Beads into a chromatographic column, balancing with a binding buffer (0.15M NaCl,20 mM Na 2HPO4 times the volume of the column, pH 7.0), adding the incubated cell culture supernatant into the balanced rProtein G Beads, and collecting effluent; the effluent was re-applied to the column and the secondary effluent was collected. Washing with 10 column volumes of wash buffer (0.15M NaCl,20 mM Na 2HPO4, pH 7.0) removed non-specifically adsorbed proteins. And (3) using an elution buffer (0.1M glycine, pH 3.0) with the volume of 10 times of the column volume, and collecting the elution solution to obtain the target protein component. Protein quantification was performed on the purified antibodies by BCA method, and electrophoresis detection was performed on the purified antibodies by SDS-PAGE.
Through detection, the protein purity of the antibody after affinity chromatography is higher, and the antibody can be used for in vitro activity analysis. Heavy and light chains after antibody melting, heavy chain size of about 55 KD and light chain size of about 25 KD.
The sequence information of antibodies a012, a046, a051, a073, a094, a100, a124, a129, a138, as sequenced, are shown below.
Antibody a012: the amino acid sequence of VH is EVQLLESGAEVKKPGESLKISCKGSGYSFTTYWIAWVRQMPGKGLEWMGIIYPGDSDTRYSPSFRGQVSISADKSINTAYLQWNSLKASDTAMYYCARPAVAGTPDAFDIWGQGTLVTVSS（SEQ ID No:1）;, wherein the VH CDR1 is GYSFTTYW (26-33 positions in SEQ ID No: 1), and the VH CDR2 is IYPGDSDT (51-58 positions in SEQ ID No: 1); VH CDR3 is ARPAVAGTPDAFDI (positions 97 to 110 in SEQ ID No: 1). The amino acid sequence of VL is DIQLTQSPSSVSASVGERVTITCRASQGVGNWLAWYQHKPGKVPKLLIHSASNLQSGVPSRFSGSGFGTDFTLTISSLQPEDFATYYCQQASSFPLTFGGGTKVEIK（SEQ ID No:2）;, wherein VL CDR1 is QGVGNW (27 th to 32 th positions in SEQ ID No. 2); VL CDR2 is SAS (SEQ ID No:2, positions 50-52); VL CDR3 is QQASSFPLT (positions 89-97 in SEQ ID No. 2). Nucleotide sequence encoding VH of GAGGTGCAGCTGTTGGAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCACCTACTGGATCGCTTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGAATTATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCGAGGCCAGGTTAGCATCTCAGCCGACAAGTCCATCAACACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACCTGCAGTGGCTGGTACGCCCGATGCTTTTGATATCTGGGGCCAAGGAACCCTGGTCACCGTCTCATCA（SEQ ID No:3）. nucleotide sequence encoding VL of GACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGAGAGAGTCACCATCACCTGTCGGGCGAGTCAGGGTGTTGGCAACTGGTTAGCCTGGTATCAGCACAAACCAGGGAAAGTCCCTAAACTCCTGATCCATTCTGCATCCAATTTGCAAAGTGGGGTCCCGTCACGCTTCAGCGGCAGTGGATTTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCCGAAGATTTTGCAACTTACTATTGTCAACAGGCTAGCAGTTTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA（SEQ ID No:4）.
Antibody a046: the amino acid sequence of VH is EVQLLESGGGLVQPGGSLRLSCVASGFTFSDHYMDWVRQAPGKGLEWVGRIRNKANRYTPEYAASVKGRFTFSRDDSKNSLYLQMNSLKIEDTAVYYCARVGPPVAAGHYDALDIWGQGTTVTVSS（SEQ ID No:5）;, wherein the VH CDR1 is GFTFSDHY (26-33 positions in SEQ ID No. 5) and the VH CDR2 is IRNKANRYTP (51-60 positions in SEQ ID No. 5); VH CDR3 is ARVGPPVAAGHYDALDI (positions 99 to 115 in SEQ ID No. 5). The amino acid sequence of VL is AIRMTQSPSFLSASVGDRVTITCRASQGISDYLAWYQQTPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPYTFGQGTKLEIK（SEQ ID No:6）;, wherein VL CDR1 is QGISDY (27 th to 32 th positions in SEQ ID No. 6); VL CDR2 is AAS (positions 50-52 in SEQ ID No: 6); VL CDR3 is QQANSFPYT (SEQ ID No:6, positions 89-97). Nucleotide sequence encoding VH of GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGTGACCACTACATGGACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATAAGAAACAAAGCTAACCGTTACACCCCAGAGTACGCCGCGTCTGTGAAAGGCAGATTCACCTTCTCAAGAGATGACTCAAAGAACTCACTGTATCTGCAAATGAACAGCCTGAAAATCGAGGACACGGCCGTCTATTACTGTGCTAGAGTAGGCCCGCCAGTAGCAGCTGGCCACTATGATGCTTTGGATATCTGGGGCCAGGGGACAACGGTCACCGTCTCATCA（SEQ ID No:7）. nucleotide sequence encoding VL of GCCATCCGGATGACCCAGTCTCCATCCTTCCTGTCTGCTTCTGTTGGGGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCGATTATTTAGCCTGGTATCAGCAAACACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA（SEQ ID No:8）.
Antibody a051: the amino acid sequence of VH is EVQLLESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAITPLSSAYGMDVWGQGTPVTVSS（SEQ ID No:9）;, wherein the VH CDR1 is GFTFSNAW (26-33 positions in SEQ ID No: 9) and the VH CDR2 is ISGSGGST (51-58 positions in SEQ ID No: 9); VH CDR3 is AITPLSSAYGMDV (positions 97 to 109 in SEQ ID No. 9). The amino acid sequence of VL is DIQMTQSPSSLSASVGDRVTITCRASQGISTWLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSRSGADFTLTINSLQPEDFATYYCQQTNNFPYTFGRGTKLDIK（SEQ ID No:10）;, wherein VL CDR1 is QGISTW (26 th to 33 th positions in SEQ ID No. 10); VL CDR2 is AAS (positions 50-52 in SEQ ID No. 10); VL CDR3 is QQTNNFPYT (SEQ ID No:10, positions 89-97). Nucleotide sequence encoding VH of GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGATAACACCCTTATCGTCCGCATACGGTATGGACGTCTGGGGCCAGGGAACCCCGGTCACCGTCTCATCA（SEQ ID No:11）. nucleotide sequence encoding VL of GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAGGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTAGATCTGGGGCAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCGACTTACTATTGTCAACAGACTAACAATTTCCCGTACACTTTTGGCCGGGGGACCAAGCTGGATATCAAG（SEQ ID No:12）.
Antibody a073: the amino acid sequence of VH is EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPMVRGVIMGDFDYWGQGTTVTVSS（SEQ ID No:13）;, wherein the VH CDR1 is GFTFSSYA (26-33 positions in SEQ ID No: 13), and the VH CDR2 is ISGSGGST (51-58 positions in SEQ ID No: 13); VH CDR3 is AKPMVRGVIMGDFDY (positions 97 to 111 in SEQ ID No. 13). The amino acid sequence of VL is DIQMIQSPSTLSASVGDRVTITCRASQSISIWLAWYQQKPGRAPKLLINAASNLQSGVPSRFSGSGSGTEFALTISSLQPEDFATYYCQQSYAKPTFGQGTTVELK（SEQ ID No:14）;, wherein VL CDR1 is QSISIW (27 th to 32 th positions in SEQ ID No. 14); VL CDR2 is AAS (positions 50-52 in SEQ ID No. 14); VL CDR3 is QQSYAKPT (positions 89-96 in SEQ ID No. 14). Nucleotide sequence encoding VH of GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAACCTATGGTTCGGGGAGTTATTATGGGGGACTTTGACTACTGGGGCCAAGGAACAACGGTCACCGTCTCATCA（SEQ ID No:15）. nucleotide sequence encoding VL of GACATCCAGATGATCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTATATGGTTGGCCTGGTATCAGCAGAAACCAGGGAGAGCCCCTAAACTCCTGATCAATGCTGCATCCAACCTCCAAAGTGGGGTCCCATCACGGTTCAGTGGCAGTGGATCCGGGACAGAATTCGCTCTCACCATCAGCAGTCTGCAACCTGAAGACTTTGCAACTTACTACTGTCAACAGAGTTACGCAAAACCGACGTTCGGCCAAGGGACCACGGTGGAACTCAAA（SEQ ID No:16）.
Antibody a094: the amino acid sequence of VH is EVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVSSITSSGRYTYYADSVRGRFTISRDNANNSLYLQMSSLRAEDTAVYYCATELGDYWGQGTMVTVSS（SEQ ID No:17）;, wherein the VH CDR1 is GFTFRNYA (26-33 positions in SEQ ID No. 17) and the VH CDR2 is ITSSGRYT (51-58 positions in SEQ ID No. 17); VH CDR3 is ATELGDY (positions 97 to 103 in SEQ ID No. 17). The amino acid sequence of VL is DIQMTQSPSSVSASVGDRVTITCRASQGISSWLSWYQQKPGRAPKLLISAASSLQTGVPSRFSGSGSGTHFTLTITSLQPEDFATYYCQQSDTFPITFGQGTRLEIK（SEQ ID No:18）;, wherein VL CDR1 is QGISSW (27 th to 32 th positions in SEQ ID No. 18); VL CDR2 is AAS (positions 50-52 in SEQ ID No: 18); VL CDR3 is QQSDTFPIT (SEQ ID No:18, positions 89-97). Nucleotide sequence encoding VH of GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACATTTAGGAACTATGCCATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCATCCATCACTAGCAGTGGTAGATACACATACTACGCAGACTCAGTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAATAACTCACTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGACAGAACTGGGTGACTACTGGGGCCAGGGGACAATGGTCACCGTCTCATCA（SEQ ID No:19）. nucleotide sequence encoding VL of GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGGGACAGAGTCACCATCACTTGTCGGGCGAGTCAAGGTATAAGTTCCTGGTTATCCTGGTATCAGCAGAAGCCAGGGAGAGCCCCTAAACTCCTGATCTCTGCTGCATCCAGTTTACAAACTGGCGTCCCATCGCGCTTCAGCGGCAGTGGATCTGGGACACATTTCACTCTCACCATCACCAGCCTGCAGCCTGAAGACTTTGCAACTTACTATTGTCAGCAGTCTGACACTTTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA（SEQ ID No:20）.
Antibody a100: the amino acid sequence of VH is EVQLLESGGGLVHPGGSLRLSCVASGFTFSRYSMNWVRQAPGKGLEWISYIDSRSGGISYADSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCVRDRFNWNNDDVFFDYWGQGTLVTVSS（SEQ ID No:21）;, wherein the VH CDR1 is GFTFSRYS (26-33 positions in SEQ ID No: 21), and the VH CDR2 is IDSRSGGI (51-58 positions in SEQ ID No: 21); VH CDR3 is VRDRFNWNNDDVFFDY (positions 97 to 112 in SEQ ID No. 21). The amino acid sequence of VL is NIQLTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLLIYAVSSLQSGVPSRFSGSQSGTDFSLTISSLQPEDFATYYCQQALSFPPTFGQGTKVEIK（SEQ ID No:22）;, wherein VL CDR1 is QSISTY (27 th to 32 th positions in SEQ ID No. 22); VL CDR2 is AVS (positions 50-52 in SEQ ID No. 22); VL CDR3 is QQALSFPPT (SEQ ID No:22, positions 89-97). Nucleotide sequence encoding VH of GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACACCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGTCGTTATAGTATGAATTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATTTCATACATTGATAGTCGTAGTGGTGGCATATCCTACGCAGACTCTGTGAAGGGCCGCTTCACCATCTCCAGAGACAACGCCAAGAACTCTCTGTCTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGTGAGAGATCGTTTCAACTGGAACAATGACGACGTGTTCTTTGACTACTGGGGCCAAGGAACACTGGTCACCGTCTCATCA（SEQ ID No:23）. nucleotide sequence encoding VL of AACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCTGTGTCCAGTTTGCAGAGTGGGGTCCCATCAAGGTTCAGCGGCAGTCAATCTGGGACAGATTTCTCTCTCACCATCAGCAGCCTGCAGCCTGAAGACTTTGCGACTTATTACTGTCAACAGGCTCTCAGTTTCCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA（SEQ ID No:24）.
Antibody a124: the amino acid sequence of VH is EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLTLADYYYYGMDVWGQGTTVTVSS（SEQ ID No:25）;, wherein the VH CDR1 is GFTFSSYS (26-33 positions in SEQ ID No: 25) and the VH CDR2 is ISSSSSYIY (51-59 positions in SEQ ID No: 25); VH CDR3 is ARDLTLADYYYYGMDV (positions 97 to 112 in SEQ ID No. 25). The amino acid sequence of VL is NIQLTQSPSSLSASVGDRVTITCRASQTITRYLNWYQHKPGKAPNLLIYGASILQSGVPSRFSGSYSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK（SEQ ID No:26）;, wherein VL CDR1 is QTITRY (27 th to 32 th positions in SEQ ID No. 26); VL CDR2 is GAS (SEQ ID No: positions 50-52 of 26); VL CDR3 is QQSYSTPYT (SEQ ID No:26, positions 89-97). Nucleotide sequence encoding VH of GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCACGCTCGCCGACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCATCA（SEQ ID No:27）. nucleotide sequence encoding VL of AACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCCTCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTACTAGGTATTTAAACTGGTATCAACACAAACCAGGGAAAGCCCCTAACCTCCTGATCTATGGTGCATCCATTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTTACTCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAG（SEQ ID No:28）.
Antibody a129: the amino acid sequence of VH is EVQLVESGGGVVQPGRSLRLSCAVSGLRLTNSGMHWVRQAPGKGLQWVAVLWSDGRNTDYEESVKGRFTISRDDSKNTLFLEMNTVGAEDTAMYYCVTDAGGSNSWEFFFDHWGQGTMVTVSS（SEQ ID No:29）;, wherein the VH CDR1 is GLRLTNSG (26-33 positions in SEQ ID No. 29) and the VH CDR2 is LWSDGRNT (51-58 positions in SEQ ID No. 29); VH CDR3 is VTDAGGSNSWEFFFDH (positions 97 to 112 in SEQ ID No. 29). The amino acid sequence of VL is NIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIK（SEQ ID No:30）;, wherein VL CDR1 is QGISSW (27 th to 32 th positions in SEQ ID No. 30); VL CDR2 is AAS (SEQ ID No:30, positions 50-52); VL CDR3 is QQANSFPFT (SEQ ID No:30, positions 89-97). Nucleotide sequence encoding VH of GAGGTACAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGTGTCCGGATTGAGATTAACTAATTCTGGGATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGCCTGCAATGGGTGGCAGTTTTGTGGTCTGATGGAAGGAATACAGACTATGAAGAGTCCGTGAAGGGCCGATTCACCATCTCAAGAGACGACTCCAAGAACACTCTGTTTTTGGAAATGAACACCGTGGGAGCCGAGGACACGGCTATGTATTACTGTGTGACAGATGCCGGTGGGAGCAATTCTTGGGAATTCTTCTTTGACCATTGGGGCCAAGGGACCATGGTCACCGTCTCATCA（SEQ ID No:31）. nucleotide sequence encoding VL of AACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA（SEQ ID No:32）.
Antibody a138: the amino acid sequence of VH is QVQLLESGGGLVKPGGSLRLSCAASGFTFRDYYMSWIREAPGKGLEWVASISRSGTTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEASSNYAFDYWGQGTPVTVSS（SEQ ID No:33）;, wherein the VH CDR1 is GFTFRDYY (26-33 positions in SEQ ID No. 33) and the VH CDR2 is ISRSGTTI (51-58 positions in SEQ ID No. 33); VHCDR3 is ARDEASSNYAFDY (SEQ ID No:33, positions 97-109). The amino acid sequence of VL is DIQLTQSPSSVSASVGDGVTITCRASQPIGTWVAWYQQKPGKAPKLLISGASSLQSGLPSRFSGRKSGTDFTLTISSLQPDDSATYFCQQTNTFPLTFGQGTRLEIK（SEQ ID No:34）; wherein VLCDR1 is QPIGTW (positions 27-32 of SEQ ID No: 34); VLCDR2 is GAS (SEQ ID No:34, positions 50-52); VLCDR3 is QQTNTFPLT (SEQ ID No:34, positions 89-97). Nucleotide sequence encoding VH of CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGGGACTACTACATGAGCTGGATCCGTGAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGCATCCATTAGTAGAAGTGGAACTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAGAGATGAGGCCTCCAGTAACTACGCATTTGACTACTGGGGCCAGGGGACCCCGGTCACCGTCTCATCA（SEQ ID No:35）. nucleotide sequence encoding VL of GACATCCAGTTGACCCAGTCTCCATCTTCCGTCTCTGCATCTGTAGGCGACGGAGTCACCATCACTTGTCGGGCGAGCCAGCCTATTGGCACCTGGGTAGCCTGGTATCAGCAAAAACCGGGGAAAGCCCCTAAGCTCCTGATCTCTGGTGCATCCAGTTTGCAAAGTGGACTCCCATCAAGGTTCAGCGGCCGTAAGTCTGGAACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGACGATTCTGCAACTTACTTTTGTCAACAGACTAACACTTTCCCTCTCACCTTCGGCCAAGGGACACGACTGGAGATTAAA（SEQ ID No:36）.
The heavy chain constant region of the above antibody has an amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID No:37）. and the light chain constant region has an amino acid sequence of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC（SEQ ID No:38）.
Example 3 (affinity and neutralization Activity analysis of antibodies)
1. Affinity detection of M1R protein.
Antibodies were serially diluted 2-fold with 4 gradients (1 XPBS, 0.02% tween (V/V), 0.5% BSA (W/V)) at an initial concentration of 200 nM. The analysis was performed from low to high concentration as shown in table 4 below. The settings shown in table 5 were made in the analysis software and curve fitting was performed.
TABLE 4 Table 4
TABLE 5
The results are shown in FIG. 7.
2. And (5) detecting neutralization activity.
Monkey poxvirus g9r+a1l Vero E6 cells and Vero cells were plated in 12 well plates. After serial dilutions of the antibodies in 3-fold 8 gradients, they were mixed with an equal volume of DMEM medium (200 PUF/mL) containing trans-complementing defective monkey poxvirus or vaccinia virus live virus (VACV) and incubated at 37 ℃ for 1h to give a mixture. Adding 100 μl of the mixture into cell plate, and culturing at constant temperature of 5% CO 2 and 37deg.C for 1 h; the plate was discarded, 1mL agar overlay (1.6 wt% agar, (2 XDMEM+pancreatin mixture (0.25% pancreatin+0.02% EDTA)) =1:1 (V/V)) was added and incubated in an incubator at 37℃for 5 days. Adding 2mL of 4% paraformaldehyde fixing solution into each well, and fixing overnight; discarding paraformaldehyde fixing solution, lightly washing with water, picking out agar cover into disinfectant containing available chlorine to kill virus in agar, lightly washing with water to remove residual 4% paraformaldehyde fixing solution, and spin-drying; and adding crystal violet for dyeing for 5min, washing with tap water, airing, photographing on an LED lamp box, counting plaques, and calculating the inhibition rate. Neutralization curves were plotted and IC50 values were calculated.
The results are shown in FIG. 8 and Table 6.
Antibodies A012, A046, A051, A073, A094, A100, A124, A129 and A138 can well neutralize trans-complementary defective monkey pox viruses and vaccinia viruses, and can be used for developing medicaments for treating infection of orthopoxviruses such as monkey pox viruses and vaccinia viruses. The neutralizing effect of the antibody A138 is best, and the IC50 of the antibody A to MPXV and VACV is respectively 0.042 mug/mL and 0.048 mug/mL.
TABLE 6
The embodiments of the present invention have been described in detail with reference to the embodiments, but the present invention is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.

Claims (13)

1. An antibody or antigen-binding fragment thereof against the M1R protein of the monkey poxvirus, characterized in that the antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region, each comprising complementarity determining regions CDR1, CDR2, CDR3;
The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26 th to 33 th, 51 th to 58 th and 97 th to 109 th positions in SEQ ID No. 33;
the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 32 th, 50 th to 52 th and 89 th to 97 th positions in SEQ ID No. 34.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises:
a911 Amino acid sequence shown as SEQ ID No. 33; or (b)
A913 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 33 and the same function as the protein shown in SEQ ID No. 33;
the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
a921 An amino acid sequence as shown in SEQ ID No. 34; or (b)
A923 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 34 and the same function as the protein shown in SEQ ID No. 34.
3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region;
the amino acid sequence of the heavy chain constant region comprises:
b11 37 of the amino acid sequence shown in SEQ ID No; or (b)
B13 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 37 and the same function as the protein shown in SEQ ID No. 37;
the amino acid sequence of the light chain constant region comprises:
b21 Amino acid sequence shown as SEQ ID No. 38; or (b)
B23 Amino acid sequence with 80%, 85% or more than 90% homology with SEQ ID No. 38 and the same function as the protein shown in SEQ ID No. 38.
4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from any one of Fab, fab ', F (ab') 2, fv, scFv, diabody, fusion antibody, and intact antibody.
5. A recombinant protein, characterized by consisting of a tag and an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.
6. A biological material associated with the antibody or antigen-binding fragment thereof of any one of claims 1 to 4, or the recombinant protein of claim 5, wherein the biological material is any one of c 1) to c 5);
c1 A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 4, or the recombinant protein of claim 5;
c2 An expression cassette comprising the nucleic acid molecule as described in c 1);
c3 A recombinant vector comprising the nucleic acid molecule as set forth in c 1) or the expression cassette as set forth in c 2);
c4 A recombinant microorganism comprising the nucleic acid molecule as described in c 1) or the expression cassette as described in c 2) or the recombinant vector as described in c 3);
c5 A transgenic cell line comprising the nucleic acid molecule as described in c 1) or the expression cassette as described in c 2) or the recombinant vector as described in c 3).
7. The biomaterial according to claim 6, wherein the nucleic acid molecule is a DNA molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a recombinant protein according to claim 5.
8. The biomaterial of claim 7, wherein the DNA molecule is any one of d 9) or d 10) and comprises a nucleotide sequence encoding the heavy chain variable region and a nucleotide sequence encoding the light chain variable region;
d9 A nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 35; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID No. 36;
d10 A DNA molecule having 80%, 85% or more than 90% homology to the nucleotide sequence defined in d 9) and encoding said antibody or antigen binding fragment thereof.
9. A conjugate, comprising: the antibody or antigen-binding fragment thereof of any one of claims 1 to 4, or the recombinant protein of claim 5;
and a coupling moiety comprising at least one of a detectable label, a drug, a toxin, a radionuclide, an enzyme.
10. The use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 4, a recombinant protein according to claim 5, a biological material according to any one of claims 6 to 8 or a conjugate according to claim 9 in any one of e 1) to e 5),
E1 Preparing a medicament for preventing and/or treating poxviruses;
e2 Non-diagnostic treatment destination to detect poxviruses;
e3 Preparing a kit for detecting poxviruses;
e4 Non-diagnostic treatment destination to detect the monkey poxvirus M1R protein;
e5 Preparing a kit for detecting the M1R protein of the monkey poxvirus;
the poxvirus is a monkey poxvirus and/or a vaccinia virus.
11. A product comprising at least one of f 1) to f 4);
f1 An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4;
f2 A recombinant protein according to claim 5;
f3 A biomaterial according to any one of claims 6 to 8;
f4 A conjugate according to claim 9;
the product is at least one selected from the group consisting of a drug, a reagent, a test plate, a chip, a test paper and a kit.
12. The product according to claim 11, wherein when the product is a medicament, it has the function indicated by g 1); the product has at least one function from g 2) to g 3) when selected from at least one of a reagent, a detection plate, a chip, a test paper and a kit;
g1 Preventing and/or treating poxviruses;
g2 Detecting the presence or amount of poxvirus;
the poxvirus is a monkey poxvirus and/or a vaccinia virus;
g3 Detecting the presence or amount of the M1R protein of the monkey poxvirus.
13. The method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a recombinant protein according to claim 5, comprising the steps of:
Obtained by culturing a recombinant microorganism or a transgenic cell line according to any one of claims 6 to 8.
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