CN105968204B - Single-domain heavy chain antibody for resisting prostate specific membrane antigen - Google Patents
Single-domain heavy chain antibody for resisting prostate specific membrane antigen Download PDFInfo
- Publication number
- CN105968204B CN105968204B CN201610074033.XA CN201610074033A CN105968204B CN 105968204 B CN105968204 B CN 105968204B CN 201610074033 A CN201610074033 A CN 201610074033A CN 105968204 B CN105968204 B CN 105968204B
- Authority
- CN
- China
- Prior art keywords
- specific membrane
- membrane antigen
- prostate specific
- chain antibody
- domain heavy
- 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
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
- B01J2220/4812—Sorbents characterised by the starting material used for their preparation the starting material being of organic character
- B01J2220/4856—Proteins, DNA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gynecology & Obstetrics (AREA)
- Pregnancy & Childbirth (AREA)
- Reproductive Health (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of genetic engineering, and particularly relates to a single-domain heavy chain antibody for prostate specific membrane antigen, which has an amino acid sequence shown in SEQ ID NO. 1 and can be used in the fields of immunodetection, antigen enrichment and purification and the like. The amino acid sequence provided by the invention can be used as a precursor, and can be transformed by random or site-directed mutagenesis technology to obtain mutants with better properties (affinity, specificity, stability and the like) for developing proteins or polypeptides for further use in medicine, industry and agriculture.
Description
Technical Field
The present invention relates to single domain heavy chain antibody technology, especially single domain heavy chain antibody or polypeptide for prostate specific membrane antigen.
Technical Field
Single domain antibodies refer to genetically engineered antibodies composed of the variable regions of common antibodies (VH or VL). A single domain heavy chain antibody (also called a nanobody, a VHH antibody) refers to a genetically engineered antibody consisting of only heavy chain antibody (heavy-chain antibody) Variable regions (Variable regions), wherein the heavy chain antibody (heavy-chain antibody) is an antibody that naturally lacks a light chain in animals such as camels and sharks. The single-domain heavy-chain antibody is the smallest known complete antigen binding fragment at present, has the characteristics of small molecular weight, good permeability and the like, and is widely applied to the fields of basic research, medical diagnosis and detection, antibody drug development and the like at present.
Prostate cancer is a malignant tumor that seriously threatens the health of old men, and early diagnosis and treatment of prostate cancer have important significance for prognosis. The Prostate Specific Membrane Antigen (PSMA) is a II-type transmembrane glycoprotein located on the membrane of prostate, consists of 750 amino acids, is divided into an intracellular region (with the amino acid sequence of 1-18), a transmembrane region (with 19-43) and an extracellular region (with 44-750), is a more sensitive and specific prostate cancer tumor marker compared with the Prostate Specific Antigen (PSA) traditionally used for clinical detection, is especially high-expressed in hormone refractory prostate cancer and prostate cancer metastasis, and has the sensitivity and specificity of 65.9 percent and 94.5 percent respectively for distinguishing prostate cancer and other types of malignant tumors. In addition, PSMA is also highly specifically expressed on tumor vascular endothelial cells in a variety of solid tumors of non-prostate origin, such as lung, bladder, gastric, pancreatic, renal, and colorectal cancers. And the polypeptide has the activity of nerve carboxyl peptidase and folate hydrolase, and an extracellular region consisting of 707 amino acids can provide a plurality of antigen epitopes, so that the polypeptide becomes an important research target in tumor immune targeted therapy and molecular imaging.
At present, it has been found that various substances capable of specifically binding to PSMA are also prepared against PSMA, including monoclonal antibodies 7E11, J591, aptamers a9 and a10, and reports of ScFv antibodies D7, Fab antibodies and humanized antibodies obtained by recombinant techniques, particularly 111 In-pendride, which has been approved by the FDA In the united states for diagnosis of prostate cancer and treatment of advanced nuclides, are based on the fact that a monoclonal antibody against PSMA is linked to a radionuclide. However, compared with single-domain heavy-chain antibodies, the ligands have the disadvantages of relatively high production cost, complex preparation process and the like.
Disclosure of Invention
The invention aims to provide a single-domain heavy chain antibody aiming at prostate-specific membrane antigen, which can be used for preparing a reagent and a tool for detecting the prostate-specific membrane antigen.
The invention provides a single domain heavy chain antibody against prostate specific membrane antigen (i.e., a single domain heavy chain antibody against prostate specific membrane antigen of the invention) having the amino acid sequence of SEQ ID No.: 1, whose amino acid sequence can be numbered and domain-partitioned by standardized antibody amino acid sequence numbering (imminogenetics, IMGT).
The present invention provides a protein or polypeptide characterized by comprising one or more than two amino acid sequences in a framework region and having at least 90% homology with one amino acid sequence.
The present invention provides a protein or polypeptide characterized by comprising one or more than two amino acid sequences in complementarity determining regions, and having at least 80% homology with one amino acid sequence.
The present invention provides a nucleic acid molecule which is characterized in that it encodes a polypeptide of SEQ ID No.: 1, the specific sequence of the nucleic acid molecule can be obtained at any time by genetic code. The sequence of the nucleic acid molecule is shown as SEQ ID No.: 2.
the invention also provides a nucleic acid molecule characterized by encoding a polypeptide of SEQ ID No.: 1, a specific sequence of the nucleic acid molecule can be obtained at any time by genetic code.
The nucleotide sequence or at least a part of the sequence provided by the invention can be expressed by a suitable expression system to obtain the corresponding protein or polypeptide. These expression systems include bacterial, yeast, filamentous fungi, animal cells, insect cells, plant cells, or cell-free expression systems.
The invention also provides a vector comprising the nucleic acid sequence. Due to the degeneracy of the genetic code, the nucleic acid sequence can vary depending on the intended use.
The invention also provides a host cell comprising the protein or expression vector.
The invention also provides a method for detecting prostate specific membrane antigen on cells, which is characterized by comprising the protein or the polypeptide. Based on the capability of the protein or polypeptide provided by the invention to be specifically combined with the prostate specific membrane antigen, a detection method of the prostate specific membrane antigen is established. Among them, preferred methods include Enzyme-linked immunosorbent assay (ELISA), Fluoroimmunoassay (FIA), immuno-chip assay, affinity chromatography and immunochromatography.
The invention aims at the application of the prostate specific membrane antigen single-domain heavy chain antibody in the immunodetection for non-disease diagnosis and treatment purposes and the enrichment and purification of thalli or antigen.
The amino acid sequence provided by the invention can be used as a precursor, and can be transformed by random or site-directed mutagenesis technology to obtain mutants with better properties (water solubility, stability, affinity, specificity and the like) for developing further proteins or polypeptides for medicine, industry and agriculture.
The invention also relates to an immunoaffinity adsorption material for the prostate specific membrane antigen, which comprises a carrier and a ligand carried on the carrier, wherein the material takes a single-domain heavy-chain antibody for the prostate specific membrane antigen as the ligand, and the single-domain heavy-chain antibody for the prostate specific membrane antigen has the amino acid sequence shown in SEQ ID No.: 1. The carrier is magnetic beads, agarose gel microspheres, silica gel microspheres or porous materials.
Some of the terms described herein have the following meanings:
homology: describing the degree of similarity of two or more amino acid sequences, the percentage of homology between a first amino acid sequence and a second amino acid sequence can be calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid sequence at the corresponding position in the second amino acid sequence by the total number of amino acids in the first amino acid sequence multiplied by [ 100% ], wherein a deletion, insertion, substitution, or addition of an amino acid in the second amino acid sequence (as compared to the first amino acid) is considered to be different. Alternatively, the percentage homology can also be obtained using known computer algorithms for sequence matching, such as NCBI Blast.
Domain (b): the basic structural unit of the tertiary structure of a protein generally has a certain function.
IMGT number: a standardized numbering scheme for The amino acid sequence of antibodies in The IMGT database (The International ImmunoGeneTiCs Datbase). Specific numbering methods can be described in the literature (Ehrenman, F., Q. Kaas, et al (2010). "IMGT/3D structure-DB and IMGT/DomainGapAlign: adataabase and a tool for immunological reagents or reagents, T cell receptors, MHC, IgSF and MhcSF." Nucleic Acids Res 38 (Database. isosse.) "D301-307. Lefranc, M.P., C.Pommie, et al (2003)" GT unique number for immunological reagents and T cell receptor domains and Igsuperfromance V-ligands "Dev. complex 27 (1): 55-www.imgt.org 25/http).
Codon (codon): also known as triplet codons, refer to nucleotide triplets corresponding to a certain amino acid. The position of insertion of this amino acid into the growing polypeptide chain is determined during translation.
The invention has the beneficial effects that: the single-domain heavy chain antibody or polypeptide for the prostate specific membrane antigen has the characteristics of specific combination with the prostate specific membrane antigen, large-scale production through a biological method, low cost, high efficiency, small molecular weight, good permeability and the like, and shows good application prospect.
Drawings
FIG. 1 shows the identification of colony PCR product electrophoresis, recombinant protein electrophoresis and Western Blot. The left Marker lane is a DNA molecular weight standard and the colony PCR fragment in lane 1 is present at the expected position. The purified protein was examined by SDS-PAGE in the middle, and a bright band appeared at the expected position. The right is a Western Blot identification chart with an anti-PSMA monoclonal antibody and an anti-His tag antibody.
Detailed Description
The present invention is further illustrated below by the preparation, analysis and use of single domain heavy chain antibodies (polypeptides), and these specific examples should not be construed in any way as limiting the scope of the invention.
Example 1
Eukaryotic expression of prostate specific membrane antigen extracellular region
Total RNA in LNCaP cells highly expressing PSMA is extracted by using an RNAioso Plus reagent, a DNA fragment encoding the PSMA extracellular region fragment is obtained by using an RT-PCR method, and the DNA fragment is inserted into a eukaryotic expression vector pRAG2a by using two restriction enzymes of Not I and BamH I and is connected into a recombinant plasmid by T4DNA ligase. The recombinant plasmid was transformed into TOP 10 competent cells by heat shock overnight and the correctly identified clones were submitted for sequencing validation. Plasmids of positive clones were extracted, DNA plasmids were transfected into HEK-293 cells using Liposome Lipofectamine 2000 for culture, supernatants were collected at different phase points and examined by SDS-PAGE electrophoresis. After a certain period of incubation, the protein was purified according to the HisTrap FF Crude kit protocol. After SDS-PAGE is carried out on the purified protein, the protein is electrically transferred to a PVDF membrane, 5 percent skim milk powder is sealed, and then PSMA antibody and His antibody are respectively added, and the mixture stays overnight at 4 ℃; after rinsing, a secondary antibody was added and incubated at room temperature for 1 hour, and rinsing was performed again, and then developing was performed by adding a developing solution (fig. 1).
Example 2
Panning and identification of anti-prostate-specific membrane antigen single domain heavy chain antibodies (i.e., anti-prostate-specific membrane antigen single domain heavy chain antibodies)
Construction and identification of a Camel-derived natural single-domain heavy-chain antibody library from a Camel-derived natural antibody phage display library (reference: Tou Zhui, xu Yang, Liu Xia, etc.; by solid-phase panning [ J)]Journal of china bioengineering, 2011, 31 (4): 31-36. "display library constructed in.") was panned against a prostate specific membrane antigen. The expression of the extracellular domain of prostate specific membrane antigen was performed as described in example 1 above, and in the first round of panning, the above synthesized PSMA extracellular domain protein was diluted to 150. mu.g/mL (coating concentrations in rounds 2-4 were 100, 50, and 50. mu.g/mL, respectively) with phosphate buffered saline (PBS, pH7.4), and 100. mu.L was added to each well of the microplate, and the wells were coated overnight at 4 ℃. PBST (containing 0.5% Tween 20) washing plate 5 times, 3% BSA-PBS at 37 degrees C blocking for 2h, washing plate 3 times, adding with 0.5% BSA-PBS incubated phage antibody library 100 u L (approximately 2X 10 binding 2X 10)11CFU), incubated at 37 ℃ for 1h, washed 5 times with PBST (3 increases from round to round), and then washed 10 times with PBS (5 increases from round to round). The adsorbed phage was then eluted with 100. mu.L of eluent (glycine-hydrochloric acid, pH 2.2) (37 ℃ C., 5min), the eluate was neutralized with 50. mu.L of Tris-HCl (1mol/L, pH 9.0), 10. mu.L of the eluate was taken for titer determination, and the remaining eluate was amplified for the next round of panning.
After four rounds of panning, the PSMA extracellular region protein with the concentration of 5 mug/mL is adopted to coat the ELISA plate, and the plate washing and sealing are the same as above. The amplified and purified phage clones were added and incubated at 37 ℃ for 15min, 100. mu.L/well and at 37 ℃ for 1 h. And adding a dilution of 1: 5000 of HRP-anti-M13 antibody, 100. mu.L/well, incubated at 37 ℃ for 1 h. PBST washing plate 5 times, adding TMB working solution 100 μ L/well, room temperature 20min, adding 50 μ L sulfuric acid (concentration 2mol/L) per well to terminate reaction, and measuring 450nm absorbance. Directly coating an enzyme label plate by using the phage antibody library amplified in the previous round as a positive control, replacing phage clone with PBS as a blank control, and measuring the binding activity of phage particles by using an indirect phase-ELISA method.
TABLE 1 Indirect phase-ELISA sample application Table
Sending the ELISA positive clone to a sequencing company for sequence determination to obtain an insert DNA sequence of the anti-prostate specific membrane antigen single-domain heavy chain antibody phage positive clone, wherein the insert DNA sequence is specifically (SEQ ID NO. 2):
ArGGCCCAGTTGCAGCTCGTGGAGTCGGGGGGAGGArTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCGCCTTCAGGAGGTATGCCATGAGCTGGGTCCGCCAGGCTCCGGGAAAGGGGCTCGAGTGGGTGTCAGAGATTAGTTATCGTGGTCGTAACACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTTCAAACGGGGCACGGCCTCAGTTCTTGCCCCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGCGGCCGC
the coded amino acid sequence is shown in SEQ ID No.: 1, and the following components:
QLQLVESGGGLVQAGGSLRLSCVASGRAFRRYAMSWVRQAPGKGLEWVSEISYRGRNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSNGARPQFLPRGQGTQVTVSS are provided. Namely, the single-domain heavy chain antibody of the invention for resisting the prostate specific membrane antigen can be specifically combined with the outer membrane region of the PSMA protein.
Example 3
ELISA and fluoroimmunoassay for PSMA-expressing cells
Gastric cancer cells MKN45 did not express PSMA, while prostate cancer cells LNCaP expressed PSMA, and using these two cells as an example, cell-level ELISA and fluorescent immunoassay were performed using the anti-prostate specific membrane antigen single domain heavy chain antibody phage positive clones obtained by panning in example 2. LNCaP cells (expressing PSMA) and MKN45 cells (not expressing PSMA), each at 1X 10, were seeded into 96-well plates4And then cultured overnight. Fixing with 4% paraformaldehyde, adding 100 μ L of 3% hydrogen peroxide solution dropwise into each well to block endogenous peroxidase activity, and incubating at 37 deg.C for 30 min. TBS plates were washed 3 times, blocked with 5% BSA-PBS, and 100. mu.L of phage positive clones of anti-prostate specific membrane antigen single domain heavy chain antibody were added and incubated for 1h at 37 ℃. Thereafter, PBS rinse, adding HRP-anti-M13 antibody, TMB working solution and OD450The measurement was the same as in example 2. The positive control used phage instead of cells and the blank control used PBS instead of added phage clones, repeated 3 times.
Cell-climbing sheets were added to each well of a 24-well plate and seeded into 4X 10 each of LNCaP cells and MKN45 cells5And then cultured overnight. Taking out the climbing film, fixing with 4% paraformaldehyde, rinsing, and dripping 3% hydrogen peroxide on the surface of the climbing film to block the activity of endogenous peroxidase. After this, 3 washes in TBS and 30min blocking with 5% BSA-PBS were performed. 100 mu L of phage clones displaying the nano antibody of the invention are dripped for incubation for 1h, and cell slide without phage clones is used as a blank control. Thereafter, the dilutions were added at 1: 2000 anti-M13 monoclonal antibody for 30 min. After washing, 30 mul of FITC-goat anti-mouse secondary antibody with dilution of 1: 200 is dropped, incubated for 30min in dark place, counterstained with DAPI staining solution, and observed under a fluorescence microscope.
The results show that: the result of the statistical difference between the measured value of MKN45 cells not expressing PSMA and the blank control shows that the phage can be non-specifically bound; however, LNCaP cytometric values were significantly higher than MKN45 cells, suggesting that this partial difference results from specific binding of PSMA and anti-prostate specific membrane antigen single domain heavy chain antibody phage positive clones. Meanwhile, cell immunofluorescence shows that LNCaP cells can be combined with single-domain heavy-chain antibody positive clones aiming at prostate specific membrane antigens, and MKN45 cells and cell creeps without anti-prostate specific membrane antigen single-domain heavy-chain antibody phage positive clones are negative, so that the selected anti-prostate specific membrane antigen single-domain heavy-chain antibody phage positive clones can be effectively and specifically combined with PSMA (prostate specific antigen) expression positive cells.
Example 4
Expression of single-domain heavy-chain antibody for resisting prostate specific membrane antigen in Escherichia coli
Extracting phagemid of the single-domain heavy chain antibody positive clone aiming at the prostate specific membrane antigen in example 2 as a template, designing a specific primer to amplify a target gene, and carrying out amplification conditions: pre-denaturation at 94 ℃ for 5 min; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 40 s; finally, the extension is carried out for 5min at 72 ℃. After the end of the detection, the detection is carried out by 1% agarose gel electrophoresis, and the target fragment is recovered by cutting the gel.
The double restriction enzyme gene fragment of the single-domain heavy chain antibody for resisting the prostate specific membrane antigen provided by the invention is cloned and connected to an expression vector pET-28a, and the recombinant plasmid is obtained after sequencing verification.
The recombinant plasmid was transformed into E.coli BL21 (Rossetta) and single colonies were picked for inducible expression. Inoculating a single colony in a test tube of an LB culture medium, carrying out shake culture at 37 ℃, and activating overnight; the next day, the cells were transferred to fresh LB liquid medium at a ratio of 1%, and cultured with shaking at 37 ℃ and 250rpm until OD was reached600After about 0.6, IPTG was added to a final concentration of 0.1mM, and induction was carried out at 37 ℃ and 250rpm for 4 hours.
Centrifuging the induced 2mL bacterial solution at 8000rpm to obtain thallus, washing the thallus for 3 times by using sterile PB S, resuspending the thallus by using 1mL sterile PBS, ultrasonically crushing the thallus on ice until the bacterial solution is clear, centrifuging cell lysate at 4 ℃, wherein the centrifugation condition is 12000rmp/min, the time is 10min, taking supernatant, adding 5 mu l of 5 xSDS supernatant sampling buffer solution, boiling the supernatant for 5min by boiling water, centrifuging, taking supernatant, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis, and purifying the supernatant by using a nickel column.
The expression quantity of the target protein (single-domain heavy chain antibody) can be further improved by optimizing induction expression conditions (such as host bacteria, expression vectors, induction time, induction temperature, IPTG concentration and the like), and a way is provided for preparing a large amount of single-domain heavy chain antibodies of the anti-prostate specific membrane antigen.
Example 5
Determination of Single Domain heavy chain antibody affinity constants against prostate specific Membrane antigen
Single domains expressed in example 4The heavy chain antibody was biotinylated using a biotinylation kit, and the affinity of the biotinylated single domain heavy chain antibody against prostate specific membrane antigen was then determined using standard competitive ELISA techniques for the recombinantly expressed PSMA protein in example 1. The method comprises the following specific steps: firstly, 1nM biotinylated single-domain heavy-chain antibody aiming at prostate specific membrane antigen is respectively incubated with 13 PSMA antigens with different concentrations (0.1 nM-100 μ M) in an EP tube for 30 min; then, 90 μ L of the mixed solution was added to an elisa plate coated with PSMA protein, which had been blocked with 3% BSA-PBST, and after incubation for 10min, the reaction solution was aspirated and washed with PBST; then, 100 μ L of HRP-labeled streptavidin with a dilution of 1: 2000 was added, incubated for 1h, and washed 5 times with PBST; use and OD of TMB working fluid450The measurement was the same as in example 2. OD by nonlinear regression analysis450Half of the maximum value, corresponding PSMA concentration, and according to the principle of antigen-antibody competitive combination experiment, the affinity constant of biotinylated single-domain heavy chain antibody aiming at prostate specific membrane antigen is 5 multiplied by 10-7and/M is about.
EXAMPLE 6 preparation of immunoaffinity adsorbing Material
1. Preparation of immunoaffinity magnetic beads
The preparation method comprises the following steps of coupling a single-domain heavy chain antibody aiming at a prostate specific membrane antigen with nano magnetic beads serving as carriers to obtain single-domain heavy chain antibody immunomagnetic beads aiming at the prostate specific membrane antigen:
1mg of carboxyl-modified magnetic beads (purchased from Stannless Baiyun nanotechnology Co., Ltd., carboxyl magnetic beads 300nm) were placed in a centrifuge tube, and 500. mu.l of activation buffer (10mM, NaH) was added2PO4pH 6.0), vortex mixing, recovering magnetic beads by a magnetic frame, and washing for 2 times by an activation buffer solution. 2mg of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added separately, mixed by vortexing, and allowed to stand for 30 min. With coupling buffer (10mM, Na)2HPO4pH7.4) of the sample, adding 1mg of a single-domain heavy-chain antibody against a prostate-specific membrane antigen dissolved in a coupling buffer solution, reacting at room temperature for 3 hours, washing the magnetic beads with the coupling buffer solution for 3 times, and adding 500. mu.l of 1% (w/v) bovine bloodAnd (3) blocking unreacted active groups by a coupling Buffer Solution of Albumin (BSA) or 1% (w/v) albumin (OVA) and reacting for 30min at room temperature. The beads were washed 3 times with coupling buffer, PBS solution (10mM, pH7.4, 0.02% w/v, Na)3N) resuspension and storage at 4 ℃.
2. And (3) preparing a single-domain heavy-chain antibody immunoaffinity adsorption material aiming at the prostate specific membrane antigen and an affinity column. The preparation method of the single-domain heavy chain antibody of the prostate specific membrane antigen by coupling agarose microspheres as a carrier comprises the following steps:
the CNBr activated dry gel was washed 10 times with 0.1M HCl and equilibrated for 5min each time. With coupling buffer (10mM, Na)2HPO4pH7.4) was added and reacted at room temperature for 4h to covalently couple the single domain heavy chain antibody against prostate specific membrane antigen to CNBr activated sepharose microspheres at room temperature for 10 washes. With coupling buffer (10mM, Na)2HP04pH7.4) was washed 2 times, and then a blocking solution was added to the reaction mixture to react at room temperature for 2 hours to block unreacted active groups. The immunoaffinity adsorption material covalently coupled with single domain heavy chain antibody against prostate specific membrane antigen was obtained by washing 3 times with 5 gel volumes of phosphate buffer (10mM, pH7.4) and acetate buffer (0.1M, pH 4.0) alternately. 0.2ml of the immunoaffinity adsorption material is put into a chromatographic column with the capacity of 1ml, washed by PBS (10mM, pH7.4) with the volume of 5-10 times of the volume of a column bed, added with 20% ethanol solution and stored at 4 ℃.
3. And (3) preparing a single-domain heavy-chain antibody immunoaffinity adsorption material aiming at the prostate specific membrane antigen and an affinity column. Silica gel microspheres are used as carriers to couple single-domain heavy chain antibodies of anti-prostate specific membrane antigens, and the specific preparation method is as follows:
taking 2g of silica gel microspheres, alternately washing the silica gel microspheres for 5-10 times by using pure water and phosphate buffer solution (PBS, 10mM, pH 6.0), suspending the silica gel microspheres by using 10ml of PBS buffer solution, adding 5mg of single-domain heavy chain antibody aiming at the prostate specific membrane antigen, uniformly mixing, adding carbodiimide (EDC) with the final concentration of 5mg/ml, rapidly and uniformly mixing, stirring at 4 ℃ for reacting for 12-24 hours to obtain the covalent coupling of the single-domain heavy chain antibody aiming at the prostate specific membrane antigenImmunoaffinity adsorption material of single-domain heavy-chain antibody. Washing 0.2ml of the immunoaffinity adsorption material in a 1ml chromatographic column with 5-10 times of column volume of PBS (10mM, pH 6), and adding 0.02% (w/v) Na3N in PBS (10mM, pH 6), stored at 4 ℃.
The ligand of the immunoaffinity adsorption material aiming at the prostate specific membrane antigen is a single-domain heavy-chain antibody, and has the amino acid sequence shown in SEQ ID No.: 1, and the ligand can specifically recognize prostate specific membrane antigen. The single-domain heavy chain antibody is easy to obtain and high in adsorption efficiency, and the aglucon can be cultured and produced in a large amount by a biological method to form the single-domain heavy chain antibody, so that complicated production methods such as artificial antibodies are avoided, and the production cost is greatly reduced. Has the characteristics of acid and alkali resistance, high temperature resistance, easy production and the like, and has important practical value for the purification and immunological detection methods with low cost and reusability of the prostate specific membrane antigen.
Claims (7)
1. The amino acid sequence of the single-domain heavy chain antibody aiming at the prostate specific membrane antigen is shown as SEQ ID NO. 1.
2. A nucleic acid molecule encoding the amino acid sequence of claim 1.
3. The nucleic acid molecule of claim 2, wherein the sequence is set forth in SEQ ID NO 2.
4. A vector comprising the nucleic acid molecule of claim 2.
5. A host cell comprising the vector of claim 4.
6. Use of the single domain heavy chain antibody against prostate specific membrane antigen of claim 1 for the preparation of reagents or materials for immunodetection, enrichment and purification of prostate specific membrane antigen.
7. An immunoaffinity adsorption material aiming at a prostate specific membrane antigen comprises a carrier and a ligand carried on the carrier, and is characterized in that the material takes a single-domain heavy-chain antibody aiming at the prostate specific membrane antigen as the ligand, and the amino acid sequence of the single-domain heavy-chain antibody aiming at the prostate specific membrane antigen is SEQ ID NO. 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610074033.XA CN105968204B (en) | 2016-02-03 | 2016-02-03 | Single-domain heavy chain antibody for resisting prostate specific membrane antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610074033.XA CN105968204B (en) | 2016-02-03 | 2016-02-03 | Single-domain heavy chain antibody for resisting prostate specific membrane antigen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105968204A CN105968204A (en) | 2016-09-28 |
| CN105968204B true CN105968204B (en) | 2020-01-21 |
Family
ID=56988664
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610074033.XA Active CN105968204B (en) | 2016-02-03 | 2016-02-03 | Single-domain heavy chain antibody for resisting prostate specific membrane antigen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105968204B (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108473590B (en) | 2016-01-12 | 2023-04-14 | 克雷森多生物制剂有限公司 | Therapeutic molecules |
| GB201607968D0 (en) | 2016-05-06 | 2016-06-22 | Crescendo Biolog Ltd | Chimeric antigen receptor |
| US11623958B2 (en) | 2016-05-20 | 2023-04-11 | Harpoon Therapeutics, Inc. | Single chain variable fragment CD3 binding proteins |
| IL263102B2 (en) | 2016-05-20 | 2023-11-01 | Harpoon Therapeutics Inc | Single domain serum albumin binding protein |
| US11535668B2 (en) | 2017-02-28 | 2022-12-27 | Harpoon Therapeutics, Inc. | Inducible monovalent antigen binding protein |
| IL300964A (en) | 2017-05-12 | 2023-04-01 | Harpoon Therapeutics Inc | Mesothelin binding proteins |
| GB201711068D0 (en) * | 2017-07-10 | 2017-08-23 | Crescendo Biologics Ltd | Therapeutic molecules binding PSMA |
| KR102429747B1 (en) | 2017-10-13 | 2022-08-05 | 하푼 테라퓨틱스, 인크. | B cell maturation antigen binding protein |
| EP3694529A4 (en) | 2017-10-13 | 2021-11-10 | Harpoon Therapeutics, Inc. | Trispecific proteins and methods of use |
| US10815311B2 (en) | 2018-09-25 | 2020-10-27 | Harpoon Therapeutics, Inc. | DLL3 binding proteins and methods of use |
| CN115768463A (en) | 2020-02-21 | 2023-03-07 | 哈普恩治疗公司 | FLT 3-binding proteins and methods of use |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010033913A1 (en) * | 2008-09-22 | 2010-03-25 | Icb International, Inc. | Antibodies, analogs and uses thereof |
-
2016
- 2016-02-03 CN CN201610074033.XA patent/CN105968204B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010033913A1 (en) * | 2008-09-22 | 2010-03-25 | Icb International, Inc. | Antibodies, analogs and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| 携载PSMA纳米抗体的超声纳米泡在靶向诊断前列腺癌中的实验研究;范校周;《中国优秀硕士学位论文全文数据库》;20150115;第12-22页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105968204A (en) | 2016-09-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105968204B (en) | Single-domain heavy chain antibody for resisting prostate specific membrane antigen | |
| CN105968201A (en) | Single-domain heavy-chain antibody aiming at prostate specific membrane antigen | |
| CN112028997B (en) | anti-CEACAM 5 nano antibody | |
| CN113150152B (en) | Specific nano antibody of human T cell surface inhibitory molecule and application thereof | |
| CN105968205B (en) | A kind of nano antibody of anti-prostate-specific membrane antigen | |
| CN105968203A (en) | Single-domain heavy chain antibody for anti-prostate specific membrane antigen extracellular region | |
| CN115286715B (en) | anti-CD3 nano antibody or antigen binding portion thereof and preparation method thereof | |
| CN114805559B (en) | Fully human anti-novel coronavirus receptor binding domain single-chain antibody No4 and application thereof | |
| WO2023024444A1 (en) | Anti-msln monoclonal internalizing antibody, preparation method therefor and application thereof | |
| CN106928358B (en) | CD105 nano antibody Nb168 | |
| CN112552408B (en) | Nano antibody targeting CAIX antigen and application thereof | |
| CN113461825A (en) | anti-PD-L2 nano antibody and application thereof | |
| CN110981959A (en) | CD47 single domain antibody, nucleotide sequence, expression vector and kit | |
| CN105524172B (en) | A kind of nano antibody of energy specific bond prostate-specific membrane antigen | |
| CN105622755B (en) | A kind of nano antibody of anti-prostate-specific membrane antigen extracellular region | |
| CN115057927B (en) | Peanut allergen Ara h1 specific nano-antibody and application thereof | |
| CN114395036B (en) | Specific nano antibody Nb2.82 for resisting bacteroides fragilis toxin protein precursor and application thereof | |
| CN110054675B (en) | Immunogenic polypeptide, anti-TTC 36 antibody CP4-3 and application | |
| CN101255194A (en) | Rabbit anti-human MK monoclonal antibody as well as hybridoma cell strain and uses thereof | |
| CN105542009A (en) | Single domain heavy-chain antibody aiming at prostate specific membrane antigen | |
| CN105504061A (en) | Nano-antibody directing at prostate-specific membrane antigen | |
| CN105968202A (en) | Single-domain heavy-chain antibody aiming at prostate specific membrane antigen extracellular region | |
| CN114478779A (en) | Humanized phosphatidylinositolglycan 3 monoclonal antibody and application thereof | |
| CN115772222A (en) | anti-CLL 1 single domain antibody and application thereof | |
| CN116023482A (en) | Neutralizing antibodies targeting coronaviruses, antigen binding fragments thereof and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |