CN111690061B - Humanized antibody against Yersinia pestis antigen F1 and application - Google Patents
Humanized antibody against Yersinia pestis antigen F1 and application Download PDFInfo
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- CN111690061B CN111690061B CN202010596195.6A CN202010596195A CN111690061B CN 111690061 B CN111690061 B CN 111690061B CN 202010596195 A CN202010596195 A CN 202010596195A CN 111690061 B CN111690061 B CN 111690061B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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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 humanized antibody against Yersinia pestis antigen F1 and its application, the light chain variable region of the antibody has SEQ ID NO 1 nucleotide sequence and SEQ ID NO 2 amino acid sequence, wherein the light chain hypervariable region sequence has SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5; the heavy chain variable region has the nucleotide sequence of SEQ ID NO.6 and the amino acid sequence of SEQ ID NO. 7, and the heavy chain hypervariable region has the nucleotide sequence of SEQ ID NO. 8, the amino acid sequence of SEQ ID NO. 9 and the amino acid sequence of SEQ ID NO. 10. The antibody is capable of specifically binding to Yersinia pestis antigen (F1).
Description
Technical Field
The invention relates to an anti-anthrax monoclonal antibody gene, a gene-coded polypeptide, a murine monoclonal antibody and a human murine chimeric antibody preparation technology, and applications of the gene and the polypeptide in preparation of anthrax therapeutic and preventive drugs.
Background
Plague is a virulent infectious disease caused by infection with Yersinia pestis (Yersinia pestis) that progresses rapidly and has a very high mortality rate if left untreated, historically resulting in the death of hundreds of millions of people. The plague of human infection is mainly due to the fact that the plague of humans can be suffered from plague after contacting with sick rodents or being bitten by infected fleas, and the plague of humans can be developed into plague septicemia or secondary plague of lung, which often causes the epidemic of the interpersonal plague. In recent years, plague has been prevalent mainly in some areas of china, africa, south america and india. While plague is currently less prevalent, yersinia pestis, which is present in natural animal hosts, often causes a small spectrum of plague outbreaks. Therefore, to avoid a wide range of plague outbreaks, an omnidirectional and multiple effective means is needed to control plagues.
At present, the prevention and treatment measures for human plague are mainly vaccination and antibiotic treatment. The vaccine must be inoculated within a certain time before exposure, multiple times of inoculation are generally needed to be effective, a large amount of antibiotics can be used for treating plague patients to control the spread of diseases, the Yersinia pestis has low resistance to the antibiotics, but the Yersinia pestis which obtains multiple resistance by transferring plasmids is separated in the Gaussa margarizan at present, and the antibiotics have certain adverse reactions and cannot be continuously used. Because of the disadvantages and limitations of vaccination and antibiotic therapy, immunological therapies with the advantages of rapidity, specificity, etc. are of great interest.
It has been shown that monoclonal antibodies (monoclonal antibodies) or polyclonal antibodies against Yersinia pestis F1 antigen, V antigen can resist subcutaneous, intranasal or aerosol challenge, for example F1 murine monoclonal antibodies F1-04-A-G1 have protective effects in mouse models of adenitis and pulmonary plague. Because of high lethality and less morbidity of the plague, the effectiveness of the novel plague vaccine in the development process needs to be proved in an animal law mode, and the effectiveness is judged by the passive protection effect of the human serum immunized by the vaccine against the infection of the yersinia pestis in the animal body. The passive immunity protection test of mice of Yersinia pestis F1 monoclonal antibody is both the validity proof of resisting plague infection and the proof of the application and method of 'animal law'.
The subject group found that the monoclonal antibody has immunoprotection effect on mice attacked by virulent pestivirus after mice are passively immunized with Yersinia pestis F1 antigen, namely, murine monoclonal antibody 4C 6. The result also indicates that the monoclonal antibody can be used as a convenient, rapid and effective plague treatment means, and provides strong evidence for developing the monoclonal antibody to treat plague in the future. Therefore, for better application in clinical treatment, the development of humanized anti-yersinia pestis F1 antibody is extremely important.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides an antibody of humanized Yersinia pestis antigen F1 and application thereof, wherein a monoclonal antibody with high affinity to plague antigen F1 is screened by a monoclonal antibody technology, amino acid sequences and nucleotide sequences of heavy chain and light chain variable regions endowed with the antibody with the specificity are obtained, and the heavy chain and light chain constant regions of the antibody are humanized and transformed, so that the antibody with specific antigen binding property, excellent toxin neutralizing activity and good animal protection function are finally obtained.
The technical scheme is as follows: a human-mouse chimeric monoclonal antibody against F1, wherein the variable region of the light chain of the antibody has the amino acid sequence shown in SEQ ID NO. 2, and the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 7.
The heavy chain variable region of the nucleic acid encoding the human-mouse chimeric monoclonal antibody has a DNA sequence shown by SEQ ID NO.6, and the light chain variable region of the nucleic acid has a DNA sequence shown by SEQ ID NO. 1.
The light chain hypervariable region has the amino acid sequences shown in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, and the heavy chain hypervariable region has the amino acid sequences shown in SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.
The monoclonal antibody is applied to the preparation of the antibody medicine for diagnosing and treating plague.
Has the advantages that: the invention provides a human-mouse chimeric monoclonal antibody hmYF202 which has high protection, high specificity and high affinity and is resistant to F1. F1 is the protein expressed by Yersinia pestis, so the monoclonal antibody hmYF202 of the invention can be applied to the research of the diagnosis, treatment and prevention of plague.
The invention takes plague antigen F1 as a target molecule to prepare murine monoclonal antibody 2D2, and prepares human murine chimeric antibody hm YF202 on the basis of YF 202. And the work doing ability of the prepared mouse monoclonal antibody YF202 and the human-mouse chimeric antibody hm YF202 can be identified. Immunological detection shows that the murine monoclonal antibody and the human murine chimeric antibody can be specifically combined with a plague F1 antigen, and in vitro neutralization experiment results prove that the YF202 and hm YF202 antibodies can effectively block the pathogenic effect of a virulent strain of Yersinia pestis. The BALB/c mouse is taken as a model, and the in vivo test proves that the protective rate of hm YF202 antibody reaches 100%.
Drawings
FIG. 1 shows the SDS-PAGE detection result of recombinant F1 protein, M, marker; 1, 2, purified F1 protein. The purity of the protein can reach more than 90 percent;
FIG. 2 shows SDS-PAGE detection of purified YF202 antibody, M, marker; 1, YF202 antibody; it can be seen that the purified antibody is very pure;
FIG. 3 is an ELISA detection result of anti-F1 monoclonal antibody YF202, which shows that the binding ability of YF202 antibody and F1 protein is strong;
fig. 4 shows the result of nucleic acid electrophoresis of the recombinant YF202 expression plasmid construct by reverse transcription PCR amplification of YF202 heavy and light chains. M, DL 2000; 1, YF202 heavy chain variable region YF202 VH; 2, YF202 light chain variable region YF202 VK;
fig. 5 shows the result of nucleic acid electrophoresis of hm YF202 recombinant expression plasmid construction, M, DL 10000; 1, recombinant expression of hm YF202 heavy chain plasmid, namely pFUSE-CHIg-hG1-hm YF 202; 2, linearizing the heavy chain template plasmid pFUSE-CHIg-hG 1; 3, hm F1-4C6 heavy chain variable region, hm YF202 VH; 4, recombinantly expressing hm YF202 light chain plasmid, namely pFUSE-CLIg-hk-hm YF 202K; 5, linearizing a light chain template plasmid pFUSE-CLIg-hk; 6, hm YF202 light chain variable region is hm YF202 VK;
FIG. 6 shows the SDS-PAGE detection of the purified antibody hm YF202, M, marker; 1, hm YF202 purified antibody; 2, control IgG; 3, culturing the supernatant; 4, flow through; 5, untransfected plasmid 293F culture supernatant;
FIG. 7 shows the result of ELISA detection of anti-F1 monoclonal antibody hm YF 202.
Detailed Description
Example 1hm preparation of YF202 antibody
Through the detection of the mouse monoclonal antibody at the earlier stage of the subject group, a YF202 hybridoma cell strain is selected to prepare a human-mouse chimeric antibody hm YF 202.
1) Amplification and verification of antibody variable region gene fragments:
culturing YF202 antibody hybridoma cells to logarithmic growth phase, and extracting total RNA of the cells by a Trizol-chloroform-isopropanol method; the total RNA of the dried cells was dissolved in 20. mu.L of water, and OD260/OD280 was measured, which was 1.9. 14. mu.L of RNA was used for reverse transcription, mRNA in total RNA was used as a template, and OligodT was used 15 As a primer, single-stranded cDNA is obtained by reverse transcription amplification.
Designing 19 VH upstream primers and 17V kappa upstream primers, 4 VH downstream primers and 3V kappa downstream primers:
v κ 5' upstream primer:
Vκ-1
5’-GGG CCC AGG CGG CCG AGC TCG AYA TCC AGC TGA CTC AGC C-3’
Vκ-2
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCW CCC AGT C-3’
Vκ-3
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGM TMA CTC AGT C-3’
Vκ-4
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGY TRACAC AGT C-3’
Vκ-5
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TRA TGA CMC AGT C-3’
Vκ-6
5’-GGG CCC AGG CGG CCG AGC TCG AYATTM AGA TRA MCC AGT C-3’
Vκ-7
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTC AGA TGA YDC AGT C-3’
Vκ-8
5’-GGG CCC AGG CGG CCG AGC TCG AYA TYC AGA TGA CAC AGA C-3’
Vκ-9
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCA WCC AGT C-3’
Vκ-10
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG WGC TSA CCC AAT C-3’
Vκ-11
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTS TRA TGA CCC ART C-3’
Vκ-12
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTK TGA TGA CCC ARA C-3’
Vκ-13
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CBC AGK C-3’
Vκ-14
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TAA CYC AGG A-3’
Vκ-15
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CCC AGW T-3’
Vκ-16
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CAC AAC C-3’
Vκ-17
5’-GGG CCC AGG CGG CCG AGC TCG AYA TTT TGC TGA CTC AGT C-3’
v kappa 3' downstream primer
VκR1
5’-AGA TGG TGC AGC CAC AGT TCG TTT KAT TTC CAG YTT GGT CCC-3’
VκR2
5’-AGA TGG TGC AGC CAC AGT TCG TTT TAT TTC CAA CTT TGT CCC-3’
VκR3
5’-AGA TGG TGC AGC CAC AGT TCG TTT CAG CTC CAG CTT GGT CCC-3’
VH 5' upstream primer
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTR MAG CTT CAG GAG TC-3’
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTB CAG CTB CAG CAG TC-3’
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAG CTG AAG SAS TC-3’
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAR CTG CAA CAR TC-3’
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTY CAG CTB CAG CAR TC-3’
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTY CAR CTG CAG CAG TC-3’
VH 7
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAC GTG AAG CAG TC-3’
VH 8
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAS STG GTG GAA TC-3’
VH 9
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AWG YTG GTG GAG TC-3’
VH 10
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAG SKG GTG GAG TC-3’
VH 11
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAM CTG GTG GAG TC-3’
VH 12
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG CTG ATG GAR TC-3’
VH 13
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAR CTT GTT GAG TC-3’
VH 14
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTR AAG CTT CTC GAG TC-3’
VH 15
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAR STT GAG GAG TC-3’
VH 16
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTT ACT CTR AAA GWG TST G-3’
VH 17
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAA CTV CAG CAR CC-3’
VH 18
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAC TTG GAA GTG TC-3’
VH 19
5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG GTC ATC GAG TC-3’
VH 3' downstream primer
VH R1
5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC CGT GGT-3’
VH R2
5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC TGT GAG AGT GGT-3’
VH R3
5’-CGA TGG GCC CTT GGT GGA GGC TGC AGA GAC AGT GAC CAG AGT-3’
VH R4
5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC TGA GGT-3’
Dissolving V kappa 5 'upstream primer, V kappa 3' downstream primer, VH 5 'upstream primer and VH 3' downstream primer to final concentration of 100 pmol/. mu.L, mixing uniformly according to the volume ratio of 1:1, and respectively amplifying V H 、V L The amplification conditions of the gene are 95 ℃ for 4 minutes, 95 ℃ for 30 seconds, 60 ℃ for 40 seconds, 72 ℃ for 45 seconds and 30 cycles; finally, extending for 10 minutes at 72 ℃, electrophoretically recovering, purifying and amplifying the gene fragment (shown in figure 4), connecting the gene fragment to a pMD-18T vector, transforming escherichia coli DH5 alpha, and obtaining the variable region sequences of a light chain and a heavy chain after sequencing, wherein the nucleotide sequence of VH is shown as SEQ ID NO. 6; the nucleotide sequence of VL is shown in SEQ ID NO. 1.
2) Design of primers for PCR and Gene amplification
Heavy chain amplification primers:
F：5’-GGTGTCCACTCGCTAGATGTGCAGCTGCAGGAATCGGGACCT-3’
R：5’-GCCCTTGGTGGATGCTGCAGAGACAGTGAC-3’
light chain amplification primers:
F：5’-ACAGACGCTCGCTGCCAAATTGTGCTCACTCAGTCTCCAG-3’
R：5’-TGCAGCCACCGTACGTTTGATTTCCAGTTTGGTC-3’
3) amplification of hm YF202 antibody heavy and light chains
The heavy chain and light chain variable regions are connected to pMD18-T vector for accurate sequencing analysis as templates, and the heavy chain and light chain genes of the human-mouse chimeric antibody are amplified by the upstream and downstream primers of the related heavy chain and light chain respectively.
(1)PCR
Reaction system:
the reaction conditions are as follows:
(2) performing 2% agarose gel electrophoresis, observing a target band under ultraviolet, cutting the gel and recovering.
(3) And purifying the target DNA fragment by using a gel recovery kit, and eluting by using deionized water.
4) Double restriction enzyme IgG expression plasmid
The IgG expression plasmids pFUSE-CHIg-hG1, pFUSE-CLIg-hk (from Invivogen) contained base coding sequences for the heavy and light chain (Kappa) constant regions of human origin of IgG 1.
(1) And carrying out double digestion on pFUSE-CHIg-hG1 and pFUSE-CLIg-hk template vectors.
Reaction system:
reaction conditions are as follows: the cleavage was carried out overnight at 37 ℃.
(2) Electrophoresis was performed on 1% agarose gel, and the gel was recovered by cutting under ultraviolet.
(3) And purifying the target DNA fragment by using a gel recovery kit, and eluting by using deionized water.
5) Infusion PCR recombinant expression plasmid
Reaction system:
reaction conditions are as follows: incubate at 50 ℃ for 15 min.
5 mul of reaction solution was taken to transform competent bacteria, spread on the corresponding resistant plates, and the next day clones were picked for sequencing. And cloning and preserving strains with correct sequencing results, carrying out amplification culture, and extracting plasmids.
As a result, an expression plasmid of hmYF202 was successfully constructed. See fig. 6.
6) Expression of hm YF202 antibody
(1) mu.L of pFUSE-CHIg-hG1-hmYF202H (i.e., 50. mu.g) was placed in 1mL of Opti-MEM medium, 250. mu.L of pFUSE-CLIg-hk-hmYF202K (i.e., 50. mu.g) was placed in 1mL of Opti-MEM medium, 200. mu.L of 293Fectin was placed in 2.8mL of Opti-MEM medium, and the three mixtures were allowed to stand at room temperature for 5 min.
(2) Then, after the two plasmid mixed solutions are uniformly mixed, 500 mu L of Opti-MEM culture medium is added and uniformly mixed, the mixed solution of the transfection reagent 293Fectin is directly added, and the mixture is uniformly mixed and then stands for 20 min. During the treatment, 293F cells were centrifuged, resuspended in 293F Expression Medium, counted and the cell viability ratio calculated with the blue plate, pipetted 90X 10 6 The cells were placed in a flask and were brought to a volume of 94mL using 293F Expression Medium.
(3) After 20min, 6mL of the DNA, 293Fectin complex was added to the prepared 293F cells.
(4) Culturing the cells in a shaking incubator with 8% CO 2 Cell supernatants were collected after 6 days at 120rmp, 37 ℃.
7) Purification of hm YF202 antibody
The collected cell supernatant was filtered through a 0.22 μm filter while the balance and the eluate were filtered through the filter. The Protein A was purified using an AKATA purifier according to standard procedures for Protein A purification, loading at 1mL/min, and eluting at 1.5 mL/min.
Results hm YF202 antibody was successfully expressed and purified. SDS-PAGE is shown in FIG. 6.
8) hm YF202 purified antibody enzyme-linked immunosorbent assay
Diluting the F1 protein with coating solution (0.1M carbonate buffer, pH9.6) to 2. mu.g/mL coated ELISA 96-well plate, adding 100. mu.L per well, and standing overnight at 4 ℃; PBST (PBS containing 0.5% Tween20) 5% skimmed milk-wash buffer blocked and incubated at 37 ℃ for 2 h; after 5 PBST washes, 100. mu.L of YF202 (2. mu.g/mL starting concentration, 15 concentration gradient dilutions) was added to each well overnight at 4 ℃; adding 100 μ L/well of goat-anti-mouse secondary antibody (Beijing Chinese fir) diluted at 1:4000 into the well, and incubating at 37 deg.C for 1 h; the peroxidase substrate color developing solution is 100 mu L/hole, the reaction is stopped by 2M sulfuric acid after 15 minutes at room temperature, and the double-wavelength 450nm/690nm is adopted for machine detection and color comparison.
The results are shown in FIG. 7: the purified monoclonal antibody hm YF202 and F1 protein have good binding activity and can recognize the conformational epitope of the protein antigen.
Sequence listing
<110> prevention and control center for east war zone disease of people liberation army in China
<120> humanized antibody against yersinia pestis antigen F1 and application
<160> 57
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gacattgtga tgacccagtc tccaaaattc atgtccacat cagtaggaga cagggtcagc 60
gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtatca acagaaacca 120
gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat 180
cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240
gaagacttgg cagagtattt ctgtcagcaa tataacagct atccgtacac gttcggaggg 300
gggaccaagc tggaaataaa acg 323
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Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
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Gln Asn Val Gly Thr Asn
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ctggaatgga tgggctacat acactacagt ggtagcacta actacaaccc atctctcaaa 180
agtcgaatct ctatcactcg agacacatcc aagaaccagt tcttcctgca gttgaattct 240
gtgactactg aggacacagc cacatattac tgtgcaagaa actatgctat ggactactgg 300
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Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr
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Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly Tyr Ser Trp His Trp
20 25 30
Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile His
35 40 45
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser
50 55 60
Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser
65 70 75 80
Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Asn Tyr Ala
85 90 95
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105
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<213> Artificial Sequence (Artificial Sequence)
<400> 15
gggcccaggc ggccgagctc gayattgtra tgacmcagtc 40
<210> 16
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gggcccaggc ggccgagctc gayattmaga tramccagtc 40
<210> 17
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gggcccaggc ggccgagctc gayattcaga tgaydcagtc 40
<210> 18
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gggcccaggc ggccgagctc gayatycaga tgacacagac 40
<210> 19
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gggcccaggc ggccgagctc gayattgttc tcawccagtc 40
<210> 20
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gggcccaggc ggccgagctc gayattgwgc tsacccaatc 40
<210> 21
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gggcccaggc ggccgagctc gayattstra tgacccartc 40
<210> 22
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gggcccaggc ggccgagctc gayattktga tgacccarac 40
<210> 23
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gggcccaggc ggccgagctc gayattgtga tgacbcagkc 40
<210> 24
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gggcccaggc ggccgagctc gayattgtga taacycagga 40
<210> 25
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gggcccaggc ggccgagctc gayattgtga tgacccagwt 40
<210> 26
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gggcccaggc ggccgagctc gayattgtga tgacacaacc 40
<210> 27
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gggcccaggc ggccgagctc gayattttgc tgactcagtc 40
<210> 28
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
agatggtgca gccacagttc gtttkatttc cagyttggtc cc 42
<210> 29
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
agatggtgca gccacagttc gttttatttc caactttgtc cc 42
<210> 30
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
agatggtgca gccacagttc gtttcagctc cagcttggtc cc 42
<210> 31
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gctgcccaac cagccatggc cctcgaggtr magcttcagg agtc 44
<210> 32
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gctgcccaac cagccatggc cctcgaggtb cagctbcagc agtc 44
<210> 33
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gctgcccaac cagccatggc cctcgaggtg cagctgaags astc 44
<210> 34
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gctgcccaac cagccatggc cctcgaggtc carctgcaac artc 44
<210> 35
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gctgcccaac cagccatggc cctcgaggty cagctbcagc artc 44
<210> 36
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gctgcccaac cagccatggc cctcgaggty carctgcagc agtc 44
<210> 37
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gctgcccaac cagccatggc cctcgaggtc cacgtgaagc agtc 44
<210> 38
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gctgcccaac cagccatggc cctcgaggtg aasstggtgg aatc 44
<210> 39
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gctgcccaac cagccatggc cctcgaggtg awgytggtgg agtc 44
<210> 40
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gctgcccaac cagccatggc cctcgaggtg cagskggtgg agtc 44
<210> 41
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gctgcccaac cagccatggc cctcgaggtg camctggtgg agtc 44
<210> 42
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gctgcccaac cagccatggc cctcgaggtg aagctgatgg artc 44
<210> 43
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gctgcccaac cagccatggc cctcgaggtg carcttgttg agtc 44
<210> 44
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gctgcccaac cagccatggc cctcgaggtr aagcttctcg agtc 44
<210> 45
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gctgcccaac cagccatggc cctcgaggtg aarsttgagg agtc 44
<210> 46
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gctgcccaac cagccatggc cctcgaggtt actctraaag wgtstg 46
<210> 47
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gctgcccaac cagccatggc cctcgaggtc caactvcagc arcc 44
<210> 48
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gctgcccaac cagccatggc cctcgaggtg aacttggaag tgtc 44
<210> 49
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gctgcccaac cagccatggc cctcgaggtg aaggtcatcg agtc 44
<210> 50
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cgatgggccc ttggtggagg ctgaggagac ggtgaccgtg gt 42
<210> 51
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
cgatgggccc ttggtggagg ctgaggagac tgtgagagtg gt 42
<210> 52
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
cgatgggccc ttggtggagg ctgcagagac agtgaccaga gt 42
<210> 53
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
cgatgggccc ttggtggagg ctgaggagac ggtgactgag gt 42
<210> 54
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
ggtgtccact cgctagatgt gcagctgcag gaatcgggac ct 42
<210> 55
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gcccttggtg gatgctgcag agacagtgac 30
<210> 56
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
acagacgctc gctgccaaat tgtgctcact cagtctccag 40
<210> 57
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tgcagccacc gtacgtttga tttccagttt ggtc 34
Claims (3)
1. A human-mouse chimeric monoclonal antibody against Yersinia pestis antigen F1, characterized in that the variable region of the light chain of said antibody has the amino acid sequence shown in SEQ ID NO. 2, and the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 7.
2. Nucleic acid encoding the human-murine chimeric monoclonal antibody according to claim 1, characterized in that the heavy chain variable region has the DNA sequence shown in SEQ ID NO.6 and the light chain variable region has the DNA sequence shown in SEQ ID NO. 1.
3. Use of the monoclonal antibody of claim 1 or 2 for the preparation of a medicament for the diagnosis and treatment of plague.
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| CN202010596195.6A CN111690061B (en) | 2020-06-28 | 2020-06-28 | Humanized antibody against Yersinia pestis antigen F1 and application |
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| CN114560932B (en) * | 2022-02-18 | 2023-09-12 | 中国人民解放军军事科学院军事医学研究院 | Plague neutralizing antibody and application thereof |
| CN117106080B (en) * | 2023-10-13 | 2024-03-01 | 青海省地方病预防控制所 | Antibodies of human origin against yersinia pestis LcrV and related products and uses thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108034005A (en) * | 2011-10-07 | 2018-05-15 | Ac免疫有限公司 | Identify the phosphorylation specific antibody of Tau |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6001358A (en) * | 1995-11-07 | 1999-12-14 | Idec Pharmaceuticals Corporation | Humanized antibodies to human gp39, compositions containing thereof |
| CN1831536B (en) * | 2005-03-08 | 2010-05-05 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunization chromatograph test paper for detecting plague yersinia and preparation method thereof |
| NZ601815A (en) * | 2008-03-31 | 2014-10-31 | Genentech Inc | Compositions and methods for treating and diagnosing asthma |
| CN101531709B (en) * | 2009-04-24 | 2012-12-05 | 中国疾病预防控制中心传染病预防控制所 | Method for cutting Yersinia pestis F1 antigen and relative application thereof |
| SG10201501342UA (en) * | 2010-02-24 | 2015-04-29 | Immunogen Inc | Folate receptor 1 antibodies and immunoconjugates and uses thereof |
| CN102206612A (en) * | 2010-10-09 | 2011-10-05 | 云南省地方病防治所 | Hybridoma cell and preparation method and application thereof |
| RS55987B1 (en) * | 2012-11-20 | 2017-09-29 | Sanofi Sa | Anti-ceacam5 antibodies and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108034005A (en) * | 2011-10-07 | 2018-05-15 | Ac免疫有限公司 | Identify the phosphorylation specific antibody of Tau |
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