CN112321708B - Peanut allergen Arah3 specific nano antibody and application thereof - Google Patents
Peanut allergen Arah3 specific nano antibody and application thereof Download PDFInfo
- Publication number
- CN112321708B CN112321708B CN202011313082.7A CN202011313082A CN112321708B CN 112321708 B CN112321708 B CN 112321708B CN 202011313082 A CN202011313082 A CN 202011313082A CN 112321708 B CN112321708 B CN 112321708B
- Authority
- CN
- China
- Prior art keywords
- ser
- gly
- ala
- artificial sequence
- arg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- 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]
-
- 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
- C07K2317/567—Framework region [FR]
-
- 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
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- 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
-
- 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
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a peanut allergen Arah3 specific nano antibody and application thereof, wherein the specific nano antibody is at least one of a nano antibody P84, a nano antibody P99, a nano antibody P72, a nano antibody P43, a nano antibody P91 or a nano antibody P178. The peanut allergen Arah3 specific nano antibody has the advantages of short preparation period, low cost and high stability, and can be used for construction of an immunodetection system.
Description
Technical Field
The invention belongs to the field of immunodetection, and particularly relates to a peanut allergen Arah3 specific nano antibody and application thereof.
Background
The development of traditional immunodetection techniques for food allergen components based on screening and production of monoclonal or polyclonal antibodies requires injection of high purity allergen proteins during animal immunization and longer screening and production periods for antibodies. A Heavy Chain antibody (HCAbs) naturally occurs in the peripheral blood of camelids and naturally lacks the light and Heavy Chain first constant regions (CH1) as compared to conventional monoclonal Antibodies. Cloning and expressing the heavy chain variable region of the heavy chain antibody to obtain the antigen recognition and binding domain of the heavy chain antibody, which is called Nanobody (Nb). The nanobody has structural stability and antigen binding activity equivalent to those of the original heavy chain antibody, and is the smallest antibody unit capable of binding to a target antigen known at present. The traditional immunization process needs to prepare high-purity allergen protein, the traditional monoclonal antibody has long preparation period and high cost, and compared with the traditional monoclonal antibody fragment, the nano antibody has many advantages. The nano antibody has extremely high solubility, is not easy to aggregate and precipitate, has very high stability, can keep the antigen binding activity under the denaturing conditions of high temperature, strong acid, strong alkali and the like, and is suitable for prokaryotic and eukaryotic expression systems. At present, the nano antibody is widely applied to the basic scientific research fields of developing therapeutic antibody drugs, diagnostic reagents (colloidal gold method, enzyme-linked immunosorbent assay, electrochemiluminescence method), affinity purification matrixes, immunological research and the like.
Disclosure of Invention
In view of the above, the invention provides a peanut allergen Arah3 specific nano antibody and application thereof, aiming at overcoming the defects in the prior art.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
the peanut allergen Arah3 specific nano antibody is at least one of nano antibody P84, nano antibody P99, nano antibody P72, nano antibody P43, nano antibody P91 or nano antibody P178.
Further, the specific nanobody comprises 4 framework regions FR1, FR2, FR3, FR4 and 3 complementarity determining regions CDR1, CDR2, CDR 3;
for nanobody P84: the amino acid sequence of FR1 is shown as SEQ ID NO.1, the amino acid sequence of FR2 is shown as SEQ ID NO.2, the amino acid sequence of FR3 is shown as SEQ ID NO.3, the amino acid sequence of FR4 is shown as SEQ ID NO.4, the amino acid sequence of CDR1 is shown as SEQ ID NO.5, the amino acid sequence of CDR2 is shown as SEQ ID NO.6, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 7;
for nanobody P99: the amino acid sequence of FR1 is shown as SEQ ID NO.8, the amino acid sequence of FR2 is shown as SEQ ID NO.9, the amino acid sequence of FR3 is shown as SEQ ID NO.10, the amino acid sequence of FR4 is shown as SEQ ID NO.11, the amino acid sequence of CDR1 is shown as SEQ ID NO.12, the amino acid sequence of CDR2 is shown as SEQ ID NO.13, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 14;
for nanobody P72: the amino acid sequence of FR1 is shown as SEQ ID NO.15, the amino acid sequence of FR2 is shown as SEQ ID NO.16, the amino acid sequence of FR3 is shown as SEQ ID NO.17, the amino acid sequence of FR4 is shown as SEQ ID NO.18, the amino acid sequence of CDR1 is shown as SEQ ID NO.19, the amino acid sequence of CDR2 is shown as SEQ ID NO.20, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 21;
for nanobody P43: the amino acid sequence of FR1 is shown as SEQ ID NO.22, the amino acid sequence of FR2 is shown as SEQ ID NO.23, the amino acid sequence of FR3 is shown as SEQ ID NO.24, the amino acid sequence of FR4 is shown as SEQ ID NO.25, the amino acid sequence of CDR1 is shown as SEQ ID NO.26, the amino acid sequence of CDR2 is shown as SEQ ID NO.27, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 28;
for nanobody P91: the amino acid sequence of FR1 is shown as SEQ ID NO.29, the amino acid sequence of FR2 is shown as SEQ ID NO.30, the amino acid sequence of FR3 is shown as SEQ ID NO.31, the amino acid sequence of FR4 is shown as SEQ ID NO.32, the amino acid sequence of CDR1 is shown as SEQ ID NO.33, the amino acid sequence of CDR2 is shown as SEQ ID NO.34, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 35;
for nanobody P178: the amino acid sequence of FR1 is shown as SEQ ID NO.36, the amino acid sequence of FR2 is shown as SEQ ID NO.37, the amino acid sequence of FR3 is shown as SEQ ID NO.38, the amino acid sequence of FR4 is shown as SEQ ID NO.39, the amino acid sequence of CDR1 is shown as SEQ ID NO.40, the amino acid sequence of CDR2 is shown as SEQ ID NO.41, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 42;
the 4 framework regions and 3 complementarity determining regions of the specific nanobody are arranged in the sequence of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4.
Further, the amino acid sequence of the VHH of the nano antibody P84 is shown in SEQ ID NO. 43; the amino acid sequence of the VHH of the nano antibody P99 is shown in SEQ ID NO. 44; the amino acid sequence of the VHH of the nano antibody P72 is shown in SEQ ID NO. 45; the amino acid sequence of the VHH of the nano antibody P43 is shown in SEQ ID NO. 46; the amino acid sequence of the VHH of the nano antibody P91 is shown in SEQ ID NO. 47; the amino acid sequence of the VHH of the nano antibody P178 is shown in SEQ ID NO. 48.
The peanut allergen Arah3 specific nano antibody is applied to food allergen immunoassay.
The application of the peanut allergen Arah3 specific nano antibody and the application of the specific nano antibody in food allergen epitope identification.
The peanut allergen Arah3 specific nano antibody is applied to the purification and tracing of the antibody-based allergen.
Compared with the prior art, the invention has the following advantages:
the peanut allergen Arah3 specific nano antibody has the advantages of short preparation period, low cost and high stability, and can be used for preparation of allergen specific nano antibodies in food components and construction of detection systems.
Drawings
FIG. 1 is an electrophoretogram of total peanut protein according to an embodiment of the present invention;
FIG. 2 is a bar graph of peanut allergen nanobody enrichment according to embodiments of the present invention;
FIG. 3 is a bar graph of the results of ELISA screening according to the present invention;
FIG. 4 is a graph of peanut allergen nanobody purification according to an embodiment of the present invention;
FIG. 5 is a protein electrophoresis diagram of a peanut allergen nanobody according to an embodiment of the present invention;
FIG. 6 is an immunoblot of purified peanut allergen nanobodies according to embodiments of the present invention;
FIG. 7 is an electrophoresis diagram of a Nanobody-targeted peanut allergen protein according to an embodiment of the present invention;
FIG. 8 is an immunoblot of a Nanobody-targeted peanut allergen protein according to an embodiment of the present invention;
FIG. 9 is a graph of the thermal stability of nanobodies according to embodiments of the present invention;
fig. 10 is a graph of nut and lupin protein cross-reactivity with peanut allergen protein nanobodies, in accordance with an embodiment of the present invention.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention will be described in detail with reference to examples.
Example 1
1. Extraction of peanut Total protein
The method comprises the steps of taking peanuts as raw materials, carrying out grinding and crushing, acetone degreasing, Tris-HCl salting-out crude extraction, ammonium sulfate fractional precipitation and other methods, and then carrying out centrifugation, ultrafiltration and PBS dialysis to obtain the peanut total protein. The protein concentration of the peanuts is determined by using a BCA method. The total peanut protein was characterized by SDS-PAGE gel electrophoresis, as shown in FIG. 1.
2. Alpaca immunity and nano antibody library construction based on total protein
The adult alpaca is immunized six times a week with the extracted peanut whole protein, and blood is taken from the jugular vein of the alpaca 3-4 days after the last immunization. The density gradient centrifugation method is adopted to extract the lymphocytes in the blood. Total RNA was extracted from lymphocytes using the TRIzol method. Reverse transcription is carried out by taking RNA as a template to generate cDNA. Then, VHH gene fragments were amplified by two PCRs using cDNA as a template. The first PCR was performed to amplify the fragment between the VHH and CH2 regions using CALL001 and CALL002 as primers. In a 1% agarose gel electrophoresis, two bands of approximately 900bp and 700bp were obtained, and the band at 700bp was recovered by cutting the gel using a Qiagen gel extraction kit (Qiagen). Then, second PCR was carried out using PMCF and A6E as primers and the recovered product of the first PCR as primers to obtain VHHs fragments. In 1% agarIn the glycogel electrophoresis, the VHH gene fragment is located at about 400 bp. Purification was performed using the Qiaquick PCR purification kit. And carrying out double digestion on the PCR product and the PMECs plasmid vector by using two restriction enzymes PstI and NotI, and connecting the digested PCR product and the PMECs plasmid vector under the action of T4 DNA ligase to form a recombinant plasmid containing the VHH gene fragment. Using Phenol: chloroform: isoamylolysis (25: 24: 1) purified recombinant plasmids. Then the recombinant plasmid is electrically transformed into Escherichia coli TG1 competent cells, the electrotransformation product is recovered for 1h by using SOC culture medium, the recovered bacterial liquid is coated on an LB solid plate containing 20% of glucose and ampicillin, and the plate is inversely incubated at 37 ℃ overnight. Colony counts were performed every other day and library volumes were calculated for the library. Randomly picking a single strain, and carrying out colony PCR by using the primer G III and the primer MP57 to determine the correct insertion rate of the VHH gene fragment in the nano antibody library in the recombinant plasmid. Collecting the strain on LB plate with scraper, re-suspending with PBS, washing, diluting with different concentrations, and determining OD 600 The library capacity of the library was calculated. Freezing and storing at minus 80 ℃.
3. Screening and identification of specific nanobodies
And (3) performing three rounds of panning on the nano antibody antibodies in the library by using a phage display technology of the helper phage M13K07, and performing multiple enrichment on the specific nano antibody. Adding the frozen nanobody into 2 × TY culture medium, and incubating at room temperature to exponential phase (OD) 600 0.5 to 0.6), adding 10 to 12 The helper phage M13K07 was incubated at room temperature for 30min to infect E.coli, the pellet was collected by centrifugation, resuspended, and cultured in 2 XTY medium again, and incubated overnight at room temperature to construct a phage library. Centrifuging at 8000rpm to remove thallus precipitate, mixing the obtained supernatant with PEG/NaCl solution according to the ratio of 4: 1(W/W), fully settling the phage-PEG/NaCl complex, centrifuging, and resuspending. The phage content was determined using an ultramicrospectrophotometer. To pan to specific nanobodies, we panned them using the phage ELISA method. Peanut allergen protein coatings were incubated overnight in 96-well plates. The wells were discarded and washed 5 times with PBST. Adding 250 partsMu.l of skim milk powder containing 2% was sealed in a 96-well plate and allowed to stand at room temperature for 1 hour. The wells were discarded and washed 5 times with PBST. Add resuspended phage 10 per well 11 And incubating for 1h at room temperature to ensure that the specific phage is fully combined with the antigen. The wells were discarded and washed with PBST. The first round of elutriation is washed 10 times, the second round of elutriation is washed 20-25 times, and the third round of elutriation is washed 20 times. To each well 100 μ L of TEA solution was added to destroy the specifically bound antigen and phage particles in the "+" well and the remaining background in the "-" well, respectively. Maximum value incubate 10 Min. Add 100. mu.L/well of 1M Tris to neutralize the TEA solution and transfer all free phage to a sterile centrifuge tube. Of these, 10. mu.l was used for phage enrichment, 10. mu.l was used as a backup, and the remaining 180. mu.l was re-expanded for phage culture in preparation for the next round of panning. 10 μ L of enriched phage was diluted 10-fold to 10 -7 Equal amounts of the phages at different dilutions were incubated for 30min at room temperature to de-infect E.coli. Then, the infected E.coli were applied in equal amounts sequentially to LB agar plates, incubated overnight at 37 ℃ and observed for enrichment of specific phages. The remaining phage were used for re-scale up and prepared for the next round of panning. Three rounds of panning were performed in series, and the third round did not require extensive phage culture, as shown in FIG. 2.
Randomly selecting 190 single strains in the elutriation enrichment plate, firstly smearing the single strains on an LB solid culture medium for a plurality of times to preserve the strains, then soaking the strains in an LB + Amp liquid culture medium corresponding to a 96-well plate, and incubating overnight at 37 ℃. The overnight suspension was transferred to 1ml of 2 XTY culture in a 96-deep well plate and incubated at room temperature with shaking to OD 600 1. And adding IPTG with the final concentration of 1mM to induce the expression of the nano antibody protein. After induction, the precipitate is left by centrifugation, a 96 deep-hole plate is wrapped by aluminum foil paper, and then the plate is placed at minus 80 ℃ to generate ice crystals through cold shock so as to break cell wall membranes to obtain the protein. Peanut allergen protein was coated in 96-well microplate and incubated overnight at 4 ℃. The wells were discarded and washed 5 times with PBST. Adding 250 μ l of blocking solution containing 2% skimmed milk powder to block the 96-well ELISA plate, incubating at room temperature for 1h, discarding the wellThe liquid of (4), which was washed 5 times with PBST. And (3) resuspending the precipitate obtained by cold shock by using PBS, oscillating and incubating for 2h at room temperature, centrifuging, taking the supernatant, adding the supernatant into a 96-hole enzyme label plate, and performing direct ELISA (enzyme linked immunosorbent assay) reaction to select specific strains. The murine anti-HA monoclonal antibody (1: 5000, PBS dilution) and alkaline phosphatase-labeled goat anti-murine monoclonal antibody (1: 5000, PBS dilution) were combined with the nanobody and the specific antibody binding to the antigen was detected. Measuring OD 5min, 15min, 30min, and 60min after adding color developing agent 405 And (5) nm. And selecting the strain with the absorbance value meeting the requirement. Colony PCR was performed on the selected strains using GIII and MP57 as primers and the PCR products were subjected to gene sequencing. Selecting strains with different gene sequences, performing direct ELISA again to identify the specificity of the strains, and performing plasmid extraction and plasmid sequencing in the strains at the same time. Comparing the sequencing results of the two times, specific strains were selected, and the results are shown in FIG. 3.
4. Expression and purification of specific nano antibody
Electrically transforming plasmids with different gene sequences into competent cells of Escherichia coli WK6, and culturing in TB liquid culture medium at 37 deg.C until OD 600 To between 0.6 and 0.9. IPTG was added to induce the strain to express specific proteins. The expressed antibody protein was then released using a double osmotic shock protocol. The expressed antibody protein is purified by using Ni-column affinity chromatography and size exclusion chromatography. And (3) after the filler is treated by PBS (phosphate buffer solution) in the Ni-column affinity chromatography, uniformly mixing the filler and the extracted nano antibody protein, and shaking and incubating for 1h at room temperature to combine the filler and the nano antibody protein. After loading the linker into the PD-10 column, the column was washed with PBS for contaminating proteins. Eluting nanometer antibody protein with 0.5M imidazole, 1ml each time, and measuring OD of the eluate after multiple elutions 280 nm until it is below 0.2. Size exclusion chromatography Using AKTA purification apparatus and superdex TM The eluted protein is further purified by a gel purification column. Firstly, washing and balancing the gel column and the flow pipeline by PBS, loading the sample into the gel column, washing by PBS until balancingUntil now, the liquid corresponding to the peak appearing in the middle of washing was collected, then the remaining hetero-proteins in the gel column were washed with 1M NaOH +1M NaCl, the pH value in the gel column was neutralized with 200mM Tris-HCl, and finally the gel column was stored in 20% ethanol in preparation for the next purification. The purity and molecular weight of the collected purified nanobody were identified using SDS-PAGE and immunoblotting experiments. The concentration of nanobodies was measured using a super-differential spectrophotometer, and the results are shown in FIGS. 4 to 6.
5. Confirmation of specific nano antibody targeting allergen
And (3) confirming the target protein of the specific nano antibody by using Co-immunoprecipitation (Co-IP) and high-resolution liquid chromatography mass spectrometry (LC-MS/MS). Incubating Pro A/G pre-coated Agarose gel beads (Agarose beads-Pro A/G) and Mouse anti-His tag monoclonal antibody (Mouse anti-His MAb) to form Agarose beads-Pro A/G-Mouse anti-His MAb compound, incubating nano antibody and peanut protein to form antibody-antigen compound, mixing the two compounds to combine the Mouse anti-His MAb with the nano antibody, and obtaining target protein of the nano antibody through centrifugal precipitation. Analyzing the position of the target protein of the nano antibody by a Western blot experiment, separating the compound by SDS-PAGE protein electrophoresis, cutting a target protein band according to the Western blot result, carrying out in-gel enzyme hydrolysis, carrying out LC-MS/MS analysis and identification, and comparing the sequence of the analysis result with the sequence of Uniprot DB to determine the allergen protein, wherein the result is shown in figures 7-8.
6. Nanobody affinity assay
And (3) detecting the affinity of the nano antibody to the antigen by direct ELISA. Peanut allergen antigen was coated overnight in 96-well plates at 4 ℃. One negative control was made for each nanobody. Add 200. mu.L of blocking solution (2% Milk) per well and block for 1h at room temperature. Nanobody proteins were diluted in 10000/1000/100/50/10/1/0.1/0nM gradient and added to the corresponding wells and incubated for 1h at room temperature. A mouse anti-HA monoclonal antibody (1: 3000, PBS dilution) is added as a primary antibody, an HRP-labeled goat anti-mouse monoclonal antibody (1: 3000, PBS dilution) is added as a secondary antibody, and TMB is added as a substrate for color development. After the color development was completed, 1M H was used 2 SO 4 The reaction was terminated. Measuring OD 450 . The apparent affinity of Nbs for peanut allergen proteins was estimated as the concentration of nanobodies corresponding to half the maximum signal produced.
7. Thermal stability of Nanobodies
In a real-time PCR detection system, the thermal stability of the purified nano antibody is measured by a thermal fluorescence method. The purified nanobody was concentrated to a final concentration of 2.5 mg/ml. Subsequently, 15. mu.l of nanobody protein was mixed uniformly with 5. mu.l 1/100 diluted SYPRO orange protein gel dye and 10. mu.l sterile PBS to a total volume of 30. mu.l. Each nanobody was done in triplicate. Gel dyes containing only PBS and SYPRO orange protein were used as blanks. The temperature of the real-time fluorescent PCR program was increased from 25 ℃ to 95 ℃ at a rate of 0.5 ℃/min. The raw data were analyzed using a non-linear fitting method, and the stability of the nanobody to stability was determined, and the results are shown in fig. 9.
8. Specificity of Nanobodies
Peanut allergen protein is loaded on a gel membrane through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), then the gel membrane is transferred on an NC membrane through a Western Blot experiment, the protein on the NC membrane is cut according to strips, and different nano antibodies are used for incubation for 1h at room temperature. It was washed 5 times for 3min using PBST. Adding a mouse anti-HA monoclonal antibody (diluted by PBS at a ratio of 1: 5000) as a primary antibody, adding an HRP-labeled goat anti-mouse monoclonal antibody (diluted by PBS at a ratio of 1: 5000) as a secondary antibody, and taking TMB as a substrate for color development. After the completion of color development, the reaction was terminated by rinsing with water. The bands appearing on the NC film were observed.
9. Cross-reaction of nanobodies
The cross reaction of the peanut allergen protein nano antibody to nut and lupin protein is detected. Peanuts, nuts and lupins were coated in equal amounts in 96-well microtiter plates and incubated overnight at 4 ℃. Three replicates were run for each experiment. Add 200. mu.L of blocking solution (2% Milk) per well and block for 1h at room temperature. Adding a quantitative peanut allergen nano antibody into the hole, and incubating for 1h at room temperature. Adding mouse anti-HA monoclonal antibody (1: 3000, PBS diluted) as primary antibody, and HRP-labeled goat anti-mouse monoclonal antibody (1: 3000, PBS diluted) as secondary antibodyReleased) as secondary antibody and TMB as substrate for color development. After the color development was completed, 1M H was used 2 SO 4 The reaction was terminated. Measuring OD 450 . The cross-reactivity of peanut allergen protein nanobody to nut and lupin protein was determined and the results are shown in fig. 10.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> university of southern kayak
<120> peanut allergen Arah3 specific nano antibody and application thereof
<130> 2020.11.18
<160> 48
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser
20
<210> 2
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val Ala
1 5 10 15
Ala
<210> 3
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Ser Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 4
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 5
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Cys Ala Ala Ser Gly Arg Thr Leu Ser Arg Tyr Ser
1 5 10
<210> 6
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ile Ser Arg Ser Gly Gly Ser
1 5
<210> 7
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Ala Ala Gly Ser Val Gly Thr Thr Ile Arg Gly Tyr Gly Tyr
1 5 10
<210> 8
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser
20
<210> 9
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val Ala
1 5 10 15
Ala
<210> 10
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser
1 5 10 15
Ala Lys Asn Thr Val Ser Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 11
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 12
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Cys Ala Ala Ser Gly Arg Thr Leu Ser Arg Tyr Ser
1 5 10
<210> 13
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Ile Ser Arg Ser Gly Gly Ser
1 5
<210> 14
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Ala Lys Tyr Arg Arg Asp Gly Leu Arg Ser Pro Tyr Asp Tyr
1 5 10
<210> 15
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser
20
<210> 16
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala
1 5 10 15
Gly
<210> 17
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Pro Lys Asn Thr Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp
20 25 30
Thr Ala Lys Tyr Tyr Cys
35
<210> 18
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Leu Gly Arg Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 19
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Cys Ala Ala Ser Gly Arg Thr Leu Ser Ser Tyr Ala
1 5 10
<210> 20
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Ile Ser Arg Tyr Gly Gly Arg Thr
1 5
<210> 21
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Gly Arg Arg Ala Asn Ala Asp Phe Gly Val
1 5 10
<210> 22
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser
20
<210> 23
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Gly Val Ser
1 5 10 15
Cys
<210> 24
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Ser Tyr Ala Ala Ser Val Lys Asp Arg Phe Ser Ile Ser Lys Asp Asp
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Gly Val Tyr Tyr Cys
35
<210> 25
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 26
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Cys Thr Ser Pro Lys Thr Thr Leu Asn Tyr Tyr Ser
1 5 10
<210> 27
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Ile Tyr Gly Ser Gly Gly Tyr Thr
1 5
<210> 28
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Ala Thr Tyr Tyr Thr Gly Arg Asn Ser Cys Ser Val Gly Asn Pro Ser
1 5 10 15
Trp Tyr Glu Ser
20
<210> 29
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser
20
<210> 30
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Met Gly Trp Phe Arg Gln Ala Leu Gly Lys Glu Arg Glu Tyr Val Ala
1 5 10 15
Ala
<210> 31
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Asn Tyr Ala Asp Phe Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu His Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 32
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 33
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Cys Ala Ala Ser Gly Arg Ala Phe Ala Asn His Ala
1 5 10
<210> 34
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Ile Ser Trp Ser Gly Asp Ile Thr
1 5
<210> 35
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Ala Val Gly Thr Ile Tyr Gly Gly Pro Tyr Leu Gln Ser Ser Ser Asp
1 5 10 15
Tyr Asp Tyr
<210> 36
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser
20
<210> 37
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val Ala
1 5 10 15
Ala
<210> 38
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 39
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 40
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Tyr Thr
1 5 10
<210> 41
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Ile Ser Arg Ser Gly Gly Ser Thr
1 5
<210> 42
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Ala Ala Ser Arg Gln Ala Arg Pro Leu Ala Thr Thr Met Ser Ser Tyr
1 5 10 15
Asp Arg
<210> 43
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser Arg Tyr
20 25 30
Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val
35 40 45
Ala Ala Ile Ser Arg Ser Gly Gly Ser Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Gly Ser Val Gly Thr Thr Ile Arg Gly Tyr Gly Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 44
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser Arg Tyr
20 25 30
Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val
35 40 45
Ala Ala Ile Ser Arg Ser Gly Gly Ser Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Tyr Arg Arg Asp Gly Leu Arg Ser Pro Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 45
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Arg Tyr Gly Gly Arg Thr Thr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Lys Tyr Tyr Cys
85 90 95
Gly Arg Arg Ala Asn Ala Asp Phe Gly Val Leu Gly Arg Gly Thr Gln
100 105 110
Val Thr Val Ser Ser
115
<210> 46
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ser Pro Lys Thr Thr Leu Asn Tyr Tyr
20 25 30
Ser Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Glu Gly Val
35 40 45
Ser Cys Ile Tyr Gly Ser Gly Gly Tyr Thr Ser Tyr Ala Ala Ser Val
50 55 60
Lys Asp Arg Phe Ser Ile Ser Lys Asp Asp Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys
85 90 95
Ala Thr Tyr Tyr Thr Gly Arg Asn Ser Cys Ser Val Gly Asn Pro Ser
100 105 110
Trp Tyr Glu Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 47
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Ala Asn His
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Leu Gly Lys Glu Arg Glu Tyr Val
35 40 45
Ala Ala Ile Ser Trp Ser Gly Asp Ile Thr Asn Tyr Ala Asp Phe Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu His Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Gly Thr Ile Tyr Gly Gly Pro Tyr Leu Gln Ser Ser Ser Asp
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 48
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val
35 40 45
Ala Ala Ile Ser Arg Ser Gly Gly Ser Thr Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ser Arg Gln Ala Arg Pro Leu Ala Thr Thr Met Ser Ser Tyr
100 105 110
Asp Arg Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Claims (5)
1. A peanut allergen Arah3 specific nano antibody is characterized in that: the specific nano antibody is a nano antibody P43;
the specific nanobody comprises 4 framework regions FR1, FR2, FR3, FR4 and 3 complementarity determining regions CDR1, CDR2 and CDR 3;
for nanobody P43: the amino acid sequence of FR1 is shown as SEQ ID NO.22, the amino acid sequence of FR2 is shown as SEQ ID NO.23, the amino acid sequence of FR3 is shown as SEQ ID NO.24, the amino acid sequence of FR4 is shown as SEQ ID NO.25, the amino acid sequence of CDR1 is shown as SEQ ID NO.26, the amino acid sequence of CDR2 is shown as SEQ ID NO.27, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 28;
the 4 framework regions and 3 complementarity determining regions of the specific nanobody are arranged in the sequence of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4.
2. The peanut allergen Arah 3-specific nanobody of claim 1, wherein: the amino acid sequence of the VHH of the nano antibody P43 is shown in SEQ ID NO. 46.
3. The use of the specific nanobody of peanut allergen Arah3 as claimed in claim 1, wherein: the specific nano antibody is applied to peanut allergen immunoassay.
4. The use of the specific nanobody of peanut allergen Arah3 as claimed in claim 1, wherein: the specific nano antibody is applied to the identification of peanut allergen epitope.
5. The use of the specific nanobody of peanut allergen Arah3 as claimed in claim 1, wherein: the specific nano antibody is applied to purification and tracing of peanut allergen based on the antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011313082.7A CN112321708B (en) | 2020-11-20 | 2020-11-20 | Peanut allergen Arah3 specific nano antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011313082.7A CN112321708B (en) | 2020-11-20 | 2020-11-20 | Peanut allergen Arah3 specific nano antibody and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112321708A CN112321708A (en) | 2021-02-05 |
| CN112321708B true CN112321708B (en) | 2022-09-20 |
Family
ID=74321924
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011313082.7A Active CN112321708B (en) | 2020-11-20 | 2020-11-20 | Peanut allergen Arah3 specific nano antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112321708B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115057927B (en) * | 2022-05-30 | 2024-02-20 | 南开大学 | Peanut allergen Ara h1 specific nano-antibody and application thereof |
| WO2024147891A1 (en) * | 2023-01-03 | 2024-07-11 | Harpoon Therapeutics, Inc. | Nectin4 targeting trispecific protein for treatment of cancer |
-
2020
- 2020-11-20 CN CN202011313082.7A patent/CN112321708B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| Nanobodies—Useful Tools for Allergy Treatment?;Flicker S et al.;《Frontiers in Immunology》;20200930(第11期);全文 * |
| Screening of nanobody specific for peanut major allergen Arah3 by phage display;Chen F et al.;《Journal of agricultural and food chemistry》;20190813;第67卷(第40期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112321708A (en) | 2021-02-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111018985B (en) | Application of single-domain antibody aiming at bovine serum albumin BSA | |
| CN112321708B (en) | Peanut allergen Arah3 specific nano antibody and application thereof | |
| CN105399827B (en) | Wasabi protein nanos antibody and its coded sequence and application | |
| CN112500482B (en) | Gold glucose specific nano antibody and double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) method thereof | |
| CN111793132A (en) | Monoclonal antibody of human procalcitonin and preparation method and application thereof | |
| CN111057148B (en) | Single-domain antibody aiming at bovine serum albumin BSA and derivative protein thereof | |
| CN111138533B (en) | Single domain antibody against hepatitis A virus and derived protein thereof | |
| CN110423277B (en) | Nano antibody of PD-1 and clinical application thereof | |
| CN112321709B (en) | Macadamia nut allergen Vicilin specific nano antibody and application thereof | |
| CN105524173B (en) | Nano antibody aiming at humanized antibody Fc fragment and application thereof | |
| CN112390880B (en) | Lupin allergen Lupan1 specific nano antibody and application thereof | |
| CN110372793B (en) | Nano antibody of PD-L1 and clinical application thereof | |
| CN112010969B (en) | High-affinity enhanced green fluorescent protein nano antibody and screening method of coding gene thereof | |
| CN111138532B (en) | Use of single domain antibodies against hepatitis a virus | |
| CN110396128B (en) | Preparation of anti-CD 19 nano antibody | |
| CN109734804B (en) | Nano antibody aiming at H3K64Ac/H3K64 fragment and application thereof | |
| CN113583119B (en) | Anti-staphylococcus aureus nanobody Nb56, application and kit | |
| CN113461814B (en) | Nano antibody for specifically recognizing vibrio parahaemolyticus, recombinant vector, host cell and application thereof | |
| CN112794910B (en) | anti-PD-1 nano antibody and application thereof | |
| Lin et al. | Improved affinity of a chicken single-chain antibody to avian infectious bronchitis virus by site-directed mutagenesis of complementarity-determining region H3 | |
| CN113980127A (en) | Nano antibody for resisting methicillin-resistant staphylococcus and preparation method and application thereof | |
| CN113461816A (en) | Nano antibody aiming at green fluorescent protein GFP and application thereof | |
| CN113912729B (en) | Single-domain antibody aiming at sST2, and derivative protein and application thereof | |
| CN114773462B (en) | Recombinant single-chain antibody for detecting bovine CRP protein and application thereof | |
| CN115057927B (en) | Peanut allergen Ara h1 specific nano-antibody and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |